EP4677544A2 - Systeme, vorrichtungen und verfahren zur analyse biologischer proben - Google Patents

Systeme, vorrichtungen und verfahren zur analyse biologischer proben

Info

Publication number: EP4677544A2
Authority: EP; European Patent Office
Prior art keywords: fiducial; tissue; frame; fixture; tissue fixture
Prior art date: 2023-03-10
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Pending

Application number

EP24771501.4A

Other languages

English (en)

French (fr)

Inventor

Shawn Mcguire

Shane M. MCMAHON

Arjun ACHARYA

Clay M. Thompson

Nicholas A. GEISSE

Jacob FLEMING

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Curi Bio Inc

Original Assignee

Curi Bio Inc

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2023-03-10

Filing date

2024-03-10

Publication date

2026-01-14

2024-03-10 Application filed by Curi Bio Inc filed Critical Curi Bio Inc

2026-01-14 Publication of EP4677544A2 publication Critical patent/EP4677544A2/de

Status Pending legal-status Critical Current

Links

Classifications

- G—PHYSICS
- G06—COMPUTING OR CALCULATING; COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Rigid containers without fluid transport within
- B01L3/5085—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/251—Colorimeters; Construction thereof
- G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection

Definitions

Such tissues may move spontaneously or may be moved by electrical stimuli or by other means, either in contraction or tension.
US2019/0029549A1 discloses approaches whereby tissue movement is detected with the use of a magnet embedded in a scaffold encompassed by a tissue. Changes in a magnetic field at a single magnetic field sensor adjacent to the magnet can, in theory, be correlated to the movement of the magnet by the tissue. This can allow for measurement of tissue movements with greater throughput and sensitivity than be achieved using conventional methods such as optical microscopy.
the camera-based approach disclosed in US2024/0027655 requires a separate lens array disposed between the camera and well plate to achieve the necessary resolution to image spatially-separated regions of interest (ROIs); however, the intermediate lens array renders the instrument incapable of imaging other plate geometries that are not compatible with the lens array.
Martins et al. teaches away from polymer micropillars in favor of illuminated optical fiber microprobes which double as tissue supports and illuminated beacons that facilitate imaging; however, the apparent cost and complexity of this approach renders it poorly-suited for high throughput commercial applications.
US2023/0023752 teaches away from molded polymer micropillars in favor of 3-D printed micropillars characterized by fiducial extensions from the head portion in a direction substantially parallel to the plane of a base element.
this approach is poorly suited for commercial applications where 3-D printed geometries are impractical.
the foregoing approaches focus on structural advances, they generally do not teach novel methods which may be utilized in connection with the tissue samples to reduce image processing times and improve measurement accuracy. [0008] Accordingly, there is a need for improved tissue measurement systems, devices, and methods to overcome shortcomings in magnetic and optical tissue tracking approaches.
a system comprising: a tissue testing plate, comprising: a first tissue fixture extending into a well of the tissue testing plate, the first tissue fixture comprising a first fiducial; and a second tissue fixture extending into the well of the tissue testing plate, the second tissue fixture comprising a second fiducial; an optical detector comprising a pixel array including a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit columns; and a non- Docket No.
CURI-P4WO transitory machine readable storage medium storing instructions, which when executed by a processor, causes the system to perform operations, including: imaging, with the optical detector, a plurality of frames of the first fiducial and the second fiducial during a movement of the first tissue fixture and/or the second tissue fixture, wherein each of the plurality of frames is imaged with each pixel circuit row at different time points during the movement of the first tissue fixture and/or the second tissue fixture; identifying at least one of the first fiducial or the second fiducial in a first frame of the plurality of frames by identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of at least a portion of the pixel circuit rows of the first frame; and identifying the at least one of the first fiducial or the second fiducial in a second frame of the plurality of frames by identifying the at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of at least
Clause 2 The system of clause 1, wherein the at least one edge extends at an angle with respect to the pixel circuit rows.
Clause 3 The system of clause 2, wherein the at least one edge is linear.
Clause 4 The system of clause 2 or clause 3, wherein the angle of the at least one edge is greater than 0 degrees and less than 90 degrees.
Clause 5 The system of any one of clauses 2-4, wherein the angle of the at least one edge is greater than or equal to approximately 30 degrees and less than or equal to approximately 75 degrees.
Clause 6 The system of any one of clauses 2-5, wherein the angle of the at least one edge is greater than or equal to approximately 40 degrees and less than or equal to approximately 60 degrees.
Clause 7 The system of any one of clauses 2-6, wherein the angle of the at least one edge is approximately 45 degrees.
Clause 8 The system of any one of clauses 2-7, wherein the at least one of the first fiducial or the second fiducial comprises parallel continuous edges extending at the angle, and wherein the identification of the at least one of the first fiducial or the second fiducial in the first frame and in the second frame comprises identifying the parallel continuous edges.
Clause 9 The system of any one of clauses 2-8, wherein the at least one of the first fiducial or the second fiducial comprises a plurality of markers, each of the markers comprising Docket No.
Clause 12 The system of clause 11, wherein the identification of the at least one of the first fiducial or the second fiducial in the second frame comprises identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest.
Clause 13 The system of clause 12, wherein the at least a portion of pixel circuit rows of the second frame in which the at least one edge of the at least one of the first fiducial or the second fiducial is identified corresponds to the predicted second region of interest.
Clause 14 The system of clause 12 or clause 13, wherein the system is further configured to perform a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest and to determine a sample parameter of a biological sample based upon the measurement, and wherein the biological sample is disposed between the first tissue fixture and the second tissue fixture.
Clause 15 The system of any one of clauses 1-14, wherein the first fiducial optically contrasts with the first tissue fixture and the second fiducial optically contrasts with the second tissue fixture with respect to at least one of a shape, size, color, brightness, luminescence, pixel intensity or pixel density.
Clause 16 The system of any one of clauses 1-15, wherein at least one of the first fiducial or the second fiducial comprises an overmolding on an end of the respective first tissue fixture or second tissue fixture.
Clause 17 The system of any one of clauses 1-16, wherein at least one of the first fiducial or the second fiducial comprises an insert disposed within the respective first tissue fixture or second tissue fixture. Docket No. CURI-P4WO [0027]
Clause 18 The system of any one of clauses 1-17, wherein at least one of the first fiducial or the second fiducial comprises a prominence disposed on an end of the respective first tissue fixture or second tissue fixture.
Clause 19 The system of any one of clauses 1-18, wherein identifying the at least one of the first fiducial or the second fiducial in the first frame comprises identifying the at least one of the first fiducial or the second fiducial in the first frame according to a first optical threshold; and wherein identifying the at least one of the first fiducial or the second fiducial in the second frame of the plurality of frames comprises identifying the at least one of the first fiducial or the second fiducial in the second frame according to a different second optical threshold.
Clause 20 The system of any one of clauses 1-19, wherein the tissue testing plate comprises a mounting lid, a first post assembly from which the first tissue fixture extends, and a second post assembly from which the second tissue fixture extends; and a body couplable to the mounting lid, wherein the body defines the well, wherein the first post assembly and the second post assembly couple to the mounting lid or the body.
a system comprising: a tissue testing plate, comprising: a first tissue fixture extending into a well of the tissue testing plate, the first tissue fixture comprising a first fiducial; and a second tissue fixture extending into the well of the tissue testing plate, the second tissue fixture comprising a second fiducial; an optical detector comprising a pixel array including a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit columns; and a non- transitory machine readable storage medium storing instructions, which when executed by a processor, causes the system to perform operations, including: imaging, with the optical detector, a plurality of frames of the first fiducial and the second fiducial during a movement of the first tissue fixture and/or the second tissue fixture, wherein each frame is imaged with each of the plurality of pixel circuits of the pixel array; identifying at least one of the first fiducial or the second fiducial in a first frame of the plurality of frames by identifying at least one edge of the at least
Clause 22 The system of clause 21, wherein the angle of the at least one edge is greater than or equal to approximately 30 degrees and less than or equal to approximately 75 degrees. Docket No. CURI-P4WO [0032]
Clause 23 The system of clause 21 or clause 22, wherein the angle of the at least one edge is greater than or equal to approximately 40 degrees and less than or equal to approximately 60 degrees.
Clause 24 The system of any one of clauses 21-23, wherein the angle of the at least one edge is approximately 45 degrees.
Clause 25 The system of any one of clauses 21-24, wherein the at least one of the first fiducial or the second fiducial comprises parallel continuous edges extending at the angle, and wherein the identification of the at least one of the first fiducial or the second fiducial in the first frame and in the second frame comprises identifying the parallel continuous edges.
Clause 26 The system of any one of clauses 21-25, wherein the at least one of the first fiducial or the second fiducial comprises a plurality of markers, each of the markers comprising parallel continuous edges extending at the angle, and wherein the identification of the at least one of the first fiducial or the second fiducial in the first frame and in the second frame comprises identifying the parallel continuous edges of at least one of the plurality of markers.
Clause 27 The system of any one of clauses 21-26, wherein the system is further configured to identify a first region of interest in the first frame based upon the identification of the at least one of the first fiducial or the second fiducial in the first frame.
Clause 28 The system of clause 27, wherein the system is further configured to predict a second region of interest in the second frame based upon at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame.
Clause 29 The system of clause 28, wherein the identification of the at least one of the first fiducial or the second fiducial in the second frame comprises identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest.
Clause 30 The system of clause 29, wherein the system is further configured to perform a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest and to determine a sample parameter of a biological sample based upon the measurement, and wherein the biological sample is disposed between the first tissue fixture and the second tissue fixture.
Clause 31 The system of any one of clauses 21-30, wherein each of the plurality of frames is imaged with each pixel circuit row at different time points during the movement of the first tissue fixture and/or the second tissue fixture, wherein the identification of the at least one Docket No.
CURI-P4WO of the first fiducial or the second fiducial in the first frame comprises identifying the at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of at least a portion of the pixel circuit rows of the first frame, and wherein the identification of the at least one of the first fiducial or the second fiducial in the second frame comprises identifying the at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of at least a portion of the pixel circuit rows of the second frame.
Clause 32 The system of any one of clauses 21-31, wherein the first fiducial optically contrasts with the first tissue fixture and the second fiducial optically contrasts with the second tissue fixture with respect to at least one of a shape, size, color, brightness, luminescence, pixel intensity or pixel density.
Clause 33 The system of any one of clauses 21-32, wherein at least one of the first fiducial or the second fiducial comprises an overmolding on an end of the respective first tissue fixture or second tissue fixture.
Clause 34 The system of any one of clauses 21-33, wherein at least one of the first fiducial or the second fiducial comprises an insert disposed within the respective first tissue fixture or second tissue fixture.
Clause 35 The system of any one of clauses 21-34, wherein at least one of the first fiducial or the second fiducial comprises a prominence disposed on an end of the respective first tissue fixture or second tissue fixture.
Clause 36 The system of any one of clauses 21-35, wherein identifying the at least one of the first fiducial or the second fiducial in the first frame comprises identifying the at least one of the first fiducial or the second fiducial in the first frame according to a first optical threshold; wherein identifying the at least one of the first fiducial or the second fiducial in the second frame of the plurality of frames comprises identifying the at least one of the first fiducial or the second fiducial in the second frame according to a different second optical threshold.
CURI-P4WO causes the system to perform operations, including: imaging a plurality of frames of the first fiducial and the second fiducial during a movement of the first tissue fixture and/or the second tissue fixture; identifying a first region of interest in a first frame of the plurality of frames based upon identifying at least one of the first fiducial or the second fiducial in the first frame; predicting a second region of interest in a second frame of the plurality of frames based upon at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame; and identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest in the second frame.
Clause 38 The system of clause 37: wherein the instructions further include performing a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest; and determining a sample parameter of a biological sample based upon the measurement, wherein the biological sample is disposed between a first tissue fixture and a second tissue fixture.
Clause 39 The system of clause 37 or clause 38, wherein the first tissue fixture is a rigid tissue fixture and the second tissue fixture is a deformable tissue fixture.
Clause 40 The system of any one of clauses 37-39, wherein the first tissue fixture and the second tissue fixture are deformable tissue fixtures.
Clause 41 The system of any one of clauses 37-40, wherein at least one of a first centroid of the first fiducial is substantially aligned with a centroid of the first tissue fixture or a second centroid of the second fiducial is substantially aligned with a centroid of the second tissue fixture.
Clause 42 The system of any one of clauses 37-41, wherein the first fiducial optically contrasts with the first tissue fixture and the second fiducial optically contrasts with the second tissue fixture with respect to at least one of a shape, size, color, brightness, luminescence, pixel intensity or pixel density.
Clause 43 The system of any one of clauses 37-42, wherein the first fiducial and the second fiducial comprise an engineered contrast enhancement.
Clause 44 The system of any one of clauses 37-43, wherein at least one of the first fiducial or the second fiducial comprises an overmolding on an end of the respective first tissue fixture or second tissue fixture. Docket No. CURI-P4WO [0054]
Clause 45 The system of any one of clauses 37-44, wherein at least one of the first fiducial or the second fiducial comprises an insert disposed within the respective first tissue fixture or second tissue fixture.
Clause 46 The system of any one of clauses 37-45, wherein at least one of the first fiducial or the second fiducial comprises a prominence disposed on an end of the respective first tissue fixture or second tissue fixture.
Clause 47 The system of clause 46, wherein the prominence has a slash, spherical, rectangular, or cross shape.
Clause 48 The system of clause 46 or clause 47, wherein the prominence has an edge extending at an angle with respect to a plurality of pixel circuit rows of the optical detector.
Clause 49 The system of any one of clauses 37-48, wherein the optical detector comprises a camera.
Clause 50 The system of any one of clauses 37-49, further comprising a light source configured to illuminate the first fiducial and the second fiducial.
Clause 51 The system of any one of clauses 37-50, wherein the optical detector comprises a pixel array including a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit columns and each frame of the plurality of frames is imaged with each of the plurality of pixel circuits of the pixel array, and wherein each of the plurality of frames is imaged with each pixel circuit row at different time points during the movement of the first tissue fixture and/or the second tissue fixture.
Clause 52 The system of clause 51, wherein the at least one of the first fiducial or the second fiducial comprises parallel continuous edges extending at an angle with respect to the pixel circuit rows.
Clause 53 The system of clause 52, wherein the at least one of the first fiducial or the second fiducial comprises a plurality of markers, each of the markers comprising parallel continuous edges extending at the angle with respect to the pixel circuit rows.
Clause 54 The system of any one of clauses 51-53, wherein the identification of the at least one of the first fiducial or the second fiducial in the first frame comprises identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the first frame. Docket No. CURI-P4WO [0064]
Clause 55 The system of clause 54, wherein the at least one edge extends continuously at an angle with respect to the pixel circuit rows.
Clause 56 The system of any one of clauses 51-55, wherein the identification of the at least one of the first fiducial or the second fiducial within the predicted second region of interest comprises identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the second frame.
Clause 57 The system of clause 56, wherein the at least one edge extends continuously at an angle with respect to the pixel circuit rows.
Clause 58 The system of any one of clauses 37-57, wherein the identification of the first region of interest in the first frame comprises selecting an area within the first frame around a centroid of the at least one of the first fiducial or the second fiducial.
Clause 59 The system of clause 58, wherein the selection of the area within the first frame comprises filtering regions of non-interest.
Clause 60 The system of any one of clauses 37-59, wherein the at least one of the first fiducial or the second fiducial is identified in the first frame according to at least one known optical parameter of the at least one of the first fiducial or the second fiducial.
Clause 61 The system of clause 60, wherein the at least one known optical parameter comprises at least one of a size, shape, angle, color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial.
Clause 62 The system of any one of clauses 37-61, wherein the prediction of the second region of interest in the second frame comprises selecting an area within the second frame larger than the at least one of the first fiducial or the second fiducial.
Clause 63 The system of clause 62, wherein the selection of the area within the second frame comprises filtering regions of non-interest.
Clause 64 The system of clause 62 or clause 63, wherein the selected area within the second frame is not larger than two of the at least one of the first fiducial or the second fiducial.
Clause 65 The system of any one of clauses 37-64, wherein the at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame comprises a tracking parameter of the at least one of the first fiducial or the second fiducial. Docket No. CURI-P4WO [0075]
Clause 66 The system of clause 65, wherein the tracking parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, position, angle, velocity, acceleration, deceleration, color, brightness, pixel intensity, or pixel density.
Clause 67 The system of clause 65 or clause 66, wherein the tracking parameter is determined over multiple frames.
Clause 68 The system of any one of clauses 37-67, wherein the prediction of the second region of interest in the second frame is at least partially based upon a known property of the biological sample.
Clause 69 The system of any one of clauses 37-68, wherein the identification of the at least one of the first fiducial or the second fiducial within the predicted second region of interest in the second frame comprises applying at least one optical threshold to identify a set of pixels within the predicted second region of interest that meets the at least one applied optical threshold, and wherein the at least one applied optical threshold is based upon at least one known optical parameter of the at least one of the first fiducial or the second fiducial.
Clause 70 The system of clause 69, wherein the at least one known optical parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, angle, color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial.
Clause 71 The system of clause 69 or clause 70, wherein the at least one known optical parameter is measured in the first frame.
Clause 72 The system of any one of clauses 69-71, wherein the at least one known optical parameter is measured across a plurality of frames.
Clause 73 The system of any one of clauses 37-72, wherein the identification of the at least one of the first fiducial or the second fiducial within the predicted second region of interest comprises filtering objects within the predicted second region of interest that are not the at least one of the first fiducial or the second fiducial.
Clause 74 The system of any one of clauses 37-73, wherein at least one of the first tissue fixture or the second tissue fixture comprises an additional fiducial disposed thereon.
Clause 75 The system of clause 74, wherein the system is further configured to perform an additional measurement of the additional fiducial and the first fiducial or the second fiducial between the first region of interest and the second region of interest. Docket No.
a system comprising: a tissue testing plate, comprising: a first tissue fixture extending into a well of the tissue testing plate, the first tissue fixture comprising a first fiducial, wherein the first fiducial optically contrasts with the first tissue fixture; and a second tissue fixture extending into the well of the tissue testing plate, the second tissue fixture comprising a second fiducial, wherein the second fiducial optically contrasts with the second tissue fixture; an optical detector positioned to image the first fiducial and the second fiducial; and a non-transitory machine readable storage medium storing instructions, which when executed by a processor, causes the system to perform operations, including: imaging a plurality of frames of the first fiducial and the second fiducial during a movement of the first tissue fixture and/or the second tissue fixture; identifying the at least one of the first fiducial or the second fiducial in the first frame according to a first optical threshold; measuring an optical parameter of the at least one of the first
Clause 77 The system of clause 76, wherein the first fiducial optically contrasts with the first tissue fixture and the second fiducial optically contrasts with the second tissue fixture with respect to at least one of a shape, size, angle, color, brightness, luminescence, pixel intensity or pixel density.
Clause 78 The system of clause 76 or clause 77, wherein the first fiducial and the second fiducial comprise an engineered contrast enhancement.
Clause 79 The system of any one of clauses 76-78, wherein at least one of the first fiducial or the second fiducial comprises an overmolding on an end of the respective first tissue fixture or second tissue fixture.
Clause 80 The system of any one of clauses 76-79, wherein at least one of the first fiducial or the second fiducial comprises an insert disposed within the respective first tissue fixture or second tissue fixture.
Clause 81 The system of any one of clauses 76-80, wherein at least one of the first fiducial or the second fiducial comprises a prominence disposed on an end of the respective first tissue fixture or second tissue fixture.
Clause 82 The system of clause 81, wherein the prominence has a spherical, rectangular, or cross shape.
Clause 83 The system of any one of clauses 76-82, wherein the optical detector comprises a camera. Docket No.
Clause 86 The system of any one of clauses 76-85, wherein the measured optical parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, angle, color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial.
Clause 87 The system of any one of clauses 76-86, wherein the measured optical parameter is measured across a plurality of frames.
Clause 88 The system of any one of clauses 76-87, wherein the optical detector comprises a pixel array including a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit columns and each frame of the plurality of frames is imaged with each of the plurality of pixel circuits of the pixel array, and wherein each of the plurality of frames is imaged with each pixel circuit row at different time points during the movement of the first tissue fixture and/or the second tissue fixture.
Clause 89 The system of clause 88, wherein the at least one of the first fiducial or the second fiducial comprises parallel continuous edges extending at an angle with respect to the pixel circuit rows.
Clause 90 The system of clause 89, wherein the at least one of the first fiducial or the second fiducial comprises a plurality of markers, each of the markers comprising parallel continuous edges extending at the angle with respect to the pixel circuit rows.
Clause 91 The system of any one of clauses 88-90, wherein the identification of the at least one of the first fiducial or the second fiducial in the first frame comprises identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the first frame.
Clause 92 The system of clause 91, wherein the at least one edge extends continuously at an angle with respect to the pixel circuit rows.
a system comprising: a tissue assembly, comprising: a mounting lid comprising a plate comprising a plurality of through holes; a first post assembly comprising a Docket No. CURI-P4WO first body comprising a plurality of first tissue fixtures, each first tissue fixture comprising a first fiducial thereon, wherein each first fiducial optically contrasts with the respective first tissue fixture; a second post assembly comprising a second body comprising a plurality of second tissue fixtures, each second tissue fixture comprising a second fiducial thereon, wherein each second fiducial optically contrasts with the respective second tissue fixture; wherein the plurality of first tissue fixtures and the plurality of second tissue fixtures are arranged in pairs of tissue fixtures, each pair of tissue fixtures comprising a first tissue fixture of the plurality of first tissue fixtures and a second tissue fixture of the plurality of second tissue fixtures arranged at respective sides of the plurality of through holes of the mounting lid; and a body comprising a plurality of wells, where
Clause 94 The system of clause 93, wherein the first tissue fixture is a rigid tissue fixture and the second tissue fixture is a deformable tissue fixture.
Clause 95 The system of clause 93 or clause 94, wherein the first tissue fixture and the second tissue fixture are deformable tissue fixtures.
Clause 96 The system of any one of clauses 93-95, wherein at least one of a first centroid of the first fiducial is substantially aligned with a centroid of the first tissue fixture or a second centroid of the second fiducial is substantially aligned with a centroid of the second tissue fixture.
Clause 97 The system of any one of clauses 93-96, wherein the first fiducial optically contrasts with the first tissue fixture and the second fiducial optically contrasts with the second tissue fixture with respect to at least one of a shape, size, angle, color, brightness, luminescence, pixel intensity or pixel density.
Clause 98 The system of any one of clauses 93-97, wherein the first fiducial and the second fiducial comprise an engineered contrast enhancement.
Clause 99 The system of any one of clauses 93-98, wherein at least one of the first fiducial or the second fiducial comprises an overmolding on an end of the respective first tissue fixture or second tissue fixture.
Clause 100 The system of any one of clauses 93-99, wherein at least one of the first fiducial or the second fiducial comprises an insert disposed within the respective first tissue fixture or second tissue fixture. Docket No. CURI-P4WO [0110]
Clause 101 The system of any one of clauses 93-100, wherein at least one of the first fiducial or the second fiducial comprises a prominence disposed on an end of the respective first tissue fixture or second tissue fixture.
Clause 102 The system of clause 101, wherein the prominence has a spherical, rectangular, or cross shape.
Clause 103 The system of any one of clauses 93-102, wherein at least one of the first fiducial or the second fiducial comprises parallel continuous edges extending at an angle with respect to a pixel array of the optical detector.
Clause 104 The system of clause 103, wherein the at least one of the first fiducial or the second fiducial comprises a plurality of markers, each of the markers comprising parallel continuous edges extending at the angle.
Clause 105 The system of any one of clauses 93-104, wherein the optical detector comprises a camera.
Clause 106 The system of any one of clauses 93-105, wherein the light source is integrated within a stimulation lid configured to be mounted on the mounting lid.
Clause 107 A tissue assembly, comprising: a mounting lid comprising a plate comprising a plurality of through holes; a first post assembly comprising a first body comprising a plurality of first tissue fixtures, each first tissue fixture comprising a first fiducial thereon, wherein each first fiducial optically contrasts with the respective first tissue fixture; a second post assembly comprising a second body comprising a plurality of second tissue fixtures, each second tissue fixture comprising a second fiducial thereon, wherein each second fiducial optically contrasts with the respective second tissue fixture; wherein the plurality of first tissue fixtures and the plurality of second tissue fixtures are arranged in pairs of tissue fixtures, each pair of tissue fixtures comprising a first tissue fixture of the plurality of first tissue fixtures and a second tissue fixture of the plurality of second tissue fixtures arranged at respective sides of the
Clause 108 The tissue assembly of clause 107, wherein the first tissue fixture is a rigid tissue fixture and the second tissue fixture is a deformable tissue fixture.
Clause 109 The tissue assembly of clause 107 or clause 108, wherein the first tissue fixture and the second tissue fixture are deformable tissue fixtures. Docket No. CURI-P4WO [0119]
Clause 110 The tissue assembly of any one of clauses 107-109, wherein at least one of a first centroid of the first fiducial is substantially aligned with a centroid of the first tissue fixture or a second centroid of the second fiducial is substantially aligned with a centroid of the second tissue fixture.
Clause 111 The tissue assembly of any one of clauses 107-110, wherein the first fiducial optically contrasts with the first tissue fixture and the second fiducial optically contrasts with the second tissue fixture with respect to at least one of a shape, size, angle, color, brightness, luminescence, pixel intensity or pixel density.
Clause 112 The tissue assembly of any one of clauses 107-111, wherein the first fiducial and the second fiducial comprise an engineered contrast enhancement.
Clause 113 The tissue assembly of any one of clauses 107-112, wherein at least one of the first fiducial or the second fiducial comprises an overmolding on an end of the respective first tissue fixture or second tissue fixture.
Clause 114 The tissue assembly of any one of clauses 107-113, wherein at least one of the first fiducial or the second fiducial comprises an insert disposed within the respective first tissue fixture or second tissue fixture.
Clause 115 The tissue assembly of any one of clauses 107-114, wherein at least one of the first fiducial or the second fiducial comprises a prominence disposed on an end of the respective first tissue fixture or second tissue fixture.
Clause 116 The tissue assembly of clause 115, wherein the prominence has a spherical, rectangular, or cross shape.
Clause 117 The tissue assembly of any one of clauses 107-116, wherein at least one of the first fiducial or the second fiducial comprises an angled rectangle or parallelogram.
Clause 118 The tissue assembly of clause 117, wherein the at least one of the first fiducial or the second fiducial comprises a plurality of markers, each of the markers comprising an angled rectangle or parallelogram.
Clause 119 A method of determining a sample parameter of a biological sample disposed between a first tissue fixture and a second tissue fixture, comprising: imaging a plurality of frames of a first fiducial disposed on the first tissue fixture and a second fiducial disposed on the second tissue fixture during a movement of the first tissue fixture and/or the second tissue fixture; identifying a first region of interest in a first frame of the plurality of frames based upon Docket No.
CURI-P4WO identifying at least one of the first fiducial or the second fiducial in the first frame; predicting a second region of interest in a second frame of the plurality of frames based upon at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame; identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest in the second frame; performing a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest; and determining a sample parameter of the biological sample based upon the measurement.
Clause 120 The method of clause 119, further comprising: illuminating the first fiducial and the second fiducial with a light source.
Clause 121 The method of clause 119 or clause 120, wherein each frame of the plurality of frames is imaged with a plurality of pixel circuit rows and imaging of the plurality of frames comprises imaging with each pixel circuit row of the plurality of pixel circuit rows at different time points during movement of the first tissue fixture and/or the second tissue fixture.
Clause 122 The method of clause 121, wherein the identification of the at least one of the first fiducial or the second fiducial in the first frame comprises identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the plurality of pixel circuit rows of the first frame.
Clause 123 The method of clause 122, wherein the at least one edge extends continuously at an angle with respect to the plurality of pixel circuit rows.
Clause 124 The method of any one of clauses 121-123, wherein the identification of the at least one of the first fiducial or the second fiducial within the predicted second region of interest comprises identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the plurality of pixel circuit rows of the second frame.
Clause 125 The method of clause 124, wherein the at least one edge extends continuously at an angle with respect to the plurality of pixel circuit rows.
Clause 126 The method of any one of clauses 119-125, wherein the identification of the first region of interest in the first frame comprises selecting an area within the first frame around a centroid of the at least one of the first fiducial or the second fiducial.
Clause 127 The method of clause 126, wherein the selection of the area within the first frame comprises filtering regions of non-interest. Docket No. CURI-P4WO [0137]
Clause 128 The method of any one of clauses 119-127, wherein the prediction of the second region of interest in the second frame comprises selecting an area within the second frame larger than the at least one of the first fiducial or the second fiducial.
Clause 129 The method of clause 128, wherein the selection of the area within the second frame comprises filtering regions of non-interest.
Clause 130 The method of clause 128 or clause 129, wherein the selected area within the second frame is not larger than two of the at least one of the first fiducial or the second fiducial.
Clause 131 The method of any one of clauses 119-130, wherein the at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame comprises a tracking parameter of the at least one of the first fiducial or the second fiducial.
Clause 132 The method of clause 131, wherein the tracking parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, angle, position, velocity, acceleration, deceleration, color, brightness, pixel intensity, or pixel density.
Clause 133 The method of clause 131 or clause 132, wherein the tracking parameter is determined over multiple frames.
Clause 134 The method of any one of clauses 119-133, wherein the prediction of the second region of interest in the second frame is at least partially based upon a known property of the biological sample.
Clause 135 The method of any one of clauses 119-134, wherein the identification of the at least one of the first fiducial or the second fiducial within the predicted second region of interest in the second frame comprises applying at least one optical threshold to identify a set of pixels within the predicted second region of interest that meets the at least one applied optical threshold, and wherein the at least one applied optical threshold is based upon at least one known optical parameter of the at least one of the first fiducial or the second fiducial.
Clause 136 The method of clause 135, wherein the at least one known optical parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, angle, color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial.
Clause 137 The method of clause 135 or clause 136, wherein the at least one known optical parameter is measured in the first frame. Docket No. CURI-P4WO [0147]
Clause 138 The method of any one of clauses 135-137, wherein the at least one known optical parameter is measured across a plurality of frames.
Clause 139 The method of any one of clauses 119-138, wherein the identification of the at least one of the first fiducial or the second fiducial within the predicted second region of interest comprises filtering objects within the predicted second region of interest that are not the at least one of the first fiducial or the second fiducial.
Clause 140 The method of any one of clauses 119-139, further comprising: performing an additional measurement of an additional fiducial disposed on at least one of the first tissue fixture or the second tissue fixture and the respective first fiducial or the second fiducial between the first region of interest and the second region of interest to determine the sample parameter of the biological sample based upon the additional measurement.
Clause 141 The method of any one of clauses 119-140, further comprising: applying a first optical threshold to identify the at least one of the first fiducial or the second fiducial in the first frame of the plurality of frames; identifying the at least one of the first fiducial or the second fiducial in the first frame according to the first optical threshold; measuring an optical parameter of the at least one of the first fiducial or the second fiducial in the first frame; determining a second optical threshold for identifying the at least one of the first fiducial or the second fiducial in the second frame of the plurality of frames based on the measured optical parameter; and applying the second optical threshold for identifying the at least one of the first fiducial or the second fiducial in the second frame of the plurality of frames; wherein the identification of the at least one of the first fiducial or the second fiducial in the predicted second region of interest in the second frame comprises identifying the at least one of the first fiducial or the second fiducial according to the second optical threshold.
Clause 142 The method of clause 141, wherein the second optical threshold is applied within the predicted second region of interest to identify a set of pixels within the predicted second region of interest that meets the second optical threshold.
Clause 143 The method of clause 141 or clause 142, wherein the measured optical parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, angle, color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial.
Clause 144 The method of any one of clauses 141-143, wherein the measured optical parameter is measured across a plurality of frames. Docket No.
FIG. 1A illustrates a perspective view of a system for analyzing a biological sample, according to an example of the present disclosure.
FIG.1B illustrates a side view of the system of FIG.1A.
FIG. 2A illustrates a schematical elevation view of a pair of tissue fixtures and a tissue sample disposed within a well, according to an example of the present disclosure.
FIG. 2B illustrates a schematical bottom plan view of the pair of tissue fixtures of FIG. 2A.
FIG. 2C illustrates a schematical elevation view of a tissue fixture of FIG.
FIG. 2D illustrates a schematical elevation view of a tissue fixture of FIG. 2A with a spherical extension, according to an example of the present disclosure.
FIG. 2E illustrates a schematical bottom plan view of a tissue fixture of FIG. 2A with a cross shape extension, according to an example of the present disclosure.
FIG. 2F illustrates a schematical elevation view of a tissue fixture of FIG. 2A with an insert, according to an example of the present disclosure.
FIG.2G illustrates an enlarged schematical elevation view of a tissue fixture of FIG. 2A with an angled fiducial, according to an example of the present disclosure.
FIG.2H illustrates an enlarged schematical elevation view of a tissue fixture of FIG. 2A with a plurality of angled fiducials, according to an example of the present disclosure.
FIG. 3 illustrates a perspective view of a mounting lid and first and second post assemblies, according to an example of the present disclosure.
FIG. 4 illustrates a perspective view of a multi-well plate, according to an example of the present disclosure.
FIG. 5 shows two image frames of tissues in a well, wherein each image frame has an overlay identifying one or more regions of interest (ROI) identified by systems of the present disclosure.
ROI regions of interest
FIG.6 is an image of a well featuring fiducials as well as distractor objects to be filtered computationally by systems of the present disclosure. Docket No. CURI-P4WO [0169]
FIG.7 schematically illustrates an exemplary optical detector including a pixel array according to an example of the present disclosure.
FIG.8A illustrates pixel representations of a moving angled fiducial and a linear regression of active pixels corresponding thereto.
FIG.8B illustrates selectively exposing pixel circuit rows of the pixel representation of FIG.8A.
FIG.9A compares spatial resolution for fiducials having different angles.
FIG.9B compares spatial resolution for multi-marker fiducials having different angles.
FIG.10A illustrates pixel representations and compares temporal position resolution of a moving fiducial according to different methods of the present disclosure.
FIG.10B illustrates pixel representations and compares temporal velocity resolution of a moving fiducial according to different methods of the present disclosure.
FIG.10C illustrates pixel representations and compares temporal acceleration resolution of a moving fiducial according to different methods of the present disclosure.
FIG.11 is a representative graphical user interface of the present disclosure featuring an image of a multi-well array procured at a single time point, wherein two wells comprise two information overlays.
FIG.10A illustrates pixel representations and compares temporal position resolution of a moving fiducial according to different methods of the present disclosure.
FIG.10B illustrates pixel representations and compares temporal velocity resolution of a moving fiducial according to different methods of the present disclosure.
FIG.10C illustrates pixel representations and compares temporal acceleration resolution of a moving fiducial according to different methods of the present
FIG. 12 illustrates another representative graphical user interface of the present disclosure featuring an output video of a tissue and synchronous information regions displaying measured tissue properties.
FIG. 13A illustrates a flow chart for a method according to an example of the present disclosure.
FIG.13B illustrates a flow chart for further steps of the method of FIG.13A.
FIG.13C illustrates a flow chart for further steps of the method of FIG.13A.
FIG.13D illustrates a flow chart for further steps of the method of FIG.13A.
FIG.13E illustrates a flow chart for further steps of the method of FIG.13A.
FIG. 14A illustrates a flow chart for a method according to an example of the present disclosure.
FIG.14B illustrates a flow chart for further steps of the method of FIG.14A.
Docket No. CURI-P4WO FIG. 15 illustrates a flow chart for a method according to an example of the present disclosure.
FIG. 16A illustrates a flow chart for a method according to an example of the present disclosure.
FIG.16B illustrate a flow chart for further steps of the method of FIG.16A.
DETAILED DESCRIPTION [0189]
This disclosure provides for systems, assemblies, devices, and methods for analyzing biological samples utilizing computer vision and algorithmic analysis of recorded image data. In one representative application, such systems enable accurate, high-throughput positional analysis of biological samples in a multi-well format.
the optical systems described herein advantageously avoid magnetic crosstalk between wells, which contributes to measurement errors. Additionally, because some embodiments of such systems utilize a single, relatively low resolution optical detector to image a large number of biological samples, the systems disclosed herein scale more efficiently than magnetometer based systems. As compared to known optical tissue analysis systems, the systems disclosed herein utilize novel fiducials which optically contrast against tissue fixtures, facilitating accurate positional measurement of the biological samples. Optionally, the systems utilize algorithmic image processing techniques that leverage the fiducials to more accurately and quickly determine properties of the biological samples.
biological samples may include, for example, any natural or engineered (synthetic) musculoskeletal tissues including skeletal muscle, smooth muscle, cardiac muscle, tendons, and ligaments. Biological samples may also include non-musculoskeletal tissue types, including pulmonary, tracheal, intestinal, hepatic and neuromuscular, and tumors. The foregoing biological samples may be prepared into specimens suitable for analysis in tissue analysis systems as described herein.
FIG. 1A and FIG. 1B illustrate a representative system 100 for analyzing biological samples utilizing computer vision and algorithmic analysis of recorded image data, according to an example of the present disclosure. The system 100 is designed to analyze an array of biological samples which may optionally be cultured adherent cells or 3D cultured tissues.
the systems and devices may be manufactured, used, and sold separately from the biological samples. Docket No. CURI-P4WO [0192]
the 3D cultured tissues may be grown, cast, or otherwise prepared in a way that produces tissues suspended, supported (as in a medium), or otherwise disposed, between two tissue fixtures (e.g., posts) of the system, as described below with respect to FIG. 2A and FIG. 2B.
the tissue fixtures include pairs of tissue fixtures (e.g., at least one pair including a rigid tissue fixture and a deformable tissue fixture or at least one pair including two deformable tissue fixtures).
the tissue fixtures may be treated to enhance cell adhesion, such as by plasma processing, chemical processing, deposition of a biological matrix, or any other method to promote cell adhesion.
the tissue fixtures may be posts, hooks, clamps, or other fixture type capable of attaching to a tissue specimen via tissue growth around such fixture or mechanical engagement.
the system 100 includes a tissue assembly 102, an optical detector 104, and a light source configured to illuminate the tissue fixture, which is optionally part of a stimulation lid 112 incorporating the light source, as will be discussed in further detail below.
the tissue assembly 102 includes a mounting lid 114, which has the tissue fixture disposed thereon, and a multi-well plate 116 or plate body defining a plurality of wells, e.g., a 48 or 96 well plate.
the tissue fixture extend into the multi-well plate 116 from the mounting lid 114.
a pair of tissue fixture is disposed in each well of the multi-well plate 116.
examples of the present disclosure may include numerous pairs of rigid and deformable tissue fixtures or pairs of deformable tissue fixtures, e.g., 48 or 96 pairs of tissue fixtures.
a representative tissue assembly is described in PCT Publication No. WO2021/173887, published February 9, 2021 and entitled DEVICES AND METHODS FOR THE GENERATION AND EVALUATION OF ENGINEERED TISSUES, which is hereby incorporated by reference in its entirety for all purposes.
One or more tissue fixture may be at least partially formed of a rigid material which does not move or moves minimally in response to force produced by the suspended tissue. Nevertheless, in some examples, the rigid post may move under the operation of the system, e.g., as a result of a force mechanically applied to the rigid post by the system.
the rigid tissue fixture may be manufactured of polystyrene, polypropylene, ceramic, titanium or any other material which is biologically compatible or which may be rendered biologically compatible with the tissue and has sufficient stiffness to resist deformation.
the rigid tissue fixture may comprise a shell and a core disposed within the shell, wherein the shell comprises a higher index of refraction Docket No.
One or more tissue fixture may be at least partially manufactured from a deformable material which moves in response to force produced by the suspended tissue.
the deformable tissue fixture may be manufactured from polydimethylsiloxane (PDMS), various silicone formulations, TPE, polyacrylamide gel, or any other material which is biologically compatible with the tissue and which is sufficiently flexible to be perceptibly deformed by the force generated by the tissue.
both tissue fixtures of each pair are manufactured from the deformable material.
the term “rigid tissue fixture” / “rigid post” and “deformable tissue fixture” / “deformable post” may be defined absolutely and/or relatively.
each rigid tissue fixture e.g., first tissue fixture 224
has a greater force-to- displacement relationship e.g., at one point along the length of the post (e.g., a greater stiffness at the distal end) and/or a different Young's modulus than the deformable tissue fixture (e.g., second tissue fixture 228) in the same well.
each rigid tissue fixture has a stiffness of about 1,000 N/m to about 10,000 N/m, for example about 10N/m to about 30N/m (e.g., about 12N/m or 24N/m).
each deformable tissue fixture has a stiffness of about 0.1N/m to about 5N/m, e.g., about 2N/m.
the system 100 may include a light source 118 configured to illuminate the tissue fixture and the fiducials disposed thereon, as will be discussed in further detail below.
the light source is integrated with the stimulation lid 112 which sits atop the tissue assembly 102 and which provides electrical stimuli to the biological samples suspended between the tissue fixtures; however, this configuration is representative.
Representative stimulation plates include those described in PCT Publication No. WO2021/173887.
the light source does not form part of the tissue assembly 102 or stimulation lid 112.
the light source may include an individual light or array of Docket No.
the light source or light sources may be disposed within the system 100 between the optical detector 104 and the multi-well plate 116.
the light source may be supported within the system above the optical detector 104 and below the multi-well plate 116 to direct light upwardly to the multi- well plate 116.
the light source may be disposed within the system alongside or below the optical detector.
the light source may be coherent or incoherent, produced by an LED, an array of LEDs laser, laser diode, arc lamp or any other suitable apparatus.
the light source may include a plurality of discrete light sources.
an array of LEDs can be incorporated into a lid and/or electrical stimulation lid that covers a multi-well plate 116 and isolates lights from each LED to its corresponding well and tissue.
the LEDs can be individually operated such that specific wells of the multi-well plate 116 can receive light based on a given experimental protocol, thus, reducing phototoxicity in tissues in wells not being interrogated simultaneously.
the light source may optionally pass through a wavelength selection filter such as an absorption filter, a thin film interference filter, dichroic mirror, prism, monochromator, any other method of wavelength selection, or any combination of one or more such elements.
the light source may include a single monolithic source illuminating one, some or all of the samples, or an array of light sources, each of which illuminates one, some or all of the samples.
the light source may illuminate the biological sample directly or be directed to the sample by a mirror, lens, fiber optic, photonic chip, other optical assembly or any combination, in whole, or in part, thereof.
the system 100 further includes at least one optical detector 104, or optical detector assembly containing at least one optical detector.
the optical detector may be a single camera sensor or an array of detectors, each of which may be a camera, diode, PMT, SiPMT, multipixel photon counter, or other such photon detector.
Each optical detector 104 may collect light from the biological sample directly or the light may be directed to the optical detector 104 by a mirror, lens, fiber optic, photonic chip, other optical assembly or any combination, in whole, or in part, thereof.
the optical detector 104 may be configured to simultaneously image all of the tissue fixture of the system, e.g., have a field of view that encompasses all tissue fixture.
Docket No. CURI-P4WO the optical detector 104 may be configured to image an entire multi-well plate 116, e.g., 24, 48, 96, 384, etc. number of wells. In the illustrated example, the optical detector 104 is configured to capture the entire multi-well plate 116.
the system 100 may comprise multiple optical detectors 104 configured to image separate portions of the multi-well plate 116, e.g., 1 ⁇ 4 of the number of wells, 1 ⁇ 2 of the number of wells, etc.
the system 100 may comprise optical detectors 104 corresponding to each well of the multi-well plate 116, e.g., 96 optical detectors for a 96 well plate.
the optical detector 104 may have a spatial resolution between 0.5 and 50 ⁇ m per pixel in order that a single image may be processed into a plurality of spatially separated regions, each corresponding to one well.
the optical detector 104 may have a frame rate between 1 and 1000 frames per second.
the optical detector 104 has a frame rate at least as great as a contraction frequency of a biological sample to be analyzed, e.g., an Engineered Cardiac Tissue. However, this is not an essential feature, as embodiments of the systems and methods described below image each well (and biological sample) with an effective frame rate that exceeds the nominal frame rate of the optical detector 104.
the optical detector 104 is a camera based upon one or more CMOS image sensors, e.g., having a standard lens with a high numerical aperture, for example a Ximea CB654MG-GP-X8G3 camera with a Canon EF 50 mm 1:1.8 STM lens .
optical detector 104 is advantageous in that it better captures contrasting brightness between the fiducials and the tissue fixture and surrounding structures and does not require a lens array for accurate imaging of the fiducials. Spatial distortion resulting from such a configuration may be corrected during the algorithmic analysis performed by the processor 106 of the system 100. Further, a CMOS image sensor enables super-frame rate imaging and analysis of the wells according to the methods described below.
CURI-P4WO logic or various combinations thereof, which when executed by the processor 106 (e.g., general processing units, graphical processing units, application specific integrated circuits), performs operations, e.g., imaging a plurality of frames of the fiducials with the optical detector 104 during a movement of the first tissue fixture and the second tissue fixture.
the instructions 110 may include logic embodying any whole or part of any method described herein.
the processor 106, storage medium 108, and instructions 110 may be embodied in whole or in part in the system 100 and/or other computing device.
the system 100 may include one or more communications or electrical interfaces having circuits configured to enable communication within the system 100 and optionally with a remote server, base station, or other network element via the internet, cellular network, RF network, Personal Area Network (PAN), Local Area Network, Wide Area Network, or other network.
the communications interface may be configured to communicate using wireless protocols (e.g., WIFI®, WIMAX®, BLUETOOTH®, ZIGBEE®, Cellular, Infrared, Nearfield, etc.) and/or wired protocols (Universal Serial Bus or other serial communications such as RS- 234, RJ-45, etc., parallel communications bus, etc.).
wireless protocols e.g., WIFI®, WIMAX®, BLUETOOTH®, ZIGBEE®, Cellular, Infrared, Nearfield, etc.
wired protocols Universal Serial Bus or other serial communications such as RS- 234, RJ-45, etc., parallel communications bus, etc.
the communications interface includes circuitry configured to initiate a discovery protocol that allows the device and other network element to identify each other and exchange control information.
the communications interface has circuitry configured to a discovery protocol and to negotiate one or more pre-shared keys.
the communications interface alternatively or additionally includes circuitry configured to initiate a discovery protocol that allows an enterprise server and the device to exchange information.
a storage medium is a tangible machine-readable storage medium that includes any mechanism that provides (i.e., stores) information in a non-transitory form accessible by a machine (e.g., a computer, network device, personal digital assistant, manufacturing tool, any device with a set of one or more processors, etc.).
the optical detector may include a plurality of pixel circuit rows, and imaging the plurality of frames may include imaging with each pixel circuit row of the optical detector at different timepoints during the movement of the biological sample.
Determining the sample parameter may be based upon at least one fiducial tracking parameter of at least one of the first fiducial or the second fiducial (e.g., at least one of a size, a shape, a color, a brightness and a fiducial separation distance). For example, for each fiducial, the size, shape, color, brightness and/or distance from other fiducials may be calculated and updated for each image frame to improve fiducial location accuracy - via computer vision - for the next frame.
Determining the sample parameter may include modifying and/or adaptively thresholding the at least one fiducial tracking parameter during imaging, e.g., to improve segmentation accuracy and help track the same object over time.
FIG. 2A and FIG. 2B show schematic side elevation and bottom views, respectively of an example pair of tissue fixtures that includes a first tissue fixture 224 and a second tissue fixture Docket No. CURI-P4WO 228 of the system 100 shown in FIG. 1A and FIG. 1B.
the first tissue fixture 224 is a rigid tissue fixture, as described above
the second tissue fixture 228 is a tissue fixture 224 and the second tissue fixture 228 are deformable tissue fixtures.
the first tissue fixture 224 and the second tissue fixture 228 are disposed on and extend from a tissue testing plate 218, e.g., the mounting lid 114 discussed above with reference to FIG. 1A and FIG. 1B, and extend into a well 220, e.g., a well of the multi-well plate 116 discussed above with reference to FIG.1A and FIG.1B.
a biological sample 222 e.g., a tissue specimen such as an Engineered tissue (ET), extends around and between the first tissue fixture 224 and the second tissue fixture 228 within the well 220.
the biological sample 222 may be cultured on the tissue fixture 224, 228.
the first tissue fixture 224 includes a first fiducial 226 and the second tissue fixture 228 includes a second fiducial 230.
the fiducials 226, 230 are manufactured into or onto the distal (bottom) ends of the first tissue fixture 224 and the second tissue fixture 228, i.e., facing the optical detector 104.
Each fiducial 226, 230 is generally an engineered structure that has one or more properties that is optically identifiable by the systems described herein.
the fiducial may have an identifiable area, aspect ratio, size, shape (a circularity, a roundness, a rectangularity), a color, a feature, and/or a brightness.
each fiducial has one or more properties that optically contrast with the respective first tissue fixture 224 and second tissue fixture 228 on which the fiducial 226, 230 is positioned in order to facilitate computer vision of the tissue fixture.
the term “optically contrasts” may mean, for example, any one or more of: the fiducial has a size, a shape, a color, feature, or a brightness that is identifiable by the optical detector, for example because it differs (e.g., by at least a threshold value which is optically distinguishable by the systems described herein) from the respective tissue fixture of which the fiducial forms a part and/or with the biological sample disposed upon said tissue fixture. Accordingly, the fiducial may be an optically contrasting portion of the tissue fixture.
the term “optically contrasts” may, in some embodiments, entail a comparison between a parameter of the fiducial and the respective tissue fixture. In other embodiments, the term Docket No.
each fiducial 226, 230 has one or more engineered properties that optically contrast with a radially outer edge of the distal end, shaft, and/or base of the respective tissue fixture 224, 228. Accordingly, each fiducial 226, 230 can be imaged by the optical detector 104 and has one or more properties which facilitate its identification in an image by one or more of the algorithms described herein.
the tissue fixtures 224, 228 may be formed of tissue fixture materials made from light-colored materials, e.g. white plastic for rigid tissue fixtures and translucent PDMS for deformable tissue fixtures.
the first fiducial 226 optically contrasts with the first tissue fixture 224 and the second fiducial 230 optically contrasts with the second tissue fixture 228 with respect to at least one of the following properties: shape; size; angle (e.g., with respect to a pixel array of the optical detector); color; brightness; luminescence; pixel intensity; or pixel density.
the first fiducial 226 and/or the second fiducial 230 may have a different shape, such as rectangular, spherical, cross-shaped, etc., that is discernibly different from the shape of, e.g., square, circular, cylindrical, of the respective first tissue fixture 224 or second tissue fixture 228.
first fiducial 226 and/or the second fiducial 230 may have a different size, e.g., smaller or larger, in terms of volume or surface area visible to the optical detector 104 that is discernibly different from the size of the respective first tissue fixture 224 or second tissue fixture 228.
first fiducial 226 and/or the second fiducial 230 may have a different color, e.g., different wavelength of light that is reflected/absorbed and/or emitted therefrom that is discernibly different from the color of the respective first tissue fixture 224 or second tissue fixture 228.
the first fiducial 226 and/or the second fiducial 230 may have a different brightness, e.g., intensity of light that is reflected or emitted therefrom that is discernibly different from the brightness of the respective first tissue fixture 224 or second tissue fixture.
the first fiducial 226 and/or the second fiducial 230 is discernibly different from respective first tissue fixture 224 or the second tissue fixture 228 with respect to various combinations of the above-mentioned properties or all of the above-mentioned properties.
the optical parameters or properties of the first fiducial 226 and/or the second fiducial 230 (and thus the manner in which the optical parameters of the fiducials 226, 230 optically Docket No.
the CURI-P4WO contrast with the respective tissue fixture 224, 228) may change or be adjusted dependent upon the specific operational parameters of the system 100 and the biological sample 222 being tested, e.g., the specifications of the light source and the optical detector 104, the nature of the biological sample 222 and any growth or tissue fixture medium provided in the well 220, and/or the parameters of the biological sample 222 being tested.
the first fiducial 226 and/or the second fiducial 230 may comprise a portion, e.g., an application/overmolding, extension, insert, etc., of the respective first tissue fixture 224 or second tissue fixture 228 that has one or more optical parameters that optically contrast with the general or surrounding structure of the tissue fixture 224, 228.
the optical parameters of the first fiducial 226 and/or the second fiducial 228 are such that the first fiducial 226 and/or the second fiducial 228 can be accurately and efficiently identified and tracked through algorithmic analysis of the plurality of frames imaged by the optical detector 104 during movement of the tissue fixture 224, 228, which in turn allows for measurement of the deflection of the tissue fixture 224, 228 for purposes of determining the sample parameter(s) of the biological sample 222.
the first fiducial 226 may be manufactured directly onto the first tissue fixture 224 as an overmolding, e.g., a colored overmolding, e.g., via a two-step molding process, e.g., overmolding or other method, on the distal (bottom) face of the first tissue fixture 224.
the first fiducial 226 may also be applied to a side of the first tissue fixture 224 extending up from the distal face, as shown in FIG. 2A.
the second fiducial 230 may be manufactured directly onto the second tissue fixture 228 as an overmolding, e.g., via a two-step molding process whereby a thin layer of the PDMS - on the bottom face of the second tissue fixture 228 - is manufactured in a dark color with non-cytotoxic dye.
a centroid 234 of the first fiducial 226 is offset from a first centroid 232 of the first tissue fixture 224 and a centroid 238 of the second fiducial 230 is substantially aligned from a second centroid 236 of the second tissue fixture 228.
the centroids 234, 238 of the fiducials 226, 230 may be aligned with the respective first centroid 232 or second centroid 236 in a first direction (e.g., y or vertical direction of FIG.2B) and offset from the respective first centroid 232 or second centroid 236 in a second direction (e.g., x or horizontal direction of FIG. 2B). It is to be appreciated that the centroids 234, centroid 238 of the fiducials 226, 230 may be aligned or offset from the respective first centroid 232 and second Docket No.
the CURI-P4WO centroid 236 in any manner that provides for accurate, robust, and efficient tracking of the first centroid 232 and the second centroid 236 via the imaging and tracking of the fiducials 226, 230 by the optical detector 104 through a plurality of frames, as will be discussed below.
the biological sample 222 may extend partially or completely around and over the fiducials 226, 230.
the optical contrast provided by the fiducials 226, 230 is sufficient such that the accurate imaging and tracking of the fiducials 226, 230 in the plurality of frames is not impeded by the presence of the biological sample 222.
first tissue fixture 224 and/or the second tissue fixture 228 may be extended through the position of the biological sample 222 and/or may include features for retaining the biological sample above the position of the fiducials 226, 230 such that the biological sample 222 does not obscure the fiducials 226, 230.
the fiducials 226, 230 may include one or more engineered contrast enhancements, as described below, in order to further contrast the fiducials 226, 230 from the biological sample 222.
the first fiducial 226 optically contrasts with the general or surrounding structure of the first tissue fixture 224 and the second fiducial 230 optically contrasts with the general or surrounding structure of the second tissue fixture 228.
the first fiducial 226 has a size (e.g., smaller) and a color (e.g., black) that are discernibly different from the general or surrounding structure of the first tissue fixture 224 (i.e., the distal end of the first tissue fixture 224 is larger and lighter in color than the first fiducial 226).
the second fiducial 230 has a shape (e.g., stadium or pill-shaped), size (e.g., smaller) and a color (e.g., black) that are discernibly different from the general or surrounding structure of the second tissue fixture 228 (i.e., the distal end of the second tissue fixture 230 is more circular, larger, and lighter in color than the second fiducial 230).
the first fiducial and/or the second fiducial may comprise a prominence or an extension, e.g., a colored extension, disposed on an end of the respective first tissue fixture 224 or second tissue fixture 228.
the first tissue fixture 224 may include a first fiducial in the form of a rectangular extension 280 disposed on an end thereof having a shape (e.g., rectangular), size (e.g., smaller), and color (e.g., black) that optically contrasts with the general or surrounding structure of the first tissue fixture 224.
the first tissue fixture may include a first fiducial in the form of a spherical extension 282 disposed on an end thereof having a shape (e.g., rectangular), size (e.g., smaller), and color (e.g., black) that optically contrasts with the Docket No. CURI-P4WO general or surrounding structure of the first tissue fixture 224.
the first tissue fixture may include a first fiducial in the form of a cross shape extension 284 disposed on an end thereof having a shape (e.g., cross-shaped), size (e.g., smaller), and color (e.g., black) that optically contrasts with the general or surrounding structure of the first tissue fixture 224.
an advantage of the spherical extension 282 is that the image of the spherical extension 282 does not change in terms of size or shape as the respective first tissue fixture 224 or second tissue fixture 228 bends, thus causing the spherical extension 282 to move toward or away from the optical detector 104 during the imaging of the plurality of frames.
the first fiducial and/or the second fiducial may comprise an insert (in this example, a colored insert 286) disposed within a slot or recess formed in an end of the respective first tissue fixture 224 or second tissue fixture 228.
the insert 286 has a color or other optical parameter that optically contrasts with the respective first tissue fixture 224 or second tissue fixture 228 such that the insert 286 can be optically observed and tracked through the material of the respective first tissue fixture 224 or second tissue fixture 228.
the insert 286 has a known length and diameter to allow for accurate tracking of the insert 286 through the plurality of frames as the respective first tissue fixture 224 or second tissue fixture 228 moves and the angle of observation of the insert 286 by the optical detector 104 changes.
the fiducials 226, 230, extensions 280, 282, 284, and insert 286 described above are illustrated in FIG.2A - FIG.2F are illustrated as having a black color. It is to be appreciated that the illustrated black color is one example of a color that may be utilized in connection with the fiducials to optically contrast the fiducials with the tissue fixture 224, 228.
a different color may be used in connection with the fiducials, such as a fluorescent color (e.g., through application of a fluorescent epoxy to form or coat the fiducials).
the fiducials may optionally incorporate an engineered contrast enhancement to increase the optical contrast between the fiducials and the respective tissue fixture including, such as any one or more of: Docket No. CURI-P4WO i. fluorescent labeling with small molecule fluorescent dyes, quantum dots, upconverting nanoparticles, or other fluorescent markers; ii. gold, silver, silica, polymer, or other nanoparticles with high scattering cross section; iii.
the inclusion of the additional fiducial 288 on the first tissue fixture 224 and/or the second tissue fixture 228 allows for additional measurements to be performed between the additional fiducial 288 and the respective first fiducial 226 or second fiducial 230 disposed on the same tissue fixture 224, 228 as the additional fiducial 288 during movement of the first tissue fixture 224 and/or the second tissue fixture 228, in order to determine one or more sample parameters of the biological sample 222.
Such additional measurements may be useful when relative movement of the fiducials 226, 230 between frames is too large to allow for simple algorithmic calculation of the sample parameters of the biological sample 222.
FIG. 2B is also representative of a view of the first tissue fixture 224, the second tissue fixture 228, the first fiducial 226, the second fiducial 230, and the biological sample 222 in an at rest state (i.e., no contraction of the biological sample 222 or movement of the fiducials 226, 230) as seen from the bottom of the well 220 looking upward toward the biological sample 222 and the tissue testing plate 218.
the view of FIG. 2B extends within a horizontal (x) and vertical (y) plane that is parallel or substantially parallel to the optical detector 104, as shown in FIG.1A and FIG.
the optical detector 104 is configured to image the well 220, the first tissue fixture 224, the second tissue fixture 228, the first fiducial 226, the second fiducial 230, and the biological sample 222 with a pixel array comprising a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit column, as will be discussed in further detail below with reference to FIG. 7.
the optical detector 104 is Docket No.
the first fiducial and/or the second fiducial may comprise an angled fiducial 290 having linear and parallel continuous edges 292 that extend at an interior angle A with respect to the horizontal direction (x), e.g., the direction of one or more pixel circuit rows of the optical detector 104, as described below with respect to FIG.7.
an angle A of a fiducial may be alternatively described as an interior angle with respect to a pixel circuit column of the optical detector (e.g., as 90 degrees minus angle A).
the angled fiducial 290 may comprise a prominence or an extension from the respective first tissue fixture 224 or second tissue fixture 228 (e.g., as described above with reference to FIG. 2C-FIG. 2E) or an insert positioned within the respective first tissue fixture 224 or second tissue fixture 228 (e.g., as described above with reference to FIG. 2F).
the angled fiducial 290 may have a rectangular or parallelogram shape.
the angle A is greater than 0 degrees and less than 90 degrees, more particularly greater than or equal to approximately 30 degrees and less than or equal to approximately 75 degrees, more particularly greater than or equal to approximately 40 degrees and less than or equal to approximately 60 degrees, more particularly approximately 45 degrees.
the angled fiducial 290 has a shape (e.g., angled rectangle or parallelogram), size (e.g., smaller), and color (e.g., black) that optically contrasts with the general or surrounding structure of the first tissue fixture 224.
the shape and angle of the angled fiducial 290 may be configured to facilitate subpixel spatial resolution and/or super frame rate resolution of the angled fiducial 290, as will be discussed in further detail below with reference to FIG.8A - FIG.8B.
the first fiducial and/or the second fiducial may comprise a fiducial 294 comprised of a plurality of angled markers 296 each having linear and parallel continuous edges 298 extending at an angle A with respect to the horizontal direction (x), e.g., the direction of the pixel circuit rows of the optical detector 104.
the plurality of angled markers 296 may be aligned in the vertical (y) direction. According to the example shown in FIG. 2H, there are three angled markers 296 disposed on the first tissue fixture 224. It is to be appreciated that any suitable number of angled markers 296 (e.g., fewer than three or more than three) may be provided on the first tissue fixture 224 depending on the exact configuration of the first tissue fixture 224 (or second tissue fixture 228), the technical parameters of the system 100, and the testing being performed.
the angle A is greater than 0 degrees and less than 90 degrees, more particularly greater than or equal to approximately 30 degrees and less than or equal to approximately 75 degrees, more particularly greater than or equal to approximately 40 degrees and less than or equal to approximately 60 degrees, more particularly approximately 45 degrees.
each of the angled markers 296 has a shape (e.g., angled rectangle or parallelogram), size (e.g., smaller), and color (e.g., black) that optically contrasts with the general or surrounding structure of the first tissue fixture 224.
FIG. 3 illustrates further details of the mounting lid 114 of the tissue assembly 102 described above with reference to FIG. 1A and FIG. 1B.
the mounting lid 114 includes a plate 340 having a first side 342 and a second side 344 with a plurality of through holes 346 extending through the plate 340 from the first side 342 to the second side 344.
a first post assembly 348 includes a first body 350, which includes a plurality of first tissue fixtures 352 disposed thereon and extending in a distal direction (downward) from the first body 350.
Each of the first tissue fixtures 352 includes a first fiducial thereon corresponding to any one of the fiducials described above with reference to FIG. 2A - FIG. 2H.
a second post assembly 354 includes a second body 356, which includes a plurality of second tissue fixtures 358 disposed thereon and extending in a distal direction (downward) from the second body 356. Docket No.
each include a first tissue fixture 352 of the plurality of first tissue fixtures 352 and a second tissue fixture 358 of the plurality of second tissue fixtures 358 arranged at respective sides of the plurality of through holes 346 of the mounting lid 114.
Further structural details and examples of the mounting lid 114 and the post assemblies 348, 354 are provided in WO2021/173887, which has been incorporated by reference in its entirety. [0244] It shall be appreciated that the mounting lid 114 is described as one representative structure for supporting the tissue fixtures within wells of a plate; however, the present disclosure is not limited to the structure below.
FIG. 4 illustrates further details of the multi-well plate 116 of the tissue assembly 102 described above with reference to FIG. 1A and FIG. 1B.
the multi-well plate 116 includes a plurality of wells 460. Each of the wells 460 of the multi-well plate 116 is configured to receive at least one of the pairs of tissue fixtures 352, 358 of the mounting lid 114. Further structural details and examples of the multi-well plate 116 are provided in WO2021/173887, which has been incorporated by reference in its entirety.
the systems of the present disclosure are configured to determine sample parameters of biological samples based upon images captured by the optical detector, which captures a plurality of images of the multi-well plate 116. Each captured image includes every well of the multi-well plate 116, and the image is subdivided by post-processing steps into spatially distinct sub-images corresponding to each well.
determining the sample parameter of the biological sample within a single well may include identifying one (or more) region(s) of interest 562 and/or filtering out a region of non-interest in one or more frames.
FIG. 5 shows two image frames of biological Docket No.
the region of interest 562 excludes a portion of the underlying tissue fixture.
Identifying the region of interest 562 may be based upon a change of at least one of the fiducial 564 (e.g., the first fiducial or the second fiducial described above) relative to one or more previous frames.
the change may be a spatial and/or temporal change, such as a velocity of one of the fiducials 564.
the location of region of interest 562 may be a function of the velocity (speed and/or direction) of the fiducial 564 and/or centroid 566 from one or more previous frames.
Identifying the region of interest 562 for a given frame may be based upon a predicted region of interest 568 which itself is based upon the region of interest 562 for at least one previous frame. For example, using the previous example, velocity information computed by the processor may be used to predict optimal region of interest location for algorithmic tracking of the fiducial in the following frame, because the fiducial in frame N+1 is likely to lie along a directional vector.
FIG. 5 shows an example of a region-of-interest (ROI) 562 able to be identified in subsequent frames which surrounds the fiducial 564 and its centroid 566, and which is smaller than the distal end of the underlying tissue fixture.
ROI region-of-interest
Use of a smaller region of interest e.g., which excludes a portion of the underlying tissue fixture, and/or utilizing a prediction method as described above may reduce computation requirements for a given biological sample.
use of the smaller, more accurate region of interest prediction method may improve tracking accuracy insofar as fewer potential distractor Docket No. CURI-P4WO objects are in the smaller region of interest being analyzed by the algorithm.
Using small region of interest prediction methods may also reduce the time to compute the new location of the fiducial, which is advantageous for high throughput systems where many biological samples are being simultaneously tracked in a multi-well plate by a single optical detector or an array of optical detectors.
the system 100 may be configured so as to identify a region of interest 562 within a first or earlier frame N of the plurality of frames imaged by the optical detector 104 as an area of the frame surrounding the centroid 566 of the fiducial 564. Due to contraction of the biological sample, such as through a natural contraction, electrical stimulation, or other stimulation, the location of the centroid 566 of the fiducial 564 will have moved, as indicated by the arrow 572, between the first or earlier frame N and the second or subsequent frame N+1.
the system 100 may be configured to predict a predicted region of interest 568 in the subsequent frame N+1 based upon at least one parameter (e.g., size, shape, position, velocity, acceleration, deceleration, color, brightness, pixel intensity, or pixel density) as determined by the system 100 from the earlier frame N or from multiple earlier imaged frames including the earlier frame N (e.g., frame N, frame N-1, frame N-2, etc.).
the system 100 is configured to then attempt to identify the fiducial 564 within the predicted region of interest 568 in the subsequent frame N+1.
the predicted region of interest 568 may vary its size, position, and/or orientation depending on the at least one parameter.
the system may iterate the location and/or size of the predicted region of interest 568 until the fiducial 564 is identified.
determining the sample parameter of the biological sample may include filtering out a region of non-interest in one or more frames.
FIG. 6 is an image showing a first fiducial 674 and a second fiducial 676, as well as a plurality of unnecessary distractor objects 678 to be filtered computationally, such as when region-of-interest is relatively large.
the algorithms employed by the system 100 may filter out the region of non-interest (e.g., a region containing one or more distractor objects 678), for example based upon at least one of an area, an aspect ratio, an angle (e.g., with reference to the pixel array of the optical detector 104), a circularity, a roundness, a rectangularity, or an edge of the distractor objects or region of non-interest.
a distractor object 678 may include a feature in an imaged frame other than the fiducial, e.g., an inclusion in the biological medium.
the selected area of the region of interest 562 and/or the predicted region of interest 568 may be sufficiently small so as to be twice or less than twice the size of the fiducials 674, 676, such that distractor objects 678 having a size greater than or equal to the fiducials 674, 676 cannot fit within the region of interest 562 and/or the predicted region of interest 568 and are thus filtered as regions of non-interest.
FIG.7 schematically illustrates an optical detector 700 suitable for use with any system of the present disclosure, and which may be operably connected to a processor and storage medium as described with respect to FIG. 1A.
Optical detector 700 includes a pixel array 702, bitlines 712, a control circuit 710, a readout circuit 706, and function logic 708.
the pixel array 702 is a two-dimensional (2D) array including a plurality of pixel circuits 704 (e.g., Pl, P2, ...
each pixel circuit 704 may include one or more photodiodes that photogenerate image charges in response to incident light.
the image charges generated in the one or more photodiodes are transferred to a floating diffusion included in each pixel circuit 704 and converted to an image signal, which is then read out from each pixel circuit 704 by the readout circuit 706 through the column bitlines 712.
the readout circuit 706 may be configured to read out the image signals through the column bitlines 712.
the readout circuit 706 may include current sources, routing circuitry, and comparators that may be included in analog to digital converters or otherwise.
the digital image data values generated by the analog to digital converters in the readout circuit 706 may then be received by the function logic 708.
the function logic 708 may store the digital image data or even manipulate the digital image data by applying post image effects (e.g., crop, rotate, adjust brightness, adjust contrast, or otherwise).
the control circuit 710 is coupled to the pixel array 702 to control operation of the plurality of photodiodes in the pixel array 702.
the control circuit 710 may expose the pixel circuits 704, e.g., the pixel circuit rows, at different time points.
the control circuit 710 may expose substantially all of the pixel circuits 704, e.g., all pixel circuit rows.
exposing the pixel circuit rows at different time points advantageously enables super frame rate resolution that enables imaging and analysis of biological samples at an effectively faster frame rate than the nominal frame rate of the optical detector 700.
image acquisition is synchronized with lighting effects such as a flash.
the optical detector 700 is implemented on a single semiconductor wafer. In another example, the optical detector 700 is on stacked semiconductor wafers.
the pixel array 702 is implemented on a pixel wafer, and the readout circuit 706, the control circuit 710 and the function logic 708 are implemented on an application specific integrated circuit (ASIC) wafer, where the pixel wafer and the ASIC wafer are stacked and interconnected by bonding (hybrid bonding, oxide bonding, or the like) or one or more through substrate vias (TSVs).
ASIC application specific integrated circuit
the pixel array 702 and the control circuit 710 are implemented on a pixel wafer, and the readout circuit 706, and the function logic 708 are implemented on an Docket No.
the system 100 may incorporate one or more features that increase the effective spatial and/or temporal resolution of the optical detector 104, thus overcoming limitations of known tissue analysis systems. Accordingly, the fiducials and methods described herein enable high fidelity measurement of biological sample parameters such as contraction velocity and peak contraction (force).
an optional aspect of any embodiment of the present disclosure includes the use of an angled fiducial on one or more tissue fixtures, representative examples of which include the angled fiducial 290 described above with reference to FIG. 2G and the angled markers 296 described above with reference to FIG. 2H.
the angle of the fiducial is described with respect to one or more of the pixel circuit rows and/or pixel circuit columns of the optical detector.
the angled fiducial 290 has an angle A with respect to a horizontal direction corresponding to the pixel circuit rows shown in FIG. 7.
the angled fiducial 290 could be characterized according to an angle of (90 degrees - angle A) in order to express the complementary interior angle established between the angled fiducial 290 and a vertical direction corresponding to the pixel circuit columns of FIG.7.
FIG. 8A is a diagrammatic representation of a linear regression approach to positioning a linear fiducial 808 during subpixel translation.
the fiducial 808 would be disposed on a tissue fixture as described above.
the top row of FIG. 8A shows pixel representations of a 10 x 10 pixel array 810 with the fiducial 808 of Docket No. CURI-P4WO width 1 pixel (dotted line) at an interior angle A of 80 degrees with respect to a direction of the pixel circuit rows (equivalently restated as an interior angle B of 10 degrees with respect to a direction of the pixel circuit columns).
each panel (2-5) shows the binary pixel change result of the fiducial 808 moving horizontally to the right by some fraction of a pixel width, e.g., panel 2 shows the fiducial 808 moving horizontally to the right by 0.25 of a pixel width; panel 3 shows the fiducial 808 moving horizontally to the right by 0.5 of the pixel width; panel 4 shows the fiducial 808 moving horizontally to the right by 0.75 of the pixel width; and panel 5 shows the fiducial 808 moving horizontally to the right by a full pixel width, thus retaining its original pixelated morphology, but translated.
the pixelated morphology changes even though the fiducial 808 translates by less than a full pixel.
the movement of the fiducial 808 by less than a pixel width is detectable by the optical detector even though the fiducial 808 moves by less than a single pixel width, i.e., sub-pixel spatial resolution.
the angled or diagonal fiducial enables this sub-pixel resolution, which advantageously enables the fiducial 808 to be imaged with greater spatial resolution corresponding more closely to minute contractions of the biological sample attached to the tissue fixture having the fiducial 808 disposed thereon.
FIG.8B schematically represents the pixel array 810 at the same five different time points as shown in the top half of FIG.8A.
the pixel array 810 is presented as having five pixel circuit rows 812a - 812e, for simplicity.
Row scan information for a given frame can be obtained for every pixel circuit rows 812a - 812e or a subset thereof, e.g., to reduce processing time. From FIG.8B, one can see that using, for example, exposing only pixel circuit rows 812a and 812e would result in faster processing time but reduce the resolution to one pixel width as Docket No. CURI-P4WO only the top and bottom pixel edges would be obtained. Using all of pixel circuit rows 812a - 812e - here representing every pixel of the frame - results in sufficient information to yield subpixel resolution.
Error sources in edge detection measurements to produce subpixel resolution can include: a non-rectilinear edge; a low contrast edge resulting in inaccurate pixel thresholding; and fiducial velocity that exceeds limits of pixel size and frame rate of the optical detector.
errors resulting from a non-rectilinear edge and/or a low contrast edge can be mitigated via a diagonal or angled fiducial (e.g., as shown in FIG.2H), wherein the fiducial optionally includes a plurality of markers or fiducials, e.g., multiple angled fiducials having linear and parallel continuous edges as shown in FIG.2H.
the difference between the four plots is the angle or diagonality of the fiducial with respect to the pixel circuit columns of the optical detector (also the pixel circuit rows), i.e., 0, 5, 30, and 60 degrees.
the angle referenced in FIG. 9A and FIG.9B corresponds to angle B in FIG.8A.
Directly to the right of each plot is a schematic representation of the fiducial as imaged by a 20 x 10 pixel array of the optical detector.
the optical detector cannot sense movement of the fiducial until it has moved by at least 0.5 pixel.
FIG.9B shows four plots, each corresponding to the sensed movement (y-axis) by an optical detector in relation to actual movement of a fiducial having a plurality of marks (here, five marks or fiducials per fiducial). Directly to the right of each plot is a schematic representation of the multi-marker fiducial as imaged by a pixel array of the optical detector. [0278] As shown in the top plot, which is equivalent to tracking without subpixel resolution, when the angle or diagonality of the multi-marker fiducial with respect to the pixel circuit columns is zero degrees, the optical detector cannot sense movement of the fiducial until it has moved by at least 0.5 pixel.
FIG.10A - FIG.10C illustrate a fiducial moving across pixel arrays and respectively compare the position, velocity, and acceleration resolution which may be measured depending on whether the pixel circuit rows are exposed simultaneously or at different time points (assuming the same nominal frame rate of the optical detector).
the scale of timing and position of FIG.10A - FIG.10C are representative. As will become apparent, exposing some or all of the pixel circuit rows at different time points enables greater temporal resolution.
FIG.10A - FIG.10C has an angle of zero degrees relative to the pixel circuit columns (corresponding to angle B in FIG.8A), i.e., is not a diagonal or angled fiducial as described with respect to FIG.2G, FIG.2H, or FIG.8A - FIG.9B; however, the advantages Docket No. CURI-P4WO described below of imaging the fiducial with different pixel circuit rows at different time points also apply to embodiments with diagonal or angled fiducials.
FIG.10A includes three rows and three columns of information.
the top row is a pixel representation of a fiducial having a width of one pixel (represented by the activated (white) pixels) moving from left to right across a 10 x 5 pixel array over three frames, wherein all pixel circuit rows of the pixel array are imaged simultaneously, i.e., at t 0 , t 1 , and t 2 , which respectively correspond to Frame N, N+1, and N+2 (columns 1, 2, and 3).
the middle row is the same as the top row, except that the fiducial is imaged with each pixel circuit row at different known time points as the fiducial moves, i.e., each pixel circuit row is exposed at a different time.
pixel circuit row 1 is imaged at t0, t1, and t2 pixel circuit row 2 at t0.1, t1.1, t2.1, pixel circuit row 3 at t0.2, t1.2, t2.2, and so on.
the larger circles correspond to the top row, wherein all pixel circuit rows are imaged at t0, t1, and t2.
the smaller dots correspond to the middle row, wherein the pixel circuit rows are imaged at t 0 , t 0.1 ,...t n+0.1 .
the pixel array senses the fiducial with significantly greater temporal resolution when the pixel circuit rows are exposed or imaged at different time points.
the greater positional resolution enables more accurate computation of sample parameters of the biological sample associated with the fiducial and corresponding tissue fixture, including velocity and acceleration.
the top row is a pixel representation of a fiducial having a width of one pixel (represented by the activated (white) pixels) moving from left to right across a 5 x 5 pixel array over three frames, wherein all pixel circuit rows of the pixel array are imaged simultaneously, i.e., at t0, t1, and t2, which respectively correspond to Frame N, N+1, and N+2 (columns 1, 2, and 3).
the middle row is the same as the top row, except that the fiducial is imaged with each pixel circuit row at different known time points as the fiducial moves.
pixel circuit row 1 is imaged at t 0 , t 1 , and t 2 pixel circuit row 2 at t 0.1 , t 1.1 , t 2.1 , pixel circuit row 3 at t 0.2 , t 1.2 , t 2.2 , and so on.
the larger circles and lighter dashed line correspond to the top row, wherein all pixel circuit rows are imaged at t 0 , t 1 , and t 2 .
the smaller dots and darker dashed line correspond to the middle row, wherein the pixel circuit rows are imaged at t0, t0.1,...tn+0.1.
the slopes of the two dashed lines represent the velocity of the fiducials according to the two imaging methods. As shown, the position of the fiducial is measured with greater temporal resolution when the pixel rows are imaged at different known time points, resulting in higher measured velocity (steeper slope), which has greater accuracy than if all pixel circuit rows are imaged simultaneously.
the top row of FIG.10C is a pixel representation of a fiducial having a width of one pixel (represented by the activated (white) pixels) moving from left to right across a 5 x 5 pixel array over three frames, wherein all pixel circuit rows of the pixel array are imaged simultaneously, i.e., at t0, t1, and t2, which respectively correspond to Frame N, N+1, and N+2 (columns 1, 2, and 3).
the middle row is the same as the top row, except that the fiducial is imaged with each pixel circuit row at different known time points as the fiducial moves.
pixel circuit row 1 is imaged at t 0 , t 1 , and t 2 pixel circuit row 2 at t 0.1 , t 1.1 , t 2.1 , pixel circuit row 3 at t 0.2 , t 1.2 , t 2.2 , and so on.
the smaller dots correspond to the middle row, wherein the pixel circuit rows are imaged at t0, t0.1,...tn+0.1.
the implied slopes between the more numerous dots at t0, t 0.1 ,...t n+0.1 . (corresponding to the middle row) enable much higher fidelity measurement of fiducial acceleration.
This information about the underlying biological sample could not otherwise be determined by exposing or imaging all pixel circuit rows simultaneously.
utilizing the super frame rate methods described above, with or without the angled fiducials described above enables measurement of relevant information about the biological samples that could not otherwise be determined by exposing or imaging all pixel circuit rows simultaneously, due to limitations of the optical detector such as the nominal frame rate.
FIG. 11 is an example of a representative image captured using an optical detector having a field of view that includes all wells of a multi-well plate.
FIG.11 is also a representative graphical user interface of the present disclosure featuring an image of a multi-well array procured at a single time point by the optical detector, wherein two information overlays report biological parameters. Two overlays are representative, and other examples may include information overlays on any number of wells or with respect to any number of tissue fixture pairs.
the representative display is a sample image of an entire multi-well array procured at a single time point by the optical detector.
the interface includes sample property regions overlaid on a plurality of the wells of the image.
Each of the sample property regions Docket No. CURI-P4WO displays a sample parameter, in this case tissue contraction force and beat frequency.
Other sample parameters which may be displayed include contractile force, passive tension, and calcium change.
the sample property regions may be synced temporally with images.
the sample property regions may each display different sample parameters than the representative example shown.
the interface may include one or more input regions overlaid or positioned adjacent to the image region, wherein each input region displays an input parameter such as a tissue stretch (as a percentage) or electrical stimulation (e.g., voltage).
each input region displays an input parameter such as a tissue stretch (as a percentage) or electrical stimulation (e.g., voltage).
the location and content of each display region may differ from the representative example shown.
FIG.12 is another representative graphical user interface of the present disclosure.
the interface features: an image region (e.g., a region of interest) displaying images of the biological sample and fiducials recorded by the optical detector; a sample property region (upper right) displaying a sample parameter (in this example, cardiac beat frequency); an input region (upper left) displaying external input information (in this example, stretch created via a stretching device integrated with the system); and a plot region including a time plot of the sample parameter and the external input information.
an image region e.g., a region of interest
a sample property region upper right
a sample parameter in this example, cardiac beat frequency
an input region upper left
external input information in this example, stretch created via a stretching device integrated with the system
a plot region including a time plot of the sample parameter and the external input information.
FIG. 13E provide flow charts for methods 1300 for determining a sample parameter of a biological sample disposed between a first tissue fixture and a second tissue fixture (e.g., the biological sample 222 supported between the first tissue fixture 224 and the second tissue fixture 228 discussed above with reference to FIG.2A and FIG.2B). It is to be appreciated that any or all of the steps described below with reference to FIG. 13A - FIG. 13E can be performed utilizing the system 100 discussed above with reference to FIG.1A and FIG. 1B and the above-described components thereof.
the processor 106 of the system 100 may be in communication with a non-transitory machine readable storage medium 108 (i.e., a hard drive) encoded with instructions 110 (i.e., software) that cause the processor 106 to perform operations corresponding to the method steps described below.
the instructions 110 may incorporate one or more algorithms for performing one or more measurements, calculations, and identification of system parameters that facilitate execution of the described method Docket No. CURI-P4WO steps/operations and determination of the sample parameters of each biological sample being tested within the system 100.
the processor 106 may issue commands to the other components of the system 100, such as the stimulation lid 112, the light source, and/or the optical detector 104, which may include or incorporate their own separate processors, to perform certain operations corresponding to the method steps described below.
the processor 106 may also incorporate instructions for activating the system 100 to cause a contraction or movement of the biological sample utilizing the tissue assembly 102, as described in WO2021/173887, which has been incorporated by reference in its entirety.
the processor 106, storage medium 108, and instructions 110 may not be incorporated directly into the assembly of the system 100 illustrated in FIG. 1A and FIG.1B.
the processor 106, storage medium 108, and instructions 110 may be incorporated in a connected device, such as a standalone computer or computer/server bank, directly connected or hard-wired to the other components of the system 100.
a connected device may be connected to multiple systems 100 for managing testing across multiple systems 100 simultaneously or serially.
the connected device may be remotely connected to the system 100 through a suitable remote communications protocol familiar to those having ordinary skill in the art.
one representative method 1300 includes a step 1304 of imaging a plurality of frames (e.g., with the optical detector 104 described above with reference to FIG.1A and FIG.1B) of a first fiducial disposed on a first tissue fixture and a second fiducial disposed on a second tissue fixture (e.g., the first fiducial 226 on the first tissue fixture 224 and the second fiducial 230 on the second tissue fixture 228 described above with reference to FIG. 2A and FIG.2B) during movement of the first tissue fixture and/or the second tissue fixture (e.g., as a result of a contractile force of the biological sample).
a plurality of frames e.g., with the optical detector 104 described above with reference to FIG.1A and FIG.1B
a first fiducial disposed on a first tissue fixture and a second fiducial disposed on a second tissue fixture e.g., the first fiducial 226 on the first tissue fixture 224 and the second fiducial 230
the method includes a step 1306 of identifying a first region of interest (e.g., the region of interest 562 described above with reference to FIG.5) in a first frame of the plurality of frames (e.g., an earlier frame N of analysis, not necessarily the initial frame N 0 captured during step 1304) based upon identifying at least one of the first fiducial or the second fiducial in the first frame.
the at least one of the first fiducial or the second fiducial may be identified in the first frame according to at least one known optical parameter of the at least one of the first fiducial or the second fiducial.
the at least one known optical parameter comprises at least one of a size, Docket No.
the method includes a step 1310 of predicting a second region of interest (e.g., the predicted region of interest 568 described above with reference to FIG.5) in a second frame (e.g., a later or subsequent frame N+1 of analysis) based upon at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame.
a second region of interest e.g., the predicted region of interest 568 described above with reference to FIG.5
a second frame e.g., a later or subsequent frame N+1 of analysis
the at least one parameter of the at least one of the first fiducial or the second fiducial includes a tracking parameter of the at least one of the first fiducial or the second fiducial.
the tracking parameter may include at least one of a size, shape, angle, position (fiducial separation distance), velocity, acceleration, deceleration, color, brightness, pixel intensity, or pixel density.
the tracking parameter may be determined between the first frame and the second frame or may be determined over multiple frames, i.e., algorithmic tracking of the fiducials may incorporate information from frames indexed to previous (N-1, N-2, etc.) and subsequent time points (N+2, N+3, etc.), in order to increase effective resolution.
the prediction of the second region of interest in the second frame may be at least partially based upon a known property of the biological sample.
the prediction of the second region of interest may incorporate information about the known size, elasticity, contraction frequency, and/or other sample parameter of the biological sample, which may influence and/or limit the overall movement of the first fiducial and/or the second fiducial through the plurality of frames.
the method also includes a step 1314 of identifying the at least one of the first fiducial or the second fiducial within the second region of interest in the second frame.
the second region of interest may be reset or identified (as opposed to predicted) based upon the identification of the at least one of the first fiducial or the second fiducial in the second frame, similar to the identification of the first region of interest in step 1306 for purposes of measurement and for prediction of a subsequent region of interest in a subsequent frame (e.g., frame N+2).
the identification of the at least one of the first fiducial or the second fiducial in step 1306 and/or in step 1310 may be performed with subpixel spatial resolution (e.g., as described above with reference to FIG. 8A - FIG.9B).
the method further includes a step 1318 of performing a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest and a step 1322 of determining a sample parameter of the biological sample based upon the measurement performed in step 1318.
the measurement of step 1318 may include calculation of a change in a tracking parameter of the at least one of the first fiducial or the second fiducial between the first frame and the second frame or across multiple frames.
algorithmic tracking of the fiducials may incorporate information from frames indexed to previous (N-1, N- 2, etc.) and subsequent time points (N+2, N+3, etc.), in order to increase effective resolution.
the tracking parameter of the at least one of the first fiducial or the second fiducial may be at least one of a size, shape, position (fiducial separation distance), velocity, acceleration, deceleration, color, brightness, pixel intensity, or pixel density.
the measurement of step 1318 may also include determination of a centroid (i.e., calculation of an average value of pixel locations in the x-y directions) of the at least one of the first fiducial or the second fiducial identified during step 1306 and during step 1314.
the sample parameter determined in step 1322 may be a dimensional change of the biological sample, e.g., a length or width, determined according to calculations of a deflection or deflections of the second tissue fixture and/or the first tissue fixture caused by contractions of the biological sample as facilitated through tracking of the movement of the at least one of the first fiducial or the second fiducial.
Step 1322 may include determining the dimensional change with subpixel spatial resolution and/or determining a first centroid of the Docket No. CURI-P4WO first fiducial and a second centroid of the second fiducial with subpixel resolution for each frame imaged by the optical detector.
the method 1300 may further include an optional step 1302 of illuminating the first fiducial and the second fiducial with a light source (e.g., the light source integrated with the stimulation lid 112 described above with reference to FIG.1A and FIG.1B.
the method 1300 may also include an optional step 1320 of performing an additional measurement of an additional fiducial (e.g., the additional fiducial 288 described above with reference to FIG.
a variation of method 1300 images a plurality of frames with an effective frame rate in excess of a nominal frame rate of the optical detector of the system.
the optical detector may include a plurality of pixel circuit rows, as discussed above with reference to FIG.7, and the step 1304 of imaging the pluralities of frames may include a step 1308 of imaging with each pixel circuit row of the plurality of pixel circuit rows at different time points during the movement of the first tissue fixture and/or the second tissue fixture.
the step 1310 of predicting the second region of interest in the second frame may include a step 1312 of identifying at least one of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the plurality of pixel circuit rows.
the step 1314 of identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest may include a step 1316 of identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the plurality of pixel circuit rows, i.e., reconstructing the first fiducial or the second fiducial through the plurality of pixel circuit rows based on the known time between pixel circuit row scans.
the at least one edge of the at least one of the first fiducial or the second fiducial identified in step 1312 and/or in step 1316 extends continuously, i.e.,, straight from one end thereof to an opposing end thereof, at an angle with respect to the pixel circuit rows (e.g., the angle A described above with reference to FIG.2G and FIG.2H).
the at least one of the first fiducial or the second fiducial comprises the angled fiducial Docket No. CURI-P4WO 290 or the plurality of angled markers 296 described above with reference to FIG. 2G and FIG. 2H.
tracking of the first fiducial and/or the second fiducial in order to identify the fiducial in the first frame and/or the second frame and to predict the second region of interest in the second frame may be performed with a temporal resolution faster than a frame rate of the optical detector, e.g., to maximize the resolution of the calculated dimensional change.
the step 1306 of identifying the first region of interest in the first frame may comprise a step 1324 of selecting an area within the first frame around a centroid of the at least one of the first fiducial or the second fiducial (e.g., the centroid 570 of the fiducial 564 within the region of interest 562 described above with reference to FIG.
the selection step 1324 may comprise a step 1326 of filtering one or more regions of non-interest (e.g., as discussed above with reference to FIG.5 and FIG.6).
the step 1310 of predicting the second region of interest in the second frame may include a step 1328 of selecting an area within the second frame larger than the at least one of the first fiducial or the second fiducial (e.g., as described above with reference to FIG.5).
the selection step 1328 may include filtering regions of non-interest (e.g., as discussed above with reference to FIG.5 and FIG.6).
the selection step 1328 may be performed such that the selected area within the second frame is not larger than two of the at least one of the first fiducial or the second fiducial, such that distractor objects (e.g., distractor objects 678 discussed above with reference to FIG. 6) of equal or larger size than the first fiducial or the second fiducial cannot fit with the first fiducial or the second fiducial within the predicted second region of interest.
the step 1314 of identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest in the second frame includes a step 1332 of applying at least one optical threshold to identify a set of pixels within the predicted second region of interest that meets the at least one applied optical threshold.
the at least one applied optical threshold may be based upon at least one known optical parameter of the at least one of the first fiducial or the second fiducial.
the at least one known optical parameter may include a least one of a size, shape, angle (e.g., with respect to the pixel array of the optical detector), color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial. Docket No. CURI-P4WO [0323]
the at least one known optical parameter may be measured in the first frame.
the at least one known optical parameter may be measured across a plurality of frames (e.g., the first frame N and frames N-1, N-2, etc. imaged prior to the first frame N).
the step 1314 of identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest in the second frame includes a step 1334 of filtering objects within the predicted second region of interest that are not the at least one of the first fiducial or the second fiducial (e.g., filtering distractor objects 678 within the predicted region of interest 568 that are smaller than the fiducial 564 as discussed above with reference to FIG. 5 and FIG. 6 or filtering objects that do not meet the at least one optical threshold from step 1332).
FIG. 14B provide flow charts for a method 1400 for use in analyzing a biological sample (e.g., the biological sample 222 supported between the first tissue fixture 224 and the second tissue fixture 228 discussed above with reference to FIG. 2A and FIG. 2B). It is to be appreciated that any or all of the steps described below with reference to FIG. 14A and FIG. 14B can be performed utilizing the system 100 discussed above with reference to FIG. 1A and FIG.1B and the above-described components thereof.
the processor 106 of the system 100 may be in communication with a non-transitory machine readable storage medium 108 (i.e., a hard drive) encoded with instructions 110 (i.e., software) that cause the processor 106 to perform operations corresponding to the method steps described below.
a non-transitory machine readable storage medium 108 i.e., a hard drive
instructions 110 i.e., software
the instructions 110 may incorporate one or more algorithms for performing one or more measurements, calculations, and identification of system parameters that facilitate execution of the described method steps/operations and determination of the sample parameters of each biological sample being tested within the system 100.
the processor 106 may issue commands to the other components of the system 100, such as the stimulation lid 112, the light source, and/or the optical detector 104, which may include or incorporate their own separate processors, to perform certain operations corresponding to the method steps described below.
the processor 106 may also incorporate instructions for activating the system 100 to cause a contraction or movement of the biological sample utilizing the tissue assembly 102, as described in WO2021/173887, which has been incorporated by reference in its entirety. [0327] As shown in FIG.
the method 1400 includes a step 1402 of imaging a plurality of frames of the first fiducial and the second fiducial during a movement of the first tissue fixture Docket No. CURI-P4WO and/or the second tissue fixture; a step 1404 of applying a first optical threshold to identify at least one of the first fiducial or the second fiducial in a first frame of the plurality of frames (e.g., an earlier frame N of analysis, not necessarily the initial frame N0 captured during step 1402); a step 1406 of identifying the at least one of the first fiducial or the second fiducial in the first frame according to the first optical threshold; a step 1408 of measuring an optical parameter of the at least one of the first fiducial or the second fiducial in the first frame; a step 1410 of determining a second optical threshold for identifying the at least one of the first fiducial or the second fiducial in a second frame of the plurality of frames (e.g., a later or subsequent frame N+1 of analysis)
the second optical threshold is applied within a predicted region of interest (e.g., the predicted region of interest 568 described above with reference to FIG.5 and/or according to step 1310 of the method 1300 described above with reference to FIG. 13A - FIG. 13E) to identify a set of pixels (or subpixels) within the predicted region of interest that meets the second optical threshold.
a predicted region of interest e.g., the predicted region of interest 568 described above with reference to FIG.5 and/or according to step 1310 of the method 1300 described above with reference to FIG. 13A - FIG. 13E
the measured optical parameter of the at least one of the first fiducial or the second fiducial comprises at least one of a size, shape, angle (e.g., with respect to a pixel array of the optical detector), color, brightness, luminescence, pixel intensity, or pixel density of the at least one of the first fiducial or the second fiducial.
the measured optical parameter may be measured across a plurality of frames (e.g., the first frame N and frames N-1, N-2, etc. imaged prior to the first frame N).
the method 1400 of FIG. 14A may be incorporated into the method 1300 described above with reference to FIG. 13A - FIG.
the step 1306 of identifying the first region of interest may include the step 1404 of applying the first optical threshold to identify the at least one of the first fiducial or the second fiducial in the first frame of the plurality of frames and the step 1406 of identifying the at least one of the first fiducial or the second fiducial in the first frame according to the first optical threshold.
the step 1314 of identifying the at least one of the first fiducial or the second fiducial in the predicted second region of interest in the second frame may include: the step 1408 of measuring an optical parameter of the at least one of the first fiducial or the second fiducial in the first frame; the step 1410 of determining a second optical threshold for identifying the at least one of the first fiducial or the second fiducial in the second frame of the plurality of frames based on the measured optical parameter; the step 1412 of applying the second optical parameter for identifying the at least one of the first fiducial or the second fiducial in the second frame of the plurality of frames; and the step 1414 of identifying the at least one of the first fiducial or the second fiducial according to the second optical threshold.
a variation of method 1400 images a plurality of frames with an effective frame rate in excess of a nominal frame rate of the optical detector of the system.
the optical detector comprises a pixel array including a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit column and each frame of the plurality of frames is imaged with each of the plurality of pixel circuits of the pixel array as discussed above with reference to FIG.7
the step 1402 of imaging the pluralities of frames may include a step 1416 of imaging with each pixel circuit row of the pixel array at different time points during the movement of the first tissue fixture and/or the second tissue fixture.
the step 1406 of identifying the at least one of the first fiducial or the second fiducial in the first frame may include a step 1418 of identifying at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of the first frame.
the at least one edge of the at least one of the first fiducial or the second fiducial identified in step 1418 extends continuously, i.e.,, straight from one end thereof to an opposing end thereof, at an angle with respect to the pixel circuit rows (e.g., the angle A described above with reference to FIG.2G and FIG.2H).
FIG.15 provides a flow chart for a method 1500 for use in analyzing a biological sample (e.g., the biological sample 222 supported between the first tissue fixture 224 and the second tissue fixture 228 discussed above with reference to FIG.2A and FIG.2B). It is to be appreciated that any or all of the steps described below with reference to FIG.15 can be performed utilizing the system 100 discussed above with reference to FIG.1A and FIG.1B and the above-described components thereof.
a biological sample e.g., the biological sample 222 supported between the first tissue fixture 224 and the second tissue fixture 228 discussed above with reference to FIG.2A and FIG.2B.
the processor 106 of the system 100 may be in communication Docket No. CURI-P4WO with a non-transitory machine readable storage medium 108 (i.e., a hard drive) encoded with instructions 110 (i.e., software) that cause the processor 106 to perform operations corresponding to the method steps described below.
the instructions 110 may incorporate one or more algorithms for performing one or more measurements, calculations, and identification of system parameters that facilitate execution of the described method steps/operations and determination of the sample parameters of each biological sample being tested within the system 100.
the method 1500 may also include the following optional steps: a step 1508 of identifying a first region of interest in the first frame based upon the identification of the at least one of the first fiducial or the second fiducial in the first frame; a step 1510 of predicting a second region of interest in the second frame based upon at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame; a step 1516 of identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest; a step 1518 of performing a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest; and a step 1520 of determining a sample parameter of the biological sample based upon the measurement, e.g., as described above with reference to the method 1300 of FIG.13A - FIG.
FIG. 16A and FIG. 16B provide flow charts for a method 1600 for use in analyzing a biological sample (e.g., the biological sample 222 supported between the first tissue fixture 224 and the second tissue fixture 228 discussed above with reference to FIG. 2A and FIG. 2B). It is to be appreciated that any or all of the steps described below with reference to FIG. 16AA and FIG. 16B can be performed utilizing the system 100 discussed above with reference to FIG. 1A Docket No.
the processor 106 of the system 100 may be in communication with a non-transitory machine readable storage medium 108 (i.e., a hard drive) encoded with instructions 110 (i.e., software) that cause the processor 106 to perform operations corresponding to the method steps described below.
the instructions 110 may incorporate one or more algorithms for performing one or more measurements, calculations, and identification of system parameters that facilitate execution of the described method steps/operations and determination of the sample parameters of each biological sample being tested within the system 100.
the processor 106 may issue commands to the other components of the system 100, such as the stimulation lid 112, the light source, and/or the optical detector 104, which may include or incorporate their own separate processors, to perform certain operations corresponding to the method steps described below.
the processor 106 may also incorporate instructions for activating the system 100 to cause a contraction or movement of the biological sample utilizing the tissue assembly 102, as described in WO2021/173887, which has been incorporated by reference in its entirety.
the method includes a step 1602 of imaging, with the optical detector, a plurality of frames of the first fiducial and the second fiducial during a movement of the first tissue fixture and/or the second tissue fixture.
the optical detector comprises a pixel array including a plurality of pixel circuits arranged into pixel circuit rows and pixel circuit column, as described above with reference to FIG. 7. Each frame is imaged with each of the plurality of circuits of the pixel array.
the method further includes a step 1604 of identifying at least one of the first fiducial or the second fiducial in a first frame of the plurality of frames by identifying at least one edge of the at least one of the first fiducial or the second fiducial and a step 1612 of identifying the at least one of the first fiducial or the second fiducial in a second frame of the plurality of frames by identifying the at least one edge of the at least one of the first fiducial or the second fiducial.
the at least one edge identified in step 1604 and/or in step 1612 extends continuously, i.e., straight from one end thereof to an opposing end thereof, at an angle with respect to the pixel circuit rows of the optical detector (e.g., the angle A described above with reference to FIG. 2G and FIG. 2H).
the angle of the at least one edge is greater than 0 degrees and less than 90 degrees, more particularly the angle of the at least one edge may be greater than or equal to approximately 30 degrees and less than or equal to approximately 75 degrees, more particularly the angle of the at least one edge may be greater Docket No. CURI-P4WO than or equal to approximately 40 degrees and less than or equal to approximately 60 degrees, and more particularly the angle of the at least one edge may be approximately 45 degrees.
the at least one of the first fiducial or the second fiducial comprises the angled fiducial 290 described above with reference to FIG. 2G or the plurality of angled markers 296 described above with reference to FIG. 2H.
the step 1604 of identifying the at least one edge in the first frame may comprise a step 1606 of identifying parallel continuous edges of the fiducial (or at least one of the plurality of fiducials) in the first frame and the step 1612 of identifying the at least one edge in the second frame may comprise a step 1614 of identifying the parallel continuous edges of the fiducial (or at least one of the plurality of fiducials) in the second frame.
the method 1600 may also include the following optional steps: a step 1608 of identifying a first region of interest in the first frame based upon the identification of the at least one of the first fiducial or the second fiducial in the first frame; a step 1610 of predicting a second region of interest in the second frame based upon at least one parameter of the at least one of the first fiducial or the second fiducial determined from the first frame; a step 1616 of identifying the at least one of the first fiducial or the second fiducial within the predicted second region of interest; a step 1618 of performing a measurement of the at least one of the first fiducial or the second fiducial between the first region of interest and the second region of interest; and a step 1620 of determining a sample parameter of the biological sample based upon the measurement, e.g., as described above with reference to the method 1300 of FIG.13A - FIG.
a variation of method 1600 images a plurality of frames with an effective frame rate in excess of a nominal frame rate of the optical detector of the system.
the step 1602 of imaging the plurality of frames includes a step 1622 of imaging with each pixel circuit row of the optical detector at different time points during the movement of the first tissue fixture and/or the second tissue fixture.
the step 1604 of identifying the at least one of the first fiducial or the second fiducial in the first frame includes a step 1624 of identifying the at least one edge of the at least one of the first fiducial or the second fiducial in each pixel circuit row of at least a portion of the pixel circuit rows of the first frame.
the step 1612 of identifying the at least one of the first fiducial or the second fiducial in the second frame includes a step 1626 of identifying the at least one edge of the at least one of the first fiducial or Docket No. CURI-P4WO the second fiducial in each pixel circuit row of at least a portion of the pixel circuit rows of the second frame.
the present application may also reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present application. Also in this regard, the present application may use the term “plurality” to reference a quantity or number. In this regard, the term “plurality” means any number that is more than one, for example, two, three, four, five, etc. The term “about,” “approximately,” etc., means plus or minus 5% of the stated value. The term “based upon” means “based at least partially upon.” The term “between” includes the values recited in connection therewith.

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