EP4256404A1 - Système de microscope électro-holographique capable de distinguer des cellules et des micro-organismes sur la base de la transmittance de lumière - Google Patents

Système de microscope électro-holographique capable de distinguer des cellules et des micro-organismes sur la base de la transmittance de lumière

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Publication number
EP4256404A1
EP4256404A1 EP21901159.0A EP21901159A EP4256404A1 EP 4256404 A1 EP4256404 A1 EP 4256404A1 EP 21901159 A EP21901159 A EP 21901159A EP 4256404 A1 EP4256404 A1 EP 4256404A1
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EP
European Patent Office
Prior art keywords
chamber
phase
phase data
main beam
electric field
Prior art date
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.)
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Application number
EP21901159.0A
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German (de)
English (en)
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EP4256404A4 (fr
Inventor
Rahmetullah VAROL
Abdurrahim YILMAZ
Huseyin UVET
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Yildiz Teknik Universitesi
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Yildiz Teknik Universitesi
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Publication of EP4256404A1 publication Critical patent/EP4256404A1/fr
Publication of EP4256404A4 publication Critical patent/EP4256404A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/04—Processes or apparatus for producing holograms
    • G03H1/0443—Digital holography, i.e. recording holograms with digital recording means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/04—Processes or apparatus for producing holograms
    • G03H1/08—Synthesising holograms, i.e. holograms synthesized from objects or objects from holograms
    • G03H1/0866—Digital holographic imaging, i.e. synthesizing holobjects from holograms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/1454—Optical arrangements using phase shift or interference, e.g. for improving contrast
    • 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
    • G01N21/1717—Systems in which incident light is modified in accordance with the properties of the material investigated with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance
    • G01N2021/1721—Electromodulation
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/0005—Adaptation of holography to specific applications
    • G03H2001/0033—Adaptation of holography to specific applications in hologrammetry for measuring or analysing
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/0005—Adaptation of holography to specific applications
    • G03H2001/0033—Adaptation of holography to specific applications in hologrammetry for measuring or analysing
    • G03H2001/0044—Adaptation of holography to specific applications in hologrammetry for measuring or analysing holographic fringes deformations; holographic sensors
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/0005—Adaptation of holography to specific applications
    • G03H2001/005—Adaptation of holography to specific applications in microscopy, e.g. digital holographic microscope [DHM]
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/04—Processes or apparatus for producing holograms
    • G03H1/0443—Digital holography, i.e. recording holograms with digital recording means
    • G03H2001/0454—Arrangement for recovering hologram complex amplitude
    • G03H2001/0458—Temporal or spatial phase shifting, e.g. parallel phase shifting method
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H2227/00—Mechanical components or mechanical aspects not otherwise provided for
    • G03H2227/03—Means for moving one component

Definitions

  • the present invention relates to rapid diagnostic devices based on imaging technologies, for use in the field of medicine and biomedicine.
  • circulating cancer cells are difficult to be distinguished because of their size similarity to normal blood components (having an average width of 15- 20 ⁇ m) and their relatively small number in the circulation.
  • the change in light transmittance is used to obtain a number of information about the cell by distinguishing of the cells [1], which is used to distinguish cell types, detect many diseases, and obtain information for purposes of differentiating pathogens in foods [20]. There are many methods to calculate the light transmittance [19].
  • the geometrical models of the cells are predicted by shape fitting based on the information about the thickness. Prediction of the cell shape is an obstacle to obtaining precise results [2-3].
  • Other methods of measuring the cell thickness require mechanical stimulation or chemical staining. Imaging of the cells exposed to mechanical stimulation or chemical staining in turn requires expensive microscopes and long experimental processes [4-5].
  • the integral refractive index is calculated using the cell thickness.
  • the cell is modeled as a sphere and a phase map is fitted on the sphere so as to obtain the light transmittance.
  • Kemper et al. [2, 8] calculate the integral refractive index by applying an iterative fitting approach on the entire optical path difference. Here, they solve the nonlinear least squares problem by applying the Gauss-Newton method [9].
  • the integral refractive index of the cell is assumed to be constant. Therefore, this method can only be applied on cells having a homogeneous light transmission distribution.
  • a spherical optical path difference should be used for the spherical cell morphology. In this technique, for a surface with low homogeneity, the margin of error is high because the calculation is made by surface fitting.
  • Schlirmann et al. [3] perform a fitting process using edge detection algorithms on the resulting phase images.
  • Steelman et al. [10] perform a circle fitting by applying the circular Hough transform, and generate a thickness profile.
  • a thickness profile can be calculated using both methods. However, since these methods make a calculation by surface fitting, the light transmittance of each point of the cell results from the fitted function, not the physical values, which increases the error rate.
  • the filters applied can be affected by external factors such as dust, signal noise, etc., in which case proper profiles cannot be obtained, leading to an increase in the margin of error.
  • Cardenas et al. [29] perform a measurement by a system capable of using an atomic force microscope (AFM) and a Digital Holographic Microscope (DHM) simultaneously. They are able to measure the cell thickness by simultaneous recording with DHM during the intrusion of the AFM probe.
  • AFM atomic force microscope
  • DHM Digital Holographic Microscope
  • Balberg et al. [11] use a special lattice structure to detect the type of the fixed cells. They correlate it with the measurement of the AFM probe while imaging with DHM.
  • the cell thickness can be physically measured directly by confocal fluorescence microscopy. However, this requires marking with methods such as fluorescent dyes [12, 13]. Lue et al. [4] manage to obtain the physical boundaries of the cell without marking using the reflectance confocal microscopy. This allows them to obtain a high-resolution cell thickness map by sectioning the cell. Then, by solving the thickness- RI coupling problem with DHM, they arrive the integral refractive index. Choi et al. [5], on the other hand, use the optical coherence microscopy (OCT) for the whole area to obtain a series of tomogram at 0.6 pm sections, in order to measure the thickness. This method can only be used on reflective biological structures. However, this method is not widely used because most of the biological structures are not reflective.
  • OCT optical coherence microscopy
  • the integral refractive index of the cell can be calculated by adding the light transmittance of the microchannel to the optical path difference from the cell of known thickness.
  • a special microchannel has to be produced precisely from a transparent material with known light transmittance.
  • experimental procedures are slow and highly skill-dependent, since the cell must be inserted into the channel without damage.
  • phase information of the light is used to calculate the light transmittance by interferometric methods.
  • phase maps are generated in two different ways: changing the wavelength of the medium liquid or the laser source [6, 7].
  • these interferometric solutions require lengthy processes that necessitate intervention in the experimental environment, and in many cases also the change of the test materials.
  • it is not possible to speak about a system that can be integrated into an incubator due to the fact that the aforementioned solutions require instantaneous intervention in the experimental environment and are bulky-large. The need for performing the experiments outside the incubator causes contamination on the cells and affects the accuracy of the experiment results.
  • phase maps are obtained with interferometric methods.
  • the first phase map is obtained before the operation is performed, and the second phase map is obtained after the operation is performed.
  • the integral refractive index can be calculated from these two different phase maps.
  • Rappaz et al. [6] provide for changing of the phase maps by adding a liquid medium with known light transmittance to the medium or by completely changing the medium therein.
  • the cells make oscillatory movements at micro- and nanometer levels. In this case, it causes temporary phase shifts and results in errors in calculations.
  • the average integral refractive index is calculated.
  • noise is tried to be reduced with gradient-based edge detection and morphological algorithms, which requires time and processing power.
  • the liquid medium in the medium must have the same osmotic pressure as the medium changed, it causes erroneous results.
  • the secondary medium fluid must meet the biological and chemical conditions for the cell, there is not enough suitable medium for some cell types and conditions, and the cell and the medium fluid react and cause a toxic effect for the cell.
  • [15] use a food dye with high distinctiveness as a medium, wherein relatively low toxicity values on living cells are observed. Since the laser wavelength has to be changed in said methods, if the test area is not large enough, it is not possible to switch between laser beams and the laser source must be removed to install a different laser source. This also affects the precision due to loss of time and contact with the optical experimental setup. In both cases, lasers with two different wavelengths are needed, and these lasers are expensive, causing a great deal of cost. In order to reduce the test period, the test area must be enlarged, which increases the cost.
  • Principal object of the invention is to provide solutions to the problems mentioned in the prior art.
  • Another object of the invention is to provide a low cost electro-holographic microscope apparatus yielding results with high speed and precision.
  • Another object of the invention is to propose an algorithmic method for operating the apparatus.
  • the improvement which is the subject of the present application, makes it possible to calculate the light transmittance of living cells with the holographic apparatus.
  • the phase maps of the cells can be obtained by using a quantitative phase imaging method with the holographic apparatus. By seeding the cells in the designed test setups, an electric field will be generated on the cell by means of its electrodes, and phase information will be generated under different electric field intensities. By using the resulting phase maps, the change in the light transmittance resulting from the change in the electric field intensity can be calculated in a unique manner.
  • Figure 1 is an isometric view of an exemplary embodiment of the apparatus of the present application.
  • Figure 2 is a side view (e.g., as seen from a direction -y/+y) of an exemplary embodiment of the apparatus of the present application.
  • Figure 3(a) is an isometric detail view of a vicinity of a chamber of an exemplary embodiment of the apparatus of the present application.
  • Figure 3(b) is a view (e.g., as seen from a direction +z/-z) of a chamber suitable for use in an exemplary embodiment of the apparatus of the present application.
  • Figure 4 is an isometric view of an exemplary embodiment of the apparatus of the present application.
  • the inventive improvement is a phase shift-based interferometric microscope apparatus (100).
  • Said apparatus (100) comprises a digital camera (2) and a beam source (21).
  • the apparatus (100) also comprises a beam splitter (22) suitably configured to split a beam (B) emitted from said beam source (21) into two parts, a main beam (Bl) and an equivalent phase-shifted reference beam (BR).
  • said digital camera (2) can be considered in that it has a structure suitable for recording a resultant obtained by the coincident travel of the main beam (Bl) and the reference beam (BR), and is positioned for this purpose.
  • the digital camera (2) can be considered as a highspeed camera suitable for recording interferograms.
  • the beam source (21) can be considered a laser diode.
  • Figure 1 is an isometric view of an exemplary embodiment of the apparatus of the present application.
  • the digital camera (2), the beam source (21) and the beam splitter (22) are schematically illustrated for better understanding of the orientations of the main beam (Bl) and the reference beam (BR).
  • the apparatus (100) also comprises a chamber (1) for placing a sample therein, which is configured to allow the main beam (Bl) to pass through the sample in a first direction (+z/-z).
  • sample can be considered as a biological material, e.g., a group of cells.
  • the apparatus (100) further comprises at least a pair of electrodes (11) disposed mutually opposite to each other on either side of the chamber (1), which are configured to expose a sample to an electric field when the sample is placed in the chamber (1).
  • the pair of electrodes (11) may preferably be suitably configured/ positioned to expose the sample to an electric field in a second direction (+y/-y) perpendicular to the first direction (+z/-z).
  • the pair of electrodes (11) may comprise two electrodes (111) of flat geometry disposed in such a way to constitute planes parallel to the first direction (+z/-z) (i.e., one plane that is parallel to a plane x-z obtained with a direction +x/-x and the first direction (+z/-z)).
  • the apparatus (100) also comprises a nanometer-precision stepping linear motor suitably positioned to shift the phase of the reference beam (BR).
  • the apparatus (100) also comprises a beam combiner (30) suitably configured and positioned to guide the main beam (Bl) to the digital camera (2) after it has passed through the chamber (1), and to guide the reference beam (BR) to the digital camera (2) after having been passed therethrough so as to travel coincident with the main beam (Bl).
  • a beam combiner (30) suitably configured and positioned to guide the main beam (Bl) to the digital camera (2) after it has passed through the chamber (1), and to guide the reference beam (BR) to the digital camera (2) after having been passed therethrough so as to travel coincident with the main beam (Bl).
  • the apparatus (100) may preferably include a first lens (31) suitably configured and positioned to enlarge the main beam (Bl) before it reaches the beam combiner (30).
  • the apparatus (100) may preferably include a second lens (32) which is suitably configured and positioned to enlarge the reference beam (BR) before it reaches the beam combiner (30).
  • a second lens (32) which is suitably configured and positioned to enlarge the reference beam (BR) before it reaches the beam combiner (30).
  • the apparatus (100) may preferably include a beam (B) guiding means (4) for guiding the main beam (Bl) to be passed through the chamber (1) (through the sample) in the first direction (+z/-z).
  • Figure 2 is a side view (e.g., as seen from a direction -y/+y) of an exemplary embodiment of the apparatus of the present application.
  • the beam guiding means (4) By means of the beam guiding means (4), the direction of the main beam (Bl), which is deflected, and which passes through the chamber (1) by moving in the first direction (+z/-z), and its travel in the direction of the second lens (32) to be delivered to the beam combiner (30) are symbolically shown with dashed lines.
  • Figure 3(a) is an isometric detailed view of a vicinity of a chamber (1) of an exemplary embodiment of the apparatus of the present application. Due to the angle of drawing, only one of the electrodes (111) of the pair of electrodes (11) located in the chamber (1) is visible.
  • Figure 3(b) is presented to illustrate an exemplary embodiment of the chamber (1) and the electrode pair (11) in detail.
  • Figure 3(b) is a view (e.g., as seen from a direction +z/-z) of a chamber (1) suitable for use in an exemplary embodiment of the apparatus of the present application.
  • the contours of the power lines (cables) to supply the electrodes (111) with voltage are indicated by dashed lines.
  • the beam guiding means (4) can be considered as a mirror that can be positioned at different angles, with a suitable structure for allowing the main beam (Bl) to be projected on the sample/object/cells to be monitored.
  • Figure 3(b) is a view (e.g., as seen from a direction +z/-z) of a chamber (1) suitable for use in an exemplary embodiment of the apparatus of the present application.
  • the apparatus (100) may preferably include a positioner (6) adapted to adjust/change the position of the chamber (1) with respect to the main beam (Bl) (its coordinates on the first direction (+z/-z) and on a plane (x-y) perpendicular to the first direction (+z/-z)).
  • the plane perpendicular to the first direction (+z/- z) can be considered as a plane x-y (the plane formed by the direction +x/-x and the direction +y/-y).
  • Figure 4 is an isometric view of an exemplary embodiment of the apparatus of the present application, illustrating an exemplary plane x-y conforming to this description.
  • Figure 4 illustrates, in dashed lines, an exemplary travel of the main beam (Bl) such that it will pass through the chamber (1) (and preferably through the second lens (32) to be transferred to the beam combiner (30)) via the beam guiding means (4).
  • Figure 4 also illustrates, dashed lines, transmission of the reference beam (BR) (preferably through the first lens (31)) to the beam combiner (30), the overlap of the main beam (Bl) and the reference beam (BR) by the beam combiner (30) and thus their resultant (Bl+BR) reaching the digital camera (2).
  • BR reference beam
  • Adjusting/changing the position of the chamber (1) with respect to the main beam (Bl) can also be considered as the adjustment of the coordinates of the place where the main beam (Bl) is incident on the chamber (1) on a plane (x-y) perpendicular to the first direction (+z/-z).
  • the positioner (6) can be considered in that it is adapted for adjusting/ modifying the position of the chamber (1) on the first direction (+z/-z) and the plane (x-y) perpendicular the first direction (+z/-z) with respect to the location of the main beam (Bl), e.g., the beam combiner (30) and the first beam source (21) or the beam guiding means (4).
  • the positioner (6) may be adapted to shift the chamber (1) on said plane, preferably in micrometer-scale steps (i.e., with step lengths of 100 micrometers or less), more preferably in nanometer-scale steps (i.e., with step lengths of 100 micrometers or less).
  • DPM Digital Holographic Microscopy
  • the interferometric methods such as digital holographic microscopy have been used for imaging cell morphology for a long period of time.
  • interferometry has been used to characterize ion mobility in the cell membrane.
  • optical transmittance has been demonstrated in different studies [5, 16].
  • the apparatus set forth in the present application provides a new method for cell characterization that has not been disclosed before. Using the high precision of digital holography, the effect of the electric fields applied on the cell on ion movements and subsequently on different cell functions can be modeled.
  • the holographic imaging system (apparatus (100)) of the present application has features suitable for performing a detailed characterization for the optical transmittance of the cell on a single cell.
  • Said apparatus (100) is suitable to provide a method that can be used to characterize how the varying electric fields regulate different functions of the cell, based on the images obtained.
  • the functions of the cell regulated by the electric fields, and ion channels, for example, are of great importance in cancer research and clinical practice [17, 18].
  • the apparatus of the present application is suitable, for example, to detect ion channels with high potential for clinical applications and to characterize how the cell functions are regulated based on the ion channels.
  • the apparatus of the present application will be of high value for research centers and private clinics working on clinical applications of ion channels.
  • An early diagnosis of diseases and early initiation of the treatment is one of the main factors for a healthy society. It is as important as the treatment if people consume healthy foods and the diseases are prevented before occur by detecting foodborne pathogens. Foodborne pathogens are important not only to humans but also to other animals and even plants.
  • kits for screening and rapid diagnosis are indispensable for big industries such as health and food.
  • High cost devices and long waiting times are required for various diagnostics operations. Due to the high cost devices, said possibility of diagnosis is not accessible everywhere, so diagnosis can only be performed by transferring the devices from one geographic location to another. Therefore, relatively longer waiting times are required for test results. In order to test the effects of foods on various drugs developed, innovative methods are also needed.
  • diagnostic kits with the ability to perform instantaneous or simultaneous tests without requiring high-cost devices and long waiting times, it is possible to obtain test results in a very quick manner and at low cost. Especially in terms of our country, these kits are mostly imported from abroad, and purchasing at high import prices causes economic loss. Especially in times of crisis, these kits cannot be supplied and health services are disrupted. With the apparatus (100) of the present application and a method therefor, screening and diagnostic kits can be developed.
  • an adjustable electric field is generated on the microscopic living form that is desired to be imaged.
  • the phase map of the cell is obtained by digital holography method. There occur changes in the light transmittance of the microscopic cells or organisms within the electric field, due to the transport of ions in said cell or organism and the increase in ion mobility in the cell membrane. In order to capture this change, it is necessary to obtain the phase maps before and after the electric field is delivered.
  • the phase maps In order to derive the phase maps, precise holographic methods are needed. By using the digital holography method, the light transmittance can be determined in proportion to the wavelength at which the cell is illuminated. In order to determine the integral refractive index of each illuminated point, the phase maps can also be obtained by digital holography method and the integral refractive index can be determined. However, due to the coherence of the light source, speckle noise occurs in the captured diffraction patterns. The high noise ratios in the diffraction patterns can cause errors in the calculation of the phase map of the cell.
  • diffraction patterns may be obtained by a digital camera (2) with a low signal to noise ratio, at a high speed (for example, 120 frames/second with today's technology), high resolution (for example, 1920x1080 pixel resolution with today's technology), and high quantum efficiency (for example, 78% and above with today's technology).
  • a diffraction pattern can be obtained by increasing the speed and decreasing the resolution (e.g., with a speed of 1000 fps and a resolution of 1024x128 pixels).
  • the apparatus (100) comprising said digital camera (2), a large number of diffraction patterns are captured and averaged; in addition, by using directional filters on each diffraction pattern image, both the speckle noise and the noise of the camera are suppressed.
  • Each diffraction pattern contains the depth information of the cell as a phase difference.
  • the term "object” In order to calculate the required phase information, the term “object” must be distinguished from the terms “direct current noise”, “cross”, “twin” and “object” in the diffraction patterns. This process is based on recording the diffraction patterns for four different phases of the reference wave (reference beam (BR)).
  • reference beam reference wave
  • each step of the linear motor is tens of nanometers long, the desired phase differences cannot be obtained exactly.
  • diffraction patterns are recorded for many different phase differences, providing a 2n cycle. Then, the desired images for the phase difference can be calculated with the curve fitting of the best fit.
  • the integral refractive index of the cell is deduced from the resulting diffraction patterns. All operations can be done very quickly and the integral refractive index of the cell can (essentially) be calculated and displayed in real-time.
  • the holographic apparatus (100) of the present application can be operated both inside and outside of an incubator, it also has a simple test procedure. With an automation system that can be introduced into the apparatus (100), an electric field can be applied periodically, and simultaneous images can be obtained. In this respect, the apparatus does not require physical contact with the test medium, since it does not require physical intervention.
  • the light transmittance of the cells can be calculated without requiring any equipment (e.g., a second laser or different types of microscopes) other than the test apparatus (100) of the invention.
  • the inventive apparatus (100) offers a relatively low-cost solution since it does not require a secondary laser or different types of microscopes. Therefore, the development being the subject of the present application solves the intended technical problem.
  • the present application also proposes a method (software algorithm) for use during the operation of the apparatus (100).
  • the explanations regarding the method, which resolves the interferograms obtained with the digital camera (2) as data, are as follows.
  • the phase data is calculated by the phase opening method.
  • the phase data of two different medium conditions are used, which are called as “primary phase data” and "secondary phase data".
  • the primary phase data is a phase data of a condition of the medium before the electric field is applied, whereas the secondary phase data is a phase data after the electric field is delivered for the same condition of the medium.
  • the phase shift region is determined. For this, the brightness value of a fixed pixel on the video is extracted throughout the video (recording time, time frame). Then, the regions where said brightness value provide the "2n cycle" are determined as phase shift regions. Within this time frame of the video, a phase map can be obtained by means of the phase shift interferometry. But, due to asymptotes in the arctangent operation, the values are reset between passes of the 2n cycle; the resulting error is corrected by decoding of the phase data. After obtaining the phase data ⁇ the first phase data and the second phase data) in two different conditions, the light transmittance of each pixel on the image obtained can be calculated from the optical path length (OPL) formula. The mathematical progression of the algorithm is based on the optical path length (OPL) and the light intensity.
  • the interferograms obtained as a result of each step of the phase shift are expressed in accordance with the following equation 1 (interferogram formulation):
  • Equation 1 the term "i" represents the number of squares; A(x,y), B(x,y), ⁇ ( x,y) and ⁇ represent the background brightness, modulation amplitude, angular phase information and phase shift amount obtained by the PZT motor, respectively. Since the values of A(x,y) and B(x,y) cannot be known, these parameters need to be eliminated. To this end, simplification is made by obtaining more than one interferogram by phase shifting, thus phase information is obtained. The phase information corresponds to information about a snapshot. The phase information before and after the electric field is applied is obtained separately and recorded. In the second stage, the transition from the OPL formula to light transmission is performed. The OPL is expressed in accordance with Equation 2 and Equation 3 below:
  • Equation 2 ⁇ is the phase information
  • A is the wavelength of the laser
  • n is the light transmittance of the medium
  • h is the path in which the light travels.
  • Equation 4 Phase information before the electric field is delivered:
  • phase information after the electric field is delivered is expressed according to Equation 5 below:
  • the integral refractive index is expressed according to Equation 6 below, based on said two phase information:
  • the height of the medium can also be determined.
  • Height (medium height) is expressed in accordance with equation 7 below:
  • the present application provides an electro-holographic method for distinguishing cells and microorganisms based on the light transmittance, said method comprising the following: passing a main beam (Bl) through a sample located in a chamber (1) while shifting the phase of the reference beam (BR) with a linear motor, overlapping said main beam (Bl) with the reference beam (BR) to thus obtain a resultant (Bl+BR), sending said resultant (Bl+BR) into a digital camera (2) to obtain a phase data; in a first case, recording the phase data as a primary phase data, in a second case, exposing the chamber (1) to an electric field different from the first case, and recording the resulting phase data as a secondary phase data, calculating an integral refractive index data using the primary phase data and the secondary phase data.
  • the method preferably comprises the following algorithm elements: determining a plurality of phase shift regions to analyze a plurality of interferograms obtained by a digital camera (2); accordingly, extracting the brightness value of a pixel located on a video area obtained by said digital camera (2) during a recording period; then determining those regions where said brightness value provides the 2n cycle as the phase shift regions; calculating a phase data for each phase shift region by the phase opening method using a primary phase data and a secondary phase data that differ from each other, wherein said primary phase data pertains to a condition of the medium before an electric field is delivered, the secondary phase data pertains to said condition of the medium in the presence of an electric field; obtaining a phase map by means of the phase shift interferometry during the recording period; calculating an "integral refractive index" data using a combination of the primary phase data and the secondary phase data; preferably, calculating an "medium height” data using the integral refractive index data.
  • the cells are exposed to an electric field by placing electrodes (111) on the sides of a part where the cells will be seeded and applying voltage thereto.
  • the electrodes (111) By feeding the electrodes (111) with a source of voltage, the cells can be observed between the desired electric field intensity and the light transmittance that will be generated under different electric fields can be observed.
  • the chamber (1) is positioned between the beam guiding means (4) and the second lens (32).
  • a beam (B, laser beam) supplied from a beam source (21, e.g., a laser diode) is split into two parts, namely a reference beam (BR) and an object beam (a main beam (Bl)) by the beam splitter 22.
  • the main beam (Bl) is guided (preferably by means of the beam guide (4)) and passes through a living microstructure to be monitored (a cell or micro-organism placed in the chamber (1), between the electrodes (111) forming the pair of electrodes (11); preferably then, the imaged area is enlarged by means of a second lens (32).
  • an electric field is generated between the electrodes (111) forming the electrode pair (11) through the current carrying cables, which are illustrated in Figure 3(b).
  • the electric field causes the light transmittance of the sample (microorganism or cells) in the chamber (1) to change, and a diffraction pattern is obtained.
  • the diffraction pattern is captured by the digital camera (2). It is useful to use diffraction patterns in different phases to extract the light transmittance of the sample in the chamber (1).
  • the relative position of the beam guiding means (4) and the chamber (1) with respect to each other can be changed by a positioner (6) with micronanometer precision (motorized or manually).
  • a positioner (6) with micronanometer precision (motorized or manually).
  • the captured diffraction patterns contain the phase information of the sample in the chamber (1).
  • the light transmittance of the sample is calculated.
  • the phase information of the sample (living microstructures, cell or micro-organism) remaining in the electric field of the electrodes (111) in the chamber (1) changes. Therefore, with the improvement of the present application, the light transmittance of living microstructures (cells or micro-organisms) can be determined.
  • the apparatus can operate independent from the cell shape while calculating the integral refractive index of each point.
  • the light transmittance can be calculated with a low margin of error by using real physical values.
  • a particular advantage of the present invention can be summarized as follows: with the apparatus of the present application (and the method for use thereof), it is not necessary for the cell type to be static; the apparatus and the method are suitable for application in all cell types.
  • the apparatus (100) of the invention does not require AFM and a system that must operate simultaneously.
  • the apparatus 100 (and the chamber (1)) is relatively simple and low cost. It does not matter whether a sample (biological structure) to be measured is reflective or not. Also, confocal fluorescence microscopy is not needed. Therefore, the integral refractive index can only be calculated with the DHM method, independent of the reflectivity property and the cell thickness.
  • the improvement of the invention provides a chamber (1) that is relatively easy to convert into a kit, since it does not require the use of chemical dyes.
  • an advantage of the present invention can be summarized as follows: In the method that can be performed with the apparatus of the invention, no micro-channel is needed. In addition, the chip (chamber (1)) in which cells or micro-organisms will be placed can be produced in desired dimensions. There is no limitation in size, so that flexibility can be offered in terms of sizing in the design of the chamber (1).
  • an advantage of the present invention can be summarized as follows:
  • a second laser source and a larger area are not needed.
  • the phase map can be changed, and thus the integral refractive index can be calculated.
  • opposite electrodes (111) are disposed on both sides of the chamber (1) to be prepared for the experiments. First, the phase information of the cell is obtained before the electric field is delivered, and then the phase information is obtained under the electric field.
  • the integral refractive index can be calculated with the interferometric apparatus and a voltage source. Since there is no need for different types of microscopes used in the aforementioned studies, it can be converted into a kit. Since the chamber (1) of the interferometric apparatus can be set up in the size of an incubator and there is no need to intervene in the chamber (1), it allows the experiments to be carried out either inside an incubator being a chamber (1), or outside it.
  • an advantage of the present invention can be summarized as follows: In the improvement of the present application, no intervention is made to the medium liquid corresponding to the sample to be placed in the chamber (1). Since it is not necessary to change the medium fluid, the cells do not move and the phase information obtained at two different times is obtained under the same conditions. Besides, by eliminating the need for a different medium fluid, the complexity of the experimental protocols is also reduced.

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Abstract

La présente invention concerne un appareil de type microscope interférométrique basé sur un décalage de phase (100). Ledit appareil comprend une caméra numérique (2), une source de faisceau (21), et un diviseur de faisceau (22) qui divise un faisceau (B), émis par la source de faisceau (21), en deux parties, un faisceau principal (Bl) et un faisceau de référence (BR) à décalage de phase équivalent. L'appareil (100) comprend également une chambre (1) pour placer un échantillon à l'intérieur de celle-ci, qui est configurée pour permettre au faisceau principal (Bl) de traverser l'échantillon dans une première direction (+z/-z); au moins une paire d'électrodes (11) disposées mutuellement opposées l'une à l'autre de chaque côté de la chambre (1), qui sont configurées pour exposer l'échantillon à un champ électrique; et un combineur de faisceaux (30) configuré et positionné de façon appropriée pour guider le faisceau principal (Bl) vers la caméra numérique (2) après que le faisceau a traversé la chambre (1), et pour guider le faisceau de référence (BR) vers la caméra numérique (2) une fois qu'il l'a traversé de façon à se qu'il déplace en coïncidence avec le faisceau principal (Bl). L'appareil (100) doit être utilisé pour calculer le changement de transmission de lumière provoqué par différentes intensités de champ électrique à appliquer par la paire d'électrodes (11) sur celui-ci.
EP21901159.0A 2020-12-02 2021-06-14 Système de microscope électro-holographique capable de distinguer des cellules et des micro-organismes sur la base de la transmittance de lumière Withdrawn EP4256404A4 (fr)

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TR2020/19536A TR202019536A2 (tr) 2020-12-02 2020-12-02 Hücre ve mikro organizmaların ışık geçirgenliğine göre ayırabilen elektro-holografik mikroskop sistemi.
PCT/TR2021/050597 WO2022119521A1 (fr) 2020-12-02 2021-06-14 Système de microscope électro-holographique capable de distinguer des cellules et des micro-organismes sur la base de la transmittance de lumière

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US8896840B2 (en) * 2012-04-25 2014-11-25 Canon Kabushiki Kaisha Interferometric method and digital holographic microscope
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