WO2010110096A1 - Dispositif réactionnel de mesure optique et son procédé de mesure - Google Patents

Dispositif réactionnel de mesure optique et son procédé de mesure Download PDF

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Publication number
WO2010110096A1
WO2010110096A1 PCT/JP2010/054257 JP2010054257W WO2010110096A1 WO 2010110096 A1 WO2010110096 A1 WO 2010110096A1 JP 2010054257 W JP2010054257 W JP 2010054257W WO 2010110096 A1 WO2010110096 A1 WO 2010110096A1
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WIPO (PCT)
Prior art keywords
light
liquid container
optical
reaction
light receiving
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Ceased
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PCT/JP2010/054257
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English (en)
Japanese (ja)
Inventor
秀二 田島
善直 平原
修 瀬川
秀雄 池田
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Universal Bio Research Co Ltd
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Universal Bio Research Co Ltd
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Priority to JP2011505979A priority Critical patent/JP5623385B2/ja
Publication of WO2010110096A1 publication Critical patent/WO2010110096A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/0332—Cuvette constructions with temperature control
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/062—LED's

Definitions

  • the present invention relates to a reaction optical measuring device and a measuring method thereof.
  • PCR polymerase chain reaction
  • double-stranded DNA is denatured to single-stranded, and the primer anneals to single-stranded DNA. Two molecules of DNA fragments complementary to a single strand are made. Since the DNA fragment synthesized in the next cycle also serves as a template, the DNA fragment synthesized after n cycles becomes 2 n molecules.
  • Patent Documents 1 and 2 disclose that a plurality of wells in the microplate are simultaneously optically measured and analyzed.
  • an optical fiber bundle having a common focal system for each well is provided above the liquid surface accommodated in the well, or the optical fiber bundle array corresponding to the well array can be moved. While being provided, some of the optical fibers of the optical fiber bundle may be used for excitation light irradiation, and other optical fibers may be used for guiding fluorescence to the light receiving unit (Patent Document 3).
  • the present invention has been made to solve the above problems, and its first object is to have a simple mechanism without using a complicated optical system or a precise moving mechanism. Regardless, it is an object of the present invention to provide a reaction optical measurement apparatus and an optical measurement method which are substantially free from chromatic aberration, are compact and inexpensive to manufacture.
  • the second object is to provide a reaction optical measurement device and an optical measurement method that are easy to adjust and easy to use.
  • a third object is to provide a reaction optical measurement device and an optical measurement method with high reliability.
  • a planar liquid container in which a plurality of liquid storage parts capable of storing a reaction solution containing a luminescent substance are arranged along a plane, and temperature control is performed in each liquid storage part, A principal ray of light emitted in a direction perpendicular to the plane through the openings of the plurality of liquid containers is incident at a predetermined incident angle with respect to the optical axis, and each is located at a position away from the planar liquid container.
  • a reaction optical measurement device having one or a plurality of concave mirror elements that form a real image by light emission at an imaging position and a light receiving unit that receives light from each real image at each predetermined measurement position.
  • the “reaction solution” is a solution in which the reaction is performed, and for example, there is a PCR reaction solution.
  • the “PCR reaction solution” is a solution in which a PCR reaction is performed, such as template DNA, primer, DNA polymerase, nucleotide, reaction buffer solution, and the like.
  • Luminescence includes fluorescence, phosphorescence, chemiluminescence, and the like. When the emission is fluorescence or phosphorescence, an excitation light irradiating section capable of irradiating excitation light is required in each liquid storage section. Accordingly, examples of the luminescent substance include a fluorescent substance, a phosphorescent substance, and a chemiluminescent substance.
  • the “liquid storage part” is a part that can store, store, and hold a liquid, and includes, for example, a well, a container, or a tube.
  • the “planar liquid container” refers to a microplate in which the wells are arranged in a plane, for example, a matrix, a cartridge container in which the wells are arranged in a row or row, or a plurality of tubes or containers in a row.
  • a spot-like liquid is housed in a plurality of indentations on a chip such as a DNA chip, or in an infiltration spot where liquid can infiltrate. It also includes cases where it invades or is placed.
  • the microplate has, for example, 96 wells of 9 mm pitch arranged in 8 rows ⁇ 12 columns.
  • the planar liquid container is made of, for example, a transparent sheet. It is preferable to cover and close all the wells, all the tubes or all the containers provided in the planar liquid container.
  • the planar liquid container closed with the sheet prevents condensation from the inside of the sheet, facilitates the removal of the sheet, and transmits the light.
  • a heating element that allows light to pass from above the sheet and has a through hole corresponding to each opening for heating at a predetermined constant temperature is placed on the sheet from above. It is preferable to provide it as possible.
  • the “concave mirror element” is an optical element that allows a real image by the element to form an image on the object side at a finite distance from the element, and mainly includes a spherical concave mirror element and an aspheric concave mirror element.
  • the range in which one concave mirror element can enter the principal ray of light emission therefrom to form a real image thereof is one or a plurality of liquid storage portions, or the entire planar liquid storage body.
  • the concave mirror element preferably has n (> 1) rotational symmetry (for example, a circle, a square, a rectangle, etc.) and has a necessary and sufficient size according to the range. An edge surrounding the reflection surface of the element is formed, and the optical axis is aligned with the rotational symmetry axis.
  • the concave mirror element is provided.
  • the optical system By reducing the distance from the liquid container to the image forming position, the optical system, and thus the entire apparatus, can be made compact.
  • Aspherical concave mirror element refers to a concave mirror element having different radii of curvature in two directions, and is different from a spherical concave mirror element having the same radius of curvature in two or more directions.
  • the “two directions” are, for example, a vertical direction (sagittal plane) and a horizontal direction (meridional plane) orthogonal to each other.
  • aspheric concave mirror element for example, a toroidal mirror is used.
  • the “predetermined incident angle” is an angle formed between the principal ray of light emission radiated from each liquid container in the vertical direction and the optical axis of the concave mirror element, and the predetermined incident angle is an angle of the concave mirror element.
  • the planar liquid container and the imaging position are determined so as not to overlap with each other based on the radius of curvature, the distance to each container of the planar liquid container that is the measurement target, and the imaging position.
  • the incident angle between the principal ray and the optical axis is ⁇
  • the radius of curvature of the toroidal mirror in the lateral direction is Rh
  • the longitudinal direction If the radius of curvature of the sagittal surface is Rv, the radius of curvature Rh, Rv of the toroidal mirror to be selected and the incident angle should be selected in consideration of real image conditions, the shape and area of the planar liquid container and the light receiving element ⁇ , the distance a between the center of each liquid container of the planar liquid container and the mirror center, and the distance b to each imaging position are determined.
  • Rv Rh.
  • Various concave mirror elements can be used by introducing a program for performing appropriate correction processing calculation into the control unit.
  • real-time PCR refers to a method of monitoring a nucleic acid (DNA) amplified by PCR in real time using a fluorescent substance.
  • Real-time PCR has the advantage that amplification can be observed in the middle of a temperature cycle and quantitative results can be obtained.
  • a fluorescent reagent containing a fluorescent substance there are an intercalation method, a hybridization method, and a LUX method.
  • the “intercalation method” uses the property that fluorescent substances such as SYBR (registered trademark) GREEN I and ethidium bromide enter double-stranded DNA during the extension reaction and emit fluorescence when irradiated with excitation light. It is a method of measuring quantity.
  • the “hybridization method” is a method of detecting only a target PCR product using a DNA probe labeled with a fluorescent substance in addition to a PCR primer. That is, the hybridized DNA (amount) is detected by hybridization of the fluorescently labeled DNA probe with the target PCR product.
  • the “LUX method” utilizes the property that the fluorescent signal of a fluorescent substance labeled on an oligonucleic acid is influenced by the shape (sequence, single strand, double strand, etc.) of the oligonucleic acid.
  • real-time PCR is performed using a PCR primer (LUX primer) labeled with one type of fluorescent substance and a PCR primer labeled with nothing.
  • the LUX primer is designed so that a fluorescent substance is labeled near the 3 ′ end and takes a hairpin structure with the 5 ′ end. When the LUX primer has a hairpin structure, the quenching effect is released and the fluorescence signal increases. By measuring this signal increase, the amount of PCR product can be measured.
  • Temperature control means that a target liquid or container is maintained at one or two or more set predetermined temperatures for a set time in accordance with a set order. It is.
  • the temperature control instruction is made by sending a corresponding signal based on a program.
  • Temperature control means that a heat block provided with a temperature source capable of raising or lowering the temperature of the liquid container that contains a liquid to be controlled based on an external signal or the like is formed in the planar shape.
  • the temperature source include a Peltier element, a heater, and a cooling device.
  • the “predetermined temperature” is a target temperature that an object such as a target liquid should reach.
  • the predetermined temperature to be set is, for example, a temperature cycle performed in the PCR method, that is, each temperature necessary for heat denaturation, annealing or hybridization, and elongation of DNA, each of about 94 ° C., between 50 ° C. and 60 ° C. Temperature, for example, about 50 ° C and about 72 ° C.
  • the predetermined temperature can be cooled by a temperature regulator at a transition promoting temperature lower than the predetermined temperature.
  • heating is performed at a temperature for promoting transition higher than these predetermined temperatures, thereby shortening the transition time and reducing one cycle time.
  • transition-promoting temperature is the time required to maintain each temperature, and depends on the reagent and volume used in the PCR method, the shape, material, size, thickness, etc. of the nozzle.
  • the processing time of the entire PCR method is about several minutes to several tens of minutes, for example, from several seconds to several tens of seconds.
  • the transition time is also included in the predetermined time.
  • the “measurement position” refers to a light receiving unit that is to receive light from each real image by the principal ray of light emitted from the opening of each liquid container formed on the image forming position by the concave mirror element into the light receiving unit.
  • the position for example, the position of the light receiving end of the light receiving element provided in the light receiving unit, the convex lens surface, the tip of the fiber, etc., and is set so as to be close to or coincide with each image forming position.
  • “Proximity” is, for example, a plane having an appropriately set inclination (for example, an inclination symmetrical to the planar liquid container with respect to the optical axis) or an appropriately set curvature passing through at least one light emission imaging position.
  • the measurement position is set by projecting each imaging position onto a curved surface having or a plane or curved surface that is in contact with or intersects with an imaging surface including each imaging position by each light emission. It is preferable in terms of measurement accuracy that each measurement position matches each imaging position. However, although the curved surface passing through each imaging position is a complicated shape, the structure of the device can be simplified by providing each measuring position on a simple measurement plane or measurement curved surface that is close to each imaging position. Can do. When each measurement position does not coincide with each image formation position, it is preferable that the aberration generated at each measurement position is set so as to be included in the sensitivity range of the light receiving unit or in the measurement error.
  • each measurement position is set within a distance at which light from the real image on the imaging position for each liquid storage unit can enter by the light receiving end of the light receiving unit.
  • the magnification or size of the real image is determined by the size and shape of the light receiving unit. Accordingly, the size, pitch, concave mirror size, curvature radius, distance between the planar liquid container and the concave mirror element, imaging position, incident angle, and the like are determined.
  • the light receiving unit includes a light receiving element capable of receiving a chief ray of light emission reflected by the concave mirror element, corresponding to all or a part of the liquid container of the planar liquid container. It is the reaction optical measuring device provided in each said predetermined measurement position.
  • a light receiving element is provided at the predetermined measurement position corresponding to all or a part of the liquid storage parts of the flat liquid container, for example, it is arranged in a plane as the predetermined measurement position. Preferred for simplicity.
  • a photoelectric element for example, a photoelectric element is provided, and one light receiving element is provided so as to receive a principal ray generated by light emission of the one liquid container.
  • the “photoelectric element” is an electronic element utilizing a photoelectric effect, and includes a photoelectric tube, a photomultiplier tube, a photoconductive cell, a phototransistor, a photodiode, and the like.
  • a reaction optical measuring device having a scanning mechanism that moves both of the planar liquid container or all or part of the optical system and the planar container.
  • the “optical system” includes the concave mirror element, the light receiving unit, and, if necessary, the excitation light irradiation unit. Furthermore, the aperture stop mentioned later is also included. Whether or not to move the optical system is determined by whether or not the optical system covers the entire planar liquid container. For example, when the concave mirror element forms a real image of only a part of the planar liquid container, the excitation light irradiation unit irradiates only a part of the liquid container of the planar liquid container, or When the light receiving unit receives a real image of only a part of the liquid container of the planar liquid container, it is necessary to move the corresponding optical system or the planar liquid container by the scanning mechanism.
  • the planar liquid container includes, for example, a case where a plurality of the liquid containers are arranged in a matrix.
  • a part of the planar liquid container is, for example, one liquid container part arranged in a matrix, several pieces, one row, one column, several columns, or several rows, Further, there may be a matrix such as several rows by several columns using a divisor of the number of rows or columns.
  • the container is preferably a unit obtained by dividing the planar liquid container into congruent regions.
  • the optical system including the light receiving element and the concave mirror element is integrally formed.
  • an optical system is formed through a common support frame or an optical system support plate, so that relative movement between components forming the optical system can be made unnecessary.
  • the concave mirror element has a size for forming each real image by light emission through the openings of all or part of the liquid container provided in the planar liquid container at the respective image formation positions.
  • This is a reaction optical measurement device having a shape.
  • the sixth invention is a reaction optical measurement apparatus having an excitation light irradiation unit provided with a light source and a predetermined type of excitation light filter capable of transmitting excitation light of a predetermined type of wavelength band.
  • a light bulb type light source such as a xenon lamp or a halogen lamp, or a number corresponding to the number of liquid containing parts to be irradiated or a plurality of light emitting elements corresponding to the type and number of wavelengths, for example, high-intensity LEDs
  • the “predetermined type” is one or more types depending on the type of fluorescence or phosphorescence used. Note that it is preferable for compactness to select a filter for selecting the wavelength of light received by the light receiving unit and the light source.
  • the “excitation light irradiating unit” may, for example, directly irradiate the liquid storage unit with the excitation light (first and third embodiments), or after reflecting the excitation light once with the concave mirror element.
  • the liquid container is irradiated (second embodiment) and cases where the liquid container is irradiated using a dichroic mirror or the like from the lateral direction (second embodiment).
  • the number and position of the liquid storage units existing in a range in which the concave mirror element allows its principal ray to enter and form a real image for example, the number and position of the liquid storage units existing in a range in which the concave mirror element allows its principal ray to enter and form a real image.
  • one or a plurality of small holes are formed corresponding to the above, and an end portion of the optical fiber for exciting light irradiation is provided in each hole.
  • the optical system is integrally formed including a light source and an excitation light irradiation unit provided with a predetermined type of excitation light filter capable of transmitting excitation light of a predetermined type of wavelength band. It is a reaction optical measuring device.
  • An eighth aspect of the invention is a reaction optical measurement device in which the light receiving element includes a photoelectric element and a convex lens that causes light from a real image of each liquid container to enter the photoelectric element at the measurement position.
  • a ninth aspect of the invention is a reaction optical measurement device provided with an optical filter that selectively transmits or blocks the emitted light having a wavelength specified for the light receiving unit between the concave mirror element and the light receiving unit. .
  • Optical filter is used to pass the wavelength of light labeled with a substance such as a DNA fragment whose amount or concentration is to be measured in various reactions, and to block the transmission of light having other wavelengths. belongs to.
  • a labeling substance that outputs a plurality of types of light wavelengths is used, a plurality of types of optical filters are provided, and light having each wavelength is transmitted through the optical filter so that the corresponding labeling substance can be used. The presence or amount thereof can be measured.
  • a tenth aspect of the invention is a reaction optical measurement device in which an aperture stop is provided between the concave mirror element and the light receiving unit.
  • a light receiving step for receiving light for each liquid container is required before the imaging step.
  • the light receiving step can receive light emitted through one opening of the liquid container corresponding to all or a part of the liquid container of the planar liquid container.
  • the concave mirror element by using the concave mirror element, the principal ray from each liquid storage part is incident obliquely even on a planar liquid container having a planar spread.
  • a real image of light emission from each liquid container can be formed on each imaging position on the planar liquid container side but not overlapping the planar liquid container with respect to the optical system. Therefore, since the distance from the planar liquid container to the imaging position can be substantially folded by the element, the entire apparatus can be formed compactly.
  • only one concave mirror element is provided as the optical system, no complicated lens combination is required, chromatic aberration is virtually eliminated, highly reliable measurement is possible, and the structure is simplified to reduce manufacturing costs. can do.
  • each predetermined measurement position is made to coincide with each imaging position, measurement with higher reliability can be performed.
  • each predetermined measurement position is set on a simple plane or curved surface that is close to the curved surface including each imaging position, the apparatus structure can be simplified and the manufacturing cost can be reduced. .
  • the light receiving section can reliably receive the real image at each measurement position for each liquid storage section, the light reception section can accurately detect each liquid storage section. The amount of light can be measured. Further, in the first invention or the ninth invention, when each light receiving element can receive all of the real image all at once, the structure on the mechanism is simplified because the scanning mechanism is not required.
  • the third invention when the light receiving elements are arranged so as to correspond to a part of the real image corresponding to each of the liquid storage portions, the number of the light receiving elements is reduced, and accordingly, the concave mirror element is reduced.
  • the structure can be further simplified and the cost can be reduced.
  • the structure of the apparatus can be formed compactly, and an accurate optical system can be provided.
  • the concave mirror element is formed so as to have a size and a shape for forming a real image by light emission through the openings of all or part of the liquid storage portion at the respective image forming positions. Therefore, when a real image of the entire liquid container of the planar liquid container is formed at the image formation position, it is possible to perform processing at high speed without the need for scanning for image formation. When a real image of a part of the liquid container of the planar liquid container can be formed, the whole liquid container can be imaged by scanning.
  • the excitation light filter by providing the excitation light filter, it is possible to easily irradiate a plurality of types of excitation light.
  • the structure of the apparatus can be further reduced in size and further enhanced.
  • a highly accurate optical system can be provided.
  • the measurement position can be set within a wide range with respect to the imaging position. Can be set.
  • a necessary wavelength band can be selected for the light receiving unit, measurement results corresponding to various purposes can be obtained from one liquid storage unit.
  • the planar liquid container surface is slightly in the optical axis direction.
  • the shape of the image does not change even if it moves or tilts, or even if there is a difference in the height of the liquid level in each liquid storage part or even if the height changes.
  • the depth of focus can be increased by reducing the aperture area of the stop.
  • FIG. 1 is a perspective view of a real-time PCR optical measurement apparatus according to a first embodiment of the present invention. It is a perspective view which shows one state of the inside of the real-time PCR optical measuring device based on the 1st Embodiment of this invention from the front. It is a perspective view which shows the other state inside the real-time PCR optical measuring device based on the 1st Embodiment of this invention from the front.
  • FIG. 2 is a perspective view showing the inside of the real-time PCR optical measurement apparatus according to the first embodiment of the present invention from the rear.
  • FIG. 2 is a perspective view showing an optical system inside the real-time PCR optical measurement apparatus according to the first embodiment of the present invention from the rear.
  • FIG. 2 is a plan view showing an optical path of the real-time PCR optical measurement apparatus according to the first embodiment of the present invention. It is a perspective view which shows the optical system which concerns on the 2nd Embodiment of this invention. It is the front and side perspective drawing which show the optical system which concerns on the 2nd Embodiment of this invention. It is a figure which shows the optical path of the real-time PCR optical measuring apparatus which concerns on the 2nd Embodiment of this invention. It is the side schematic and perspective view which show the light-receiving part of the optical system which concerns on the 2nd Embodiment of this invention.
  • FIG. 1 shows a perspective view of a real-time PCR optical measurement apparatus 10 as a reaction optical measurement apparatus according to an embodiment of the present invention.
  • FIG. 1 (A) shows the real-time PCR optical measurement apparatus 10.
  • 1B shows a case where the drawer 14 is closed.
  • the apparatus 10 has a height of about 50 cm and a width and a depth of about 30 cm, for example.
  • the real-time PCR optical measuring apparatus 10 is incorporated in a light-shielding casing 12 as a whole, and can accommodate a plurality of PCR reaction solutions containing a fluorescent substance as a luminescent substance (in this example, 8 rows ⁇ 12 columns).
  • 96) wells 26 as liquid storage portions arranged in a matrix of 9 mm pitch are arranged along a plane, and a planar liquid storage body in which temperature control based on the PCR method is performed in each well 26
  • a microplate 22 is provided in the drawer 14.
  • the housing 12 includes a connector portion 20 provided with a terminal for connecting to an external power source or a communication device and a USB terminal.
  • the casing 12 is provided with a ventilation port 18, and the drawer 14 is provided with a locking bar 15 and a knob 16 that can project in the left-right direction for locking the drawer 14. Rotate in both forward and reverse directions by 90 ° to switch between the vertical state and the horizontal state so that the locking bar 15 is switched out or retracted.
  • FIG. 2 is a perspective view showing the inside in detail by removing the casing 12 with the drawer 14 opened in FIG.
  • a Peltier as a temperature controller for controlling the temperature according to the PCR method for each well 26 of the microplate 22 containing the PCR reaction solution containing the fluorescent substance.
  • a thermal cycler 38 composed of a plate-shaped heating block having elements and the like is provided below the microplate 22. Further, a heat dissipating fin (not shown) is provided on the lower side of the heating block, and a cooling fan 42 is provided on the side of the outside so that outside air taken in from the ventilation port 18 in a state where the drawer 14 is closed.
  • the microplate 22 is provided so as to be cooled by blowing air toward the heat dissipating fins.
  • a right-hand orthogonal system is defined in which the normal direction of the plane formed by the microplate 22 is the Z-axis direction, the row direction is the X-axis direction, and the column direction is the Y-axis direction. To do.
  • a transparent sheet 27 (see FIG. 6) for closing each opening of each well 26 is placed on the microplate 22 and pasted on the upper side of the microplate 22. Wear and attach.
  • a heating lid 24 for preventing condensation from on the transparent sheet 27 to the inside of the transparent sheet 27 is detachably attached to the microplate 22.
  • the heating lid 24 is a block-like member having a built-in heater for heating the transparent sheet 27 to a constant temperature, and has 96 through holes at positions corresponding to the wells 26 of the microplate 22. 28 is provided.
  • the through-hole 28 can emit not only fluorescence (light emission) emitted from the microplate 22 in the main direction but also excitation light from a high-intensity LED array portion 32 as an excitation light irradiation portion described later.
  • the light can enter each well 26.
  • the heating lid 24 is used as a linear motion cam provided with an inclination 40 that descends in the forward direction (Y-axis direction) at the upper edge of both side surfaces 41 of the drawer 14 by opening and closing the drawer 14.
  • the heating lid 24 is provided with a mechanism that follows the cam.
  • the heating lid 24 can move only in the vertical direction (Z-axis direction) without moving in the front-rear direction (Y-axis direction).
  • the heating lid 24 is lifted away from the microplate 22, and when the drawer 14 is closed, the heating lid 24 is lowered and attached to the upper side of the microplate 22.
  • the through-hole 28 emits fluorescence through which the excitation light from the excitation-light irradiation unit enters or radiates each well 26 through the through-hole 28. It is formed to be possible.
  • the real-time PCR optical measurement apparatus 10 is provided with a Y moving body 31 that is mounted with an optical system and is movable in the front-rear direction (Y-axis direction).
  • the Y moving body 31 is driven via an arm 46 attached to the optical system support plate 44 of the Y moving body 31 by a timing belt 48 attached to the outside of the side frame plate 56 to which the housing 12 is attached. Is done.
  • the timing belt 48 is fed by one row of the wells 26 of the microplate 22 by the stepping motor 50 attached to the inside of the side frame plate 56, and therefore, the 96-well microplate 22 as in this example.
  • the vehicle is driven by an instruction to sequentially move 9 mm ( ⁇ y) from the first line and scan 8 lines.
  • the side frame plate 56 is provided with four sets of light-emitting elements and light-receiving elements as the scanning position detection sensor 52 so as to sandwich the arm 46 from the front and back, and each row of the microplate 22 provided on the arm 46 is shown.
  • the row position is identified by the light receiving state of the light that has passed based on the perforation position and the number of the perforated holes 52a.
  • the Y moving body 31, the timing belt 48, the stepping motor 50, the arm 46, the scanning position detection sensor 52, and the like correspond to the scanning mechanism.
  • reference numeral 54 is a motor drive board
  • reference numeral 58 is a temperature control board
  • reference numeral 60 is for converting analog data measured using the toroidal mirror 30 and the like into digital data. Board.
  • the Y moving body 31 includes a toroidal mirror 30 having a size corresponding to one row as a concave mirror element, and an edge portion surrounding the reflecting surface cut out in a rectangular shape, and a plurality of light emitting elements as the excitation light irradiation unit.
  • the high-brightness LED array portion 32 is provided in which high-brightness LEDs 32a and 32b having two types of wavelengths for one row are alternately arranged. Details of the toroidal mirror 30 will be described later. That is, the range in which the principal image of light emitted from the concave mirror element can be incident to form a real image thereof is a well as 12 liquid storage portions.
  • optical system support plate 44 provided in the Y moving body 31 along the front-rear direction (Y-axis direction) at an angle twice the predetermined incident angle ⁇ with respect to the vertical direction.
  • the optical system is attached along the optical system support frame 33 attached at an inclination, and a part of the optical system is attached along the vertical direction (Z-axis direction).
  • the light shielding cylinder 34 for protecting the optical path from stray light is attached along the inclination of the optical system support frame 33, and the light shielding cylinder 36 is attached to the optical system support frame 33 along the vertical direction. It has been.
  • the high-intensity LED array portion 32 is attached to be inclined with respect to the front-rear direction (Y-axis direction) at an angle at which excitation light can enter one row of the through-holes 28, and the toroidal mirror 30 corresponds to the above-mentioned one row.
  • the toroidal mirror 30 corresponds to the above-mentioned one row.
  • FIG. 3 shows that the drawer 14 of the real-time PCR optical measurement apparatus 10 is closed and the locking bar 15 is protruded toward the locking hole provided in the removed casing 12 to lock the drawer 14.
  • the object-side telecentric construction stop 61 is shown as an aperture stop provided above the optical radiation filter 62 by further removing the light blocking cylinder 34.
  • FIG. 4 is a perspective view of the device 10 as viewed from the rear side.
  • Reference numeral 45 denotes a light-shielding cover for keeping a part of the optical system in a light-shielding state so that light from the optical system does not affect the sensor 52 and the like, and the light-shielding cylinder 34 penetrates inside. It is provided as follows.
  • Reference numeral 64 denotes a power supply module.
  • FIG. 5 shows a part of the optical system exposed from the rear side by removing the light shielding cylinders 34 and 36 and the light shielding cover 45 in FIG. 4 from the apparatus 10. As shown in the figure, it coincides with the toroidal mirror 30 as the concave mirror element, the high-intensity LED array 32 attached so as to be adjacent to the toroidal mirror, the optical radiation filter 62, and the imaging position 75. It has 12 lenses 72 as light receiving elements 73 and photodiodes 70 as photoelectric elements provided so as to correspond to the liquid storage portions in one row provided along the measurement position 77 (75).
  • FIG. 5 (B) shows the optical system provided in the apparatus 10 shown in FIG. 5 (A) taken out, in order to excite the toroidal mirror 30 as the concave mirror element and the fluorescence. These positions are between the high-intensity LED array unit 32 that can switch and irradiate two types of excitation light, and the vertical focus and the horizontal focus between the toroidal mirror 30 and the light receiving element 73 group.
  • the object side telecentric construction stop 61 provided on the toroidal mirror 30 side of the optical radiation filter 62 and the light radiated from the microplate 22 and reflected by the toroidal mirror 30 have three types of wavelength bands.
  • FIG. 6 and 7 show an arrangement of an optical system including a microplate 22 of 9 rows and 8 rows ⁇ 12 columns and a specific toroidal mirror 30 when the incident angle ⁇ is 12 degrees.
  • the toroidal mirror 30 is an opening of 12 wells 26 for one row among 96 wells 26 serving as liquid containing portions arranged at 9 mm pitch in 8 rows ⁇ 12 columns as the planar liquid containing body.
  • the liquid level 23 of the liquid stored in the microplate 22 from the optical center of the toroidal mirror 30 (the same level of liquid is dispensed into each well 26, so the liquid level is the same)
  • the imaging position 75 (coincidence with the measurement position 77) of each light receiving element 73 where the real image of the chief ray fluorescence from each well 26 is formed is shown by calculation. .
  • FIG. 6A shows the toroidal mirror 30 and the microplate 22 cut along the meridional plane (ZY plane).
  • the width of the toroidal mirror 30 cut by the meridional surface is 18 mm, and the angle between the light beam traveling in the vertical direction from the microplate 22 as the principal light beam and the optical axis 80 of the mirror 30, that is, the incident angle ⁇ is 12 degrees.
  • the lateral radius of curvature Rh is 300 mm.
  • FIG. 6B shows the cross-sectional shape of the toroidal mirror 30 cut along the optical axis plane orthogonal to the meridional surface, the reflection of the principal ray from the center of each toroidal mirror 30, the microplate 22, and each well.
  • the projection of the image forming position 75 of each central point of the liquid surface 23 onto the ZX plane is 110 mm, and the longitudinal radius of curvature Rv is 287.03 mm.
  • Six half of the imaging position 75 projected on the ZX plane (the other six half are omitted with respect to the optical axis 80 are omitted).
  • the microplate 22 has 12 rows of wells 26 arranged at a pitch of 9 mm.
  • the imaging position 75 is determined as the Z coordinate from the side far from the optical axis 80.
  • the positions are -256.34 mm, -261.43 mm, -265.73 mm, -268.64 mm, -270.91 mm, and -272.62 mm, respectively.
  • FIG. 7 shows the imaging position 75 of the well 26 in one row and the position of the microplate 22 by the toroidal mirror 30 projected onto the XY plane.
  • the X coordinates are 43.75 mm, 36.5 mm, 28.86 mm, 20.84 mm, 12.61 mm, and 4.23 mm, respectively
  • the Y coordinates are 111.92 mm and 114.83 mm from the far side of the optical axis 80, respectively. 117.26mm, 118.94mm, 120.2mm, 121.09mm.
  • the position coordinates of each of the twelve imaging positions 75 in one row correspond to the position coordinates of the measurement position 77, and are (43.75 mm, 111.92 mm, -256.34 mm), (36.5 mm, 114.83mm, -261.43mm), (28.86mm, 117.26mm, -265.73mm), (20.84mm, 118.94mm, -268.64mm), (12.61mm, 120.2mm, -270.91mm), (4.23mm, 121.09mm , -272.62 mm).
  • the axial direction of the light receiving element 73 at each position is also shown in FIGS. Since this measurement position is translated by ⁇ y (corresponding to 9 mm in this example) in the Y-axis direction by scanning, only the Y coordinate changes, so that the nth row (n is 1 to 8)
  • the measurement position 77 in (natural number) is (43.75 mm, 111.92 mm + (n ⁇ 1) ⁇ y, ⁇ 256.34 mm), (36.5 mm, 114.83 mm + (n ⁇ 1) ⁇ y, ⁇ 261.43 mm), (28.86 mm, 117.26).
  • the knob 16 provided on the drawer 14 of the real-time optical measuring device 10 is rotated 90 degrees to be horizontal, whereby the locking bar 15 is retracted to be opened and closed, and the drawer 14 is manually opened. Then, both side surfaces 41 of the drawer 14 function as a linear motion cam that interlocks with the linear motion of the drawer 14, and the heating lid 24 is automatically lifted and detached from the upper side surface of the microplate 22, and the microplate. 22 advances from the position directly below the heating lid 24 together with the drawer 14 in the Y-axis direction and reaches a position as shown in FIG.
  • a PCR reaction solution containing DNA extracted from each specimen and a fluorescent substance is placed in each well 26 of the microplate 22, and a predetermined amount of PCR solution is equally placed in each of the 96 wells 26 using a separate dispensing device or the like. Dispensing and attaching the transparent sheet 27 to the upper side of the microplate 22 to close the opening of each well 26.
  • the drawer 14 is moved rearward to close the drawer 14, and then the knob 16 is rotated 90 degrees so that the locking bar 15 protrudes outward to lock the drawer 14 in the closed state. To do.
  • the heating plate 24 of the microplate 22 is attached to the microplate 22 from the upper side of the transparent sheet 27.
  • the temperature control according to the PCR method is performed on the lower side of the microplate 22 while preventing the condensation by heating the heating lid 24 to a predetermined constant temperature. 38 will be performed.
  • the measurement by the optical system is sequentially performed by driving the stepping motor 50 with respect to the microplate 22 to sequentially move the Y moving body 31 from the innermost row along the Y-axis direction.
  • optical system attached to the Y moving body 31 such as the tridal mirror 30 is stopped on each row by moving 9 mm toward the previous row, optical measurement is sequentially performed for eight rows.
  • the optical measurement is performed through the through holes 28 of the heating lid 24, and through the openings of the 12 wells 26 in the first row of the microplate 22.
  • Excitation light from the high-intensity LED 32a of the unit 32 is directly applied to the PCR reaction solution accommodated in the 12 wells 26 for one row.
  • the principal ray of fluorescence emitted from the well 26 in the vertical direction passes through the sheet 27 and the through hole 28 of the heating lid 24 and is reflected by the toroidal mirror 30 to construct the object side telecentric structure.
  • the board 58 passes through the diaphragm 61, the optical radiation filter 62, and the lens 72 of the twelve light receiving elements 73 at the imaging position 75, and the light received by the photodiode 70 is an analog amount of light. It is converted into an electric signal of a corresponding digital quantity and sent to an information processing apparatus provided outside, for analysis.
  • the Y moving body 31 is moved by one row in the Y-axis direction, that is, 9 mm, and stopped, and the optical measurement is performed in the same manner for all eight rows. Scanning is performed by moving the Y moving body in the Y-axis direction.
  • movement control is easy because it is only necessary to move the optical system at a constant pitch along the Y-axis direction.
  • the number of light receiving elements can be reduced.
  • the real-time PCR optical measurement apparatus 100 performs an optical measurement process on the microplate 22 as the planar liquid container as in the case of the first embodiment described above. It is.
  • the real-time PCR optical measurement apparatus 100 is similar to the real-time PCR optical measurement apparatus 10 according to the first embodiment, but mainly the differences will be described below.
  • the real-time PCR optical measurement apparatus 100 includes a toroidal mirror 130 in which an edge surrounding a reflection surface is cut into a rectangular shape as an optical system, and a xenon lamp as an excitation light irradiation unit.
  • one or a plurality of light sources 132 such as a halogen lamp, a Fresnel lens 132a, an excitation light filter 132b capable of selecting light of a predetermined wavelength (in this example, two types of excitation light), and the toroidal mirror 130
  • the dichroic mirror that reflects the light of the corresponding wavelength so as to make the excitation light simultaneously enter into the well 26 as the liquid container, while transmitting the light of the wavelength including the fluorescence from the toroidal mirror 130.
  • 132c and three types of filters 174a, 174b, and 174c capable of selecting three types of fluorescence wavelengths.
  • Reference numeral 161 denotes an object-side telecentric construction stop as an aperture stop.
  • the incident angle ⁇ formed with the optical axis of the toroidal mirror 130 with respect to the principal ray of fluorescence emitted from the microplate 22 in the vertical direction is 12 degrees as in the first embodiment. That is, the range in which the principal image of the light emitted from the concave mirror element can be incident to form a real image is the entire microplate 22 as a liquid container.
  • the light receiving portion twelve light receiving elements 173 for one row of the microplate 22 as the planar liquid container do not coincide with the image forming position 175 (see FIG. 10), and the image forming position A support substrate 171 (see FIG. 11) provided parallel to a predetermined measurement plane close to 175 is attached to a position corresponding to the predetermined measurement position 177 on the measurement plane.
  • the light receiving elements 173 for one row and the support substrate 171 to which the light receiving elements 173 are attached are movably provided.
  • the toroidal mirror 130, the light source 132, the Fresnel lens 132a and the like which are optical systems are provided so as to be stationary with respect to the measurement plane together with the microplate 22 and the like. This is different from the first embodiment in which the entire optical system is integrally moved with respect to the microplate 22 in the Y-axis direction by the Y moving body 31.
  • the entire optical system is not mounted, but the support substrate 171 to which the light receiving element 173 of the optical system is attached is attached to the predetermined measurement plane.
  • the movement distance of the Y moving body is not parallel to the 9 mm pitch of the microplate 22 but measured as shown in FIG. 10 to be described later.
  • the measurement results of all 96 wells for 8 rows can be obtained. .
  • the toroidal mirror 130 according to this embodiment has the same two radii of curvature Rh and Rv as the toroidal mirror 30 according to the first embodiment described above, and the same material.
  • the size of the toroidal mirror 30 according to the first embodiment is twelve for one row of the 8 mm ⁇ 12 rows 9 mm pitch microplate 22 as the planar liquid container as described above.
  • Fluorescent chief rays from the well 26 in the vertical direction are incident all at once, and a size capable of forming a real image at an imaging position all at a magnification of 1 (for example, 110 mm in the X-axis direction, Y-axis
  • the toroidal mirror 130 according to the present embodiment has a vertical direction from 96 all wells 26 of 9 rows of 8 rows ⁇ 12 columns of the microplate 22. Fluorescence chief rays are incident all at once and magnification In can image the real image simultaneously imaging position size (e.g., 110 mm in the X-axis direction, the size of 80mm in the Y-axis direction) with the difference with.
  • FIG. 10 shows an imaging curved surface passing through the imaging position 175.
  • FIG. 10 shows the angle between the principal ray traveling vertically from the microplate 22 of 8 rows ⁇ 12 columns with a pitch of 9 mm and the microplate 22 that is the principal ray, and the optical axis 180 of the toroidal mirror 130, that is, incident.
  • the angle ⁇ is 12 degrees
  • a specific toroidal mirror 130, and the liquid level 23 (the same height in all the wells 26) of the liquid contained in the planar liquid container from the optical center of the toroidal mirror 130 When the distance a to 290 mm is 290 mm, an imaging position 175 where the real image of the principal ray fluorescence from each well 26 is formed, and a measurement position 177 (different from the imaging position and predetermined measurement) Included in the plane).
  • the predetermined measurement position 177 passes through at least one image formation position 175a, and the image formation position on a plane having the inclination symmetrical to the microplate 22 in the Y-axis direction with respect to the optical axis 180. 175 is projected. Thereby, the microplate 22 can be easily compared with the measurement position 177 or the imaging position 175.
  • FIG. 10A shows the toroidal mirror 130 projected onto the optical axis plane orthogonal to the meridional plane (ZY plane), six half of the imaging position 175 and measurement position 177 (the remaining half are the optical axis). It is omitted because it is symmetrical with respect to 180).
  • the length of the toroidal mirror 130 cut along the optical axis surface is 110 mm, and the longitudinal radius of curvature Rv is 287.03 mm.
  • FIG. 10B shows the toroidal mirror 130 and the microplate 22 cut along the meridional plane (ZY plane).
  • the width of the toroidal mirror 130 cut along the meridional surface is 80 mm, and the lateral radius of curvature Rh is 300 mm.
  • each measurement position 177 of all the wells 26 can be specified by an orthogonal system (XY coordinate) on the measurement plane.
  • FIG. 11 shows twelve light receiving elements 173 for one row corresponding to the light receiving section, and a support substrate 171 to which the light receiving elements 173 are attached.
  • Each light receiving element 173 is provided with a lens 172 and a photodiode 170 as shown in FIG.
  • the distance between adjacent light receiving elements is 4.4 mm, 8.85 from the center of the array in the horizontal direction (Y-axis direction). It is attached at intervals of mm, 9mm, 9.28mm, 9.68mm, 10.21mm.
  • FIG. 12 shows a perspective view of the reaction optical measurement device 200.
  • FIG. 12A shows a case where the drawer 214 provided in the reaction optical measurement device 200 is opened, and FIG. The case where the drawer 214 is closed is shown.
  • the device is, for example, about 50 cm high and about 30 cm wide and deep.
  • the reaction optical measurement device 200 is incorporated in a housing 212 having a light shielding property as a whole, and can accommodate a plurality of reaction solutions containing a fluorescent material as a luminescent material (in this example, Unlike the apparatus according to the first embodiment, the matrix-like container is placed with its rows and columns interchanged and is arranged in a matrix of 12 rows ⁇ 8 columns and 9 mm pitch (96) as liquid storage units
  • the wells 226 are arranged along a plane, and a microplate 222 is provided in the drawer 214 as a planar liquid container for controlling the temperature based on a predetermined reaction process in each well 226.
  • the housing 212 includes a connector portion 259 (see FIG.
  • the housing 212 is provided with ventilation ports 218 and 219 for taking in air from the fan for temperature control of the microplate 222 and ventilation ports 213 for taking in air for cooling the electronic circuit.
  • the housing 212 is provided with an LED 217 for displaying “in operation” or the like.
  • a locking bar 215 capable of protruding in the left-right direction for locking the drawer is provided, and the knob 216 is manually rotated 90 degrees in both forward and reverse directions to switch between the vertical state and the horizontal state. Then, the locking bar 215 is switched to be pulled out or retracted.
  • FIG. 13 is a perspective view showing the inside in detail with the drawer 214 opened in FIG. 12, the optical system protective cover 260 and the like (see FIG. 14) and the casing 212 described later being removed.
  • the microplate 222 storing the reaction solution containing the fluorescent substance is stored in a substantially prismatic microplate storage recess 235, and each well 226 of the microplate 222 is stored.
  • a plate-like heating block 238 having a Peltier element or the like as a temperature controller for performing temperature control according to a predetermined reaction process is provided below the microplate 222.
  • a cooling fan (not shown) below the heating block 238 blows upward the outside air taken from the ventilation port 218 toward the heat radiating fins with the drawer 214 closed.
  • the microplate 222 is provided so as to be cooled.
  • a right-hand orthogonal system is defined in which the normal direction of the plane formed by the microplate 222 is the Z-axis direction, the row direction is the X-axis direction, and the column direction is the Y-axis direction. To do.
  • a transparent sheet 221 (see FIG. 19) for closing each opening of each well 226 is attached to the upper side of the microplate 222. Has been attached.
  • a heating lid 224 for preventing condensation on the inside of the transparent sheet 221 is detachably provided on the microplate 222.
  • the heating lid 224 is a block-like member with a built-in heater for heating the transparent sheet 221 to a constant temperature, and has 96 through holes 228 at positions corresponding to the wells 226 of the microplate 222. Is provided.
  • the through-hole 228 can emit not only fluorescence emitted from the microplate 222 in the main direction but also excitation light from an excitation light irradiation unit 232 (see FIG. 16) described later. Can be incident.
  • the heating lid 224 is provided with a notch 239 that is edged with a curved surface that is curved so as to be recessed in the Z-axis direction at the upper edge of both side surfaces 241 of the drawer 214.
  • the heating lid 224 is provided with a lid opening / closing mechanism 229 (see FIG. 15) which is used as a linear motion cam and is driven by the cam.
  • the heating lid 224 does not move in the front-rear direction (Y-axis direction) but moves only in the vertical direction (Z-axis direction), and the heating lid 224 first rises. Then, when the drawer 14 is separated from the microplate 222 and the drawer 14 is closed, the heating lid 224 is lowered and attached to the upper side of the microplate 222. When the heating lid 224 is attached, the through-hole 228 passes through the through-hole 228 and the excitation light from the light irradiation unit enters the well 226 and emits emitted fluorescence. It is formed to be possible.
  • the reaction optical measuring device 200 is provided with a Y moving body 231 mounted with an optical system so as to be movable in the front-rear direction (Y-axis direction, column direction).
  • the Y moving body 231 is slidably mounted by being guided by the two rails 227 provided on the upper edges of the two side plates 249 to which the casing 212 is to be attached.
  • the four guide members 225 are supported.
  • the Y moving body 231 is attached to a stepping motor 250 that is mounted to project inward from the side plate 249, and the side plate 249 to which the housing 212 is to be mounted on the outer side, and is rotated by the stepping motor 250.
  • the Y moving body 231 is moved by one row of the well 226 of the microplate 222 in the forward and reverse directions of the Y axis based on an instruction by the stepping motor 250 or the like, and thus, as in this example.
  • the 96-well microplate 222 it is possible to scan 12 rows by sequentially moving from the first row every 9 mm ( ⁇ y).
  • a travel range detection sensor 252 two sets of light emitting elements and light receiving elements are positioned so as to sandwich a light shielding detection plate provided on the arm 253 from the front and back, and thereby the Y axis The upper ends can be detected.
  • the Y moving body 231, the stepping motor 250, the timing belt 251, the ball screw 248, the arm 253, the travel range detection sensor 252 and the like correspond to the scanning mechanism.
  • reference numeral 254 is a motor control board
  • reference numeral 258 is a temperature control board
  • Reference numeral 252a is a code for supplying electric power to the sensor 252.
  • Reference numeral 247 denotes a wiring hole for guiding a cord bundle for supplying power to the various boards.
  • the Y moving body 231 is supported from both sides by the optical system support plate 244, has a length (along the X-axis direction) and width of one row of the well 226 of the microplate 222, and has a reflecting surface.
  • a toroidal mirror 230 as a concave mirror element whose surrounding edge is cut out in a rectangular shape, and inclined along the front-rear direction (Y-axis direction) at a double angle of the predetermined incident angle ⁇ with respect to the vertical direction (Z-axis direction).
  • the mirror-side light-shielding cylinder 234 having the length and width for protecting the optical path from stray light, and the length of one row of the well 226 of the microplate 222 and the optical system support plate 244
  • a slit 236 having a width is formed, and a slit forming plate 237 supported by the optical system support plate 244 is provided.
  • Reference numeral 276 denotes the microplate irradiation light source switching plate as will be described later. That is, the range in which the principal image of the light emitted from the concave mirror element can be incident to form a real image is eight wells as liquid storage portions.
  • FIG. 14 is a perspective view of the reaction optical measurement device 200 as viewed from the rear side with the casing 212 removed and the drawer 214 closed.
  • reference numeral 245 denotes an optical system control board provided with a photodiode 270 described later and a circuit board for taking out an analog signal of the photodiode 270 and digital conversion.
  • Reference numeral 255 denotes a motor driving IC for driving the motor
  • reference numeral 257 denotes a power supply module
  • reference numeral 259 denotes a connector portion provided with a terminal for connecting to an external power supply or a communication device or a USB terminal.
  • FIG. 15 is a perspective view in which the Y moving body 231 is further removed from FIG.
  • reference numeral 229 denotes a lid opening / closing mechanism for raising and lowering the heating lid 224 according to the opening / closing of the drawer 214.
  • FIG. 16 is a cross-sectional view showing the Y moving body 231 in detail.
  • Reference numeral 266 is optically connected to the mirror-side light-shielding cylinder 234 and attached to the optical system support plate 244 by being inclined in the front-rear direction (along the Y-axis direction) at the same angle as the cylinder 234.
  • This is a light-receiving-side light blocking cylinder.
  • the mirror-side light-shielding cylinder 234 is connected to the mirror 230 at one end so that the inclination angle of the cylinder 234 becomes ⁇ in each direction with respect to the optical axis direction of the mirror 230, and at the other end.
  • the light receiving side light shielding cylinder 266 is connected at the same inclination angle.
  • the mirror-side light-shielding cylinder 234 is sequentially selected from the plurality of types of filters 262 and can be installed at the other end, a positioning sensor 263 for the filter 262, and the cylinder 234.
  • a motor 269 attached to the outer surface for rotating the plurality of filters 262 to the end portion.
  • One end of the light receiving side light blocking cylinder 266 is optically connected to the mirror side light blocking cylinder 234, and the other end corresponds to the number of wells in one row provided on the optical system control board 245.
  • Eight photodiodes 270, convex lenses 272a, and prisms 272b are provided substantially along the X-axis direction.
  • the Y moving body 231 is provided with an excitation light irradiation unit 232.
  • the excitation light irradiation unit 232 includes the slit 236 and the slit forming plate 237, and a plurality (eight in this example) corresponding to the number of the wells 226 is provided below the slit forming plate 237.
  • High-intensity LEDs 242a each arranged in a row and different types of high-intensity LEDs 243a, and a heat sink 242b for supporting the LEDs 242a and 243a and for releasing heat generated in the LEDs 242a and 243a, 243b.
  • the excitation light irradiation unit 232 selects only one of the light beams emitted from the LEDs 242a and 243b, and selects excitation light that irradiates the wells for one row of the microplate 222 through the gap 256. It has a mechanism.
  • the selection mechanism is provided with grooved levers 242d and 243d, each of which is pivotally supported at one end, and interlocks within a predetermined angle range and rotates around the lower edge to rotate the well 226 of the excitation light from the LED 242a and 243a rows.
  • the switching wall plates 242c and 243c having a length equal to or longer than the length of one row of the wells 226 of the microplate 222 and the pins 242e and 243e provided on the levers 242d and 243d, respectively.
  • a link rod 274a provided with two grooves slidably engaged with each other, a crankshaft 274c having a groove slidably engaged with a pin 274b provided on the link rod 274a, and the crankshaft 274c And a motor 274d that rotates in both the forward and reverse directions.
  • the excitation light radiated from the high-brightness LED 243a column is transmitted to irradiate each well 226 for one row of the microplate 222, while being emitted from the high-brightness LED 242a column.
  • the state which blocked the excitation light is represented.
  • the excitation light is not switched to each well 226 of the microplate 222 by repeatedly turning on and off the light source of the high-intensity LED, the intensity of the excitation light is stable, and Deterioration of the light source LED that emits light with low power can be prevented.
  • FIG. 17A is an exploded view of the light-receiving-side light-shielding cylinder 266.
  • the fan-shaped recess forming base 264 and the photo as a light-receiving element serving as a photoelectric element by closing the fan-shaped recess forming base 264 are shown.
  • the fan-shaped recess forming base 264 is provided with an object-side telecentric construction stop 261 that is optically connected to the mirror-side light-shielding cylinder 234 at an important position of the fan-shaped recess.
  • Eight optical system elements 272 are arranged so that the real image of each well 226 is at a position where it enters each of the eight photodiodes 270.
  • FIG. 17B shows an enlarged perspective view of the optical system element 272.
  • the optical system element 272 is integrally provided with a convex lens 272a and a 90-degree prism 272b.
  • the light receiving surface (in this example, has an outer diameter of 9.1 mm) arranged on the support substrate 271 has each well 226. It is provided so that a real image is formed. That is, the light receiving surface is the measurement position 277 and the imaging position 275.
  • FIG. 18 is a configuration for making the light receiving surface coincide with the measurement position 277 and the imaging position 275, and shows a cross-sectional view of the light receiving side light shielding cylinder 266.
  • Each optical system element 272 includes: , Along the X-axis direction (column direction), the position is naturally different, but the Z′-axis direction and the Y′-axis direction are also different (the X-axis is different from the XYZ right-handed coordinate system). XY'Z 'right-handed coordinate system rotated 2 ⁇ as the center).
  • the optical element 272 and the photodiode 270 as a photoelectric element correspond to the light receiving element 273. Therefore, according to the present embodiment, a real image of each of the eight wells 226 for one row can be formed on the light receiving surface of the light receiving element 273, so that accurate and reliable light emission measurement is performed. be able to.
  • the toroidal mirror 230 is an opening portion of eight wells 226 for one row among the wells 226 as the liquid containing portions arranged at 9 mm pitch of 96 12 rows ⁇ 8 columns as the planar liquid containing body.
  • the principal ray from each well 226 when the distance a to the liquid level 223 is 250 mm.
  • the position of each light receiving element 273 where a real image of fluorescence is formed is obtained by calculation.
  • FIG. 19A shows the toroidal mirror 230 and the microplate 222 cut along the meridional plane (ZY plane).
  • ZY plane the meridional plane
  • FIG. 19B shows the toroidal mirror 230 viewed from the optical axis surface orthogonal to the meridional surface, the path after reflection of principal rays from the center of each well 226, and the center of each liquid surface 223.
  • the projection onto the Z′X plane with the light receiving surface of the light receiving element 273 on which the point is imaged is shown.
  • the light receiving elements 273 are shown as four half (the other four half are symmetric with respect to the optical axis 280 and are omitted).
  • the Z ′ coordinate of the center position of the reflecting surface of each prism 272b is the optical axis 280. From the far side, -191.2mm, -195.2mm, -197.8mm, and -199.1mm.
  • the X coordinates are 32.2 mm, 23.4 mm, 14.1 mm, and 4.7 mm from the far side from the optical axis 280, respectively.
  • the Y ′ coordinate of the center position of the reflecting surface of each prism 272b is ⁇ 2.6 mm, ⁇ 1.4 mm, ⁇ 0.5 mm, and 0 mm from the far side of the optical axis 280. Accordingly, the coordinates of the center position of the reflecting surface of each of the eight prisms 272b in one row are (32.2mm, -2.6mm, -191.2mm) from a position far from the optical axis 280 in the XY'Z 'coordinate system.
  • FIG. 19C shows a cross-sectional shape of the toroidal mirror 230 cut along the optical axis plane inclined by 15 degrees with respect to the Z axis perpendicular to the meridional plane of the toroidal mirror 230, and cut along the optical axis plane.
  • the curvature radius Rv of the toroidal mirror 230 in the vertical direction is 187.5 mm.
  • each well 226 of the microplate 222 a predetermined amount of reaction solution containing a substance extracted from each specimen and a fluorescent substance is added to each of the 96 wells 226 by a separately provided dispensing device or the like, in this example, 50 ⁇ l.
  • the reagent is equally dispensed, and the transparent sheet 227 is attached to the upper side of the microplate 222 to close the opening of each well 226.
  • the drawer 214 is moved rearward to close the drawer 214, and then the knob 216 is rotated 90 degrees so that the locking bar 215 protrudes outward to lock the drawer 214 in a closed state. To do.
  • the heating lid 224 of the microplate 222 is attached to the microplate 222 from the upper side of the transparent sheet 221.
  • the heating lid 224 is heated to a constant temperature to prevent condensation, and the temperature control according to the processing content is performed by the heating block 238 provided on the lower side of the microplate 222. Will be done.
  • the measurement by the optical system is sequentially performed by driving the stepping motor 250 with respect to the microplate 222, thereby sequentially moving the Y moving body 231 from the innermost row along the Y-axis direction.
  • the optical system attached to the Y moving body 231 such as the toroidal mirror 230 as a concave mirror element is stopped on each row by moving 9 mm toward the previous row, optical measurement is sequentially performed for 12 rows. .
  • the optical measurement is performed when it is necessary to pass through the openings of the eight wells 226 in the first row of the microplate 222 through the through hole 228 of the heating lid 224.
  • the photodiode 270 receives light through the light receiving surface at the image forming position via the object side telecentric construction diaphragm 261, the lenses 272a and the prisms 272b of the eight light receiving elements 273, and receives an amount of light that is an analog amount thereof.
  • the control circuit of the optical system control board 245 converts the electric signal into a corresponding digital quantity and sends it to an information processing apparatus provided outside for analysis.
  • movement control is easy because it is only necessary to move the optical system at a constant pitch along the Y-axis direction. Further, the number of light receiving elements can be further reduced as compared with the case of the first embodiment. Further, it is possible to perform optical measurement continuously for 12 lines without stopping at a constant pitch, and in this case, the measurement time for 12 lines can be shortened.
  • microplate was also described only for the case of 9 mm pitch of 8 rows ⁇ 12 columns, but not limited to this case, various microplates, for example, the case of 4.5 mm pitch of 16 columns ⁇ 24 rows, Needless to say, the present invention can also be applied to microplates having various pitches such as 12 rows ⁇ 16 columns and 6 rows ⁇ 8 columns.
  • optical measurement can be performed without using the scanning mechanism by arranging the light receiving elements by the number of all wells.
  • the scanning mechanism portion can be dispensed with, the scale of the apparatus can be reduced and processing can be performed at a higher speed since no scanning time is required.
  • the scanning mechanism is not limited to moving the optical system as described above, and may move the planar liquid container or both. Spatial indications such as X-axis, Y-axis, Z-axis, vertical and horizontal directions, rows and columns are for illustrative purposes only and limit the spatial direction and position of the structure. is not.
  • the present invention relates to a reaction optical measurement apparatus and method, and is capable of optically measuring various reactions by labeling. For example, it can be obtained by monitoring nucleic acid (DNA) amplified by PCR in real time.
  • the amount of DNA used as an initial template for PCR can be determined using the amplification curve of the PCR product. In particular, it can be used in various fields such as biochemical field, agricultural field, pharmaceutical field, medical field and industrial field.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un dispositif réactionnel de mesure optique et un procédé. L'objectif de l'invention est de proposer un dispositif réactionnel de mesure optique compact et très fiable et un procédé pour celui-ci. Le dispositif réactionnel de mesure est configuré de manière à comprendre un contenant de liquide plat, dans lequel une pluralité d'unités contenant du liquide capables de contenir une solution réactionnelle contenant une substance émettant de la lumière sont disposées le long d'un plan et la température de chaque unité contenant du liquide est régulée, un ou plusieurs éléments miroirs concaves, qui permettent au rayon de lumière principal de la lumière émise généré dans une direction perpendiculaire au plan de traverser à un angle prédéterminé par rapport à l'axe optique une ouverture de chacune des unités contenant du liquide et qui forme des images réelles de la lumière émise à chacune des positions de formation d'images à l'exception du contenant de liquide plat, et des unités de réception de la lumière destinées à recevoir la lumière issue de l'image réelle à des positions de mesure prédéterminées respectives.
PCT/JP2010/054257 2009-03-26 2010-03-12 Dispositif réactionnel de mesure optique et son procédé de mesure Ceased WO2010110096A1 (fr)

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JP2014529726A (ja) * 2011-08-03 2014-11-13 エッペンドルフ アクチェンゲゼルシャフト 研究室用サンプルを取り扱うための研究室用装置及び方法
JP2016512881A (ja) * 2013-03-15 2016-05-09 バイオファイアー・ディフェンス・エルエルシー 試料アレイ内の試料を実質的に同時に監視するためのコンパクト光学システム
WO2016205950A1 (fr) * 2015-06-23 2016-12-29 Metaoptima Technology Inc. Appareil pour l'imagerie de la peau
JP2018088930A (ja) * 2012-06-28 2018-06-14 フルオレセントリック，インコーポレイテッド 化学的インジケータデバイス
JP2021001802A (ja) * 2019-06-21 2021-01-07 東亜ディーケーケー株式会社 光学測定装置
CN114324242A (zh) * 2021-12-10 2022-04-12 江西超联光电科技有限公司 一种用于反射镜光学加工原位检测的装置

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US10667694B2 (en) 2015-06-23 2020-06-02 Metaoptima Technology Inc. Apparatus for imaging skin
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