WO2021070259A1 - Analysis device and analysis method - Google Patents

Analysis device and analysis method Download PDF

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Publication number
WO2021070259A1
WO2021070259A1 PCT/JP2019/039701 JP2019039701W WO2021070259A1 WO 2021070259 A1 WO2021070259 A1 WO 2021070259A1 JP 2019039701 W JP2019039701 W JP 2019039701W WO 2021070259 A1 WO2021070259 A1 WO 2021070259A1
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Prior art keywords
fluorescence
objective lens
dimensional sensor
filter unit
lens
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PCT/JP2019/039701
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French (fr)
Japanese (ja)
Inventor
洋平 花崎
裕美 日下
曽根原　剛志
達也 山下
庄司　智広
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株式会社日立ハイテク
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Priority to PCT/JP2019/039701 priority Critical patent/WO2021070259A1/en
Publication of WO2021070259A1 publication Critical patent/WO2021070259A1/en

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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

Definitions

  • This disclosure relates to an analyzer and an analysis method.
  • Next-generation sequencers are widely used as devices for analyzing nucleic acids such as DNA. Measurement by the next-generation sequencer is performed using a flow cell (sample substrate) in which a large number of minute reaction fields are fixed. The next-generation sequencer irradiates the reaction field on the flow cell with excitation light via an objective lens, and detects fluorescence from the reaction field by a two-dimensional sensor such as a CCD camera or a CMOS camera. As a result, the base information can be obtained as a fluorescence image. In this way, by performing a chemical reaction on a microreaction field fixed to a flow cell and observing fluorescence, it is possible to analyze the base sequence of the target DNA.
  • Throughput is one of the important indicators in next-generation sequencers. Throughput is the total number of bases that can be analyzed per unit time, and technological development is underway to increase this.
  • four types of fluorescent dyes corresponding to each base are used, and it is necessary to irradiate the excitation light corresponding to each dye and separate the fluorescence.
  • detection is generally performed using four types of filter sets corresponding to each dye.
  • a dichroic mirror for separating the excitation light and fluorescence, and a filter for fluorescence at least 12 types of optical elements are used and each filter set is switched. A mechanism is also needed.
  • the fluorescence observation time for example, assuming that the imaging time for one color is 200 ms and the number of panels to be measured is 140 panels, it takes about 2 minutes for fluorescence imaging alone. In order to improve the throughput, it is necessary to further shorten such shooting time.
  • Patent Document 1 describes that the fluorescence is separated by a plurality of dichroic mirrors and photographed by two two-dimensional sensors to simultaneously read two color dyes in one photographing.
  • the mechanism for switching the filter set can be simplified and the time required for fluorescence imaging can be reduced to about half.
  • Patent Document 2 a first dichroic mirror that allows fluorescence to pass through and a second dichroic mirror that separates fluorescence for each dye are arranged at an angle in the same direction with respect to the optical axis, and the optical filter is switched and 2 It is described that a plurality of fluorescent dyes are observed simultaneously by a two-dimensional sensor.
  • Patent Document 1 does not mention the focus positions of the two two-dimensional sensors. Therefore, there is a possibility that the focus may be out of focus due to chromatic aberration of the objective lens or the like, a high-quality fluorescent image cannot be obtained, and the accuracy of base identification is lowered.
  • Patent Document 2 does not mention the focus positions of the two two-dimensional sensors, and when the optical filters are switched to capture four images, the focus shifts due to chromatic aberration of the objective lens or the like. there is a possibility.
  • the present disclosure provides a technique for focusing images of a plurality of colors.
  • the analyzer of the present disclosure includes a stage on which a sample substrate that emits at least the first fluorescence, the second fluorescence, and the third fluorescence is placed, an objective lens, and the first.
  • the first two-dimensional sensor and the second two-dimensional sensor that image the third fluorescence, and the first two-dimensional sensor and the second two-dimensional sensor have the first fluorescence, the second fluorescence, and the second fluorescence.
  • a second driving device that relatively changes the distance between the objective lens and the sample substrate, the first two-dimensional sensor, the second two-dimensional sensor, the first driving device, and the second driving device.
  • a control unit that controls at least a drive device is provided, and the control unit includes a case where the image pickup is performed using the first filter unit and a case where the image pickup is performed using the second filter unit. It is characterized in that the first drive device and the second drive device are controlled so as to adjust the distance between the objective lens and the sample substrate, respectively.
  • the graph which plotted the focus value when the distance z between the incident side main plane of a synthetic lens system and a sample substrate was changed.
  • the graph which plotted the focus value when the distance z between the incident side main plane of a synthetic lens system and a sample substrate was changed.
  • FIG. 1 is a schematic view showing an analyzer according to the first embodiment.
  • the vertical direction of the paper surface is the vertical direction.
  • the analyzer of this embodiment is, for example, a nucleic acid analyzer that analyzes a nucleic acid base sequence, and controls the entire optical system 100, the stage 109 on which the sample substrate 101 is placed, and the analyzer as shown in FIG. A control unit 110 is provided.
  • the sample substrate 101 is, for example, a flow cell and has a flow path for the reaction solution.
  • the number of flow paths may be one or a plurality.
  • a nucleic acid to be read (sometimes simply referred to as a "sample") such as single-strand DNA is fixed in the flow path, and a reaction solution (reagent) is introduced by a liquid feeding mechanism (not shown).
  • the reaction solution contains a plurality of types of fluorescently labeled nucleotides (dNTPs) and polymerases, each labeled with a different fluorescent dye.
  • a reversible terminator (protecting group) that inhibits the elongation of the next base is bound to the fluorescently labeled nucleotide, whereby only one base of the fluorescently labeled nucleotide that is complementary to the nucleic acid to be read is taken up. After one base is taken up, the floating fluorescently labeled nucleotides are removed by washing. After that, fluorescence observation is performed by the optical system 100 to remove the fluorescent dye and the protecting group. By repeating the above steps as one cycle, the fluorescent color sequence is determined and the base sequence is determined.
  • the stage 109 supports the sample substrate 101 so that the sample substrate 101 is orthogonal to the optical axis of the objective lens 103.
  • the stage 109 is configured to be movable at least in the horizontal direction by a drive device (not shown).
  • the stage 109 may have a temperature control mechanism such as a heat block at a position in contact with the sample substrate 101, and the elongation reaction can be promoted by heating and cooling the sample substrate 101 as needed. Further, the stage 109 may be configured so that a plurality of sample substrates 101 can be mounted at the same time.
  • the optical system 100 includes a light source 102, an objective lens 103, a two-dimensional sensor 104A (first two-dimensional sensor), a two-dimensional sensor 104B (second two-dimensional sensor), a dichroic mirror 105 (optical element), and a filter unit 106A ( It has a first filter unit), a filter unit 106B (second filter unit), a filter unit switching mechanism 107 (first drive device), and an objective lens drive device 108 (second drive device).
  • the light source 102 emits light including excitation light having a wavelength capable of exciting the fluorescent dye bound to the sample.
  • the light source 102 for example, an Xe lamp or a white LED can be used, and not only one type of light source but also, for example, a light source in which a plurality of types of LEDs are combined may be used.
  • the filter unit 106A is a transmission filter 111A that transmits the excitation light from the light source 102, a dichroic mirror 112A that reflects the excitation light and is incident on the sample substrate 101, and transmits only the fluorescence from the sample. It has a fluorescent filter 113A.
  • the transmission filter 111A and the fluorescence filter 113A can efficiently excite the sample and remove components other than fluorescence generated from the sample, thereby increasing the contrast of the obtained fluorescence image.
  • the filter unit 106B has a transmission filter 111B, a dichroic mirror 112B, and a fluorescence filter 113B.
  • the filter units 106A and 106B have different types of target phosphors, and have different wavelengths of fluorescence to be transmitted and wavelengths to be blocked. In addition, there is no difference in the performance (ability) of transmitting the fluorescence of the target phosphor and the performance of blocking light other than the fluorescence.
  • the filter unit switching mechanism 107 is configured so that the positions of the filter units 106A and 106B can be changed, and one of these is arranged on the optical axis of the objective lens 103.
  • a motor or a solenoid can be used as the drive mechanism of the filter unit switching mechanism 107.
  • the dichroic mirror 105 is arranged on the optical axis of the objective lens 103, and the fluorescence that has passed through the filter unit 106A or 106B is incident on the two-dimensional sensors 104A and 104B. Specifically, the fluorescence transmitted through the dichroic mirror 105 is imaged on the two-dimensional sensor 104A, and the fluorescence reflected by the dichroic mirror 105 is imaged on the two-dimensional sensor 104B.
  • the dichroic mirror 105 is arranged at an angle of, for example, 45 ° with respect to the optical axis of the objective lens 103.
  • other optical elements such as a half mirror, a beam splitter, and a cold mirror may be used.
  • the two-dimensional sensors 104A and 104B acquire an image of fluorescence incident on the sensor surface and transmit the fluorescence image to the control unit 110.
  • a CCD camera or a CMOS camera can be used as the two-dimensional sensors 104A and 104B.
  • the objective lens driving device 108 is connected to the objective lens 103, and the objective lens 103 is configured to be movable in the vertical direction. This makes it possible to adjust the distance between the objective lens 103 and the sample substrate 101.
  • the objective lens driving device 108 includes, for example, a stepping motor, a stage fixed to the objective lens 103, a pulse oscillator, and the like. Instead of providing the objective lens driving device 108, a driving device capable of driving the stage 109 not only in the horizontal direction but also in the vertical direction may be used.
  • the control unit 110 irradiates light by the light source 102, switches by the filter unit switching mechanism 107, captures images by the two-dimensional sensors 104A and 104B, drives the objective lens driving device 108, drives the driving device (not shown) of the stage 109, and reacts. Controls the drive of the liquid feeding mechanism (not shown).
  • the control unit 110 executes a process of analyzing the base sequence of nucleic acid based on the fluorescence images acquired by the two-dimensional sensors 104A and 104B.
  • the control unit 110 reads a program for driving each component of the analyzer and executing analysis processing and a storage unit for storing various data, and reads the program and various data to perform the above operation. It has a processor to execute, an input unit for the user to input data and instructions, and the like.
  • Example of analysis method A method of analyzing the base sequence of the nucleic acid to be analyzed using the analyzer of the present embodiment will be described.
  • fluorescently labeled nucleotides labeled with four color fluorescent dyes (first fluorescent dye to fourth fluorescent dye) that emit the first fluorescence to the fourth fluorescence are bound to the sample one base at a time.
  • the image is taken and the base sequence is analyzed.
  • the filter unit 106A separates the first fluorescence and the second fluorescence
  • the filter unit 106B separates the third fluorescence and the fourth fluorescence.
  • FIG. 2 is a flowchart showing an analysis method according to the present embodiment.
  • the user immobilizes the nucleic acid (sample) to be analyzed on the sample substrate 101 in advance, and stores reagents such as fluorescently labeled nucleotides and polymerases in the liquid feeding mechanism. Further, in the storage unit of the control unit 110, the types of the first fluorescent dye to the fourth fluorescent dye, the focusing positions of the first fluorescence to the fourth fluorescence, and the number of bases to be analyzed in the sequence are stored. ing.
  • step S1 the control unit 110 places the sample substrate 101 on the stage 109 and moves the stage 109 so that the observation position of the sample substrate 101 is located on the optical axis of the objective lens 103. After that, the control unit 110 drives the liquid feeding mechanism to introduce the reaction liquid into the sample substrate 101, and causes the sample to synthesize only one base of the fluorescently labeled nucleotide.
  • step S2 the control unit 110 drives the filter unit switching mechanism 107 to arrange the filter unit 106A on the optical axis of the objective lens 103.
  • step S3 the control unit 110 drives the objective lens driving device 108 so that the objective lens 103 is focused between the focusing position of the first fluorescence and the focusing position of the second fluorescence.
  • the position (first position) of the objective lens 103 is adjusted.
  • step S4 the control unit 110 transmits an imaging instruction to the two-dimensional sensors 104A and 104B, the two-dimensional sensors 104A and 104B image the fluorescence image of the sample substrate 101, and the captured fluorescence image is obtained by the control unit 110.
  • the imaging time can be shortened by simultaneously imaging the two-dimensional sensors 104A and 104B, but they may be imaged separately.
  • step S5 the control unit 110 drives the filter unit switching mechanism 107 to arrange the filter unit 106B on the optical axis of the objective lens 103.
  • imaging is performed using the filter unit 106A first, but the order in which the filter units 106A and 106B are used is not limited.
  • step S6 the control unit 110 drives the objective lens driving device 108 so that the objective lens 103 is focused between the in-focus position of the third fluorescence and the in-focus position of the fourth fluorescence.
  • the position (second position) of the objective lens 103 is adjusted.
  • step S7 the two-dimensional sensors 104A and 104B capture a fluorescence image of the sample substrate 101 and transmit the captured fluorescence image to the control unit 110.
  • step S8 the control unit 110 determines which of the first fluorescent dye to the fourth fluorescent dye is bound based on the four fluorescent images captured in steps S4 and S7, and identifies the base. To do. Base identification is performed, for example, based on the signal intensity of fluorescence of four colors. Further, the control unit 110 stores the identification result in the storage unit.
  • step S9 the control unit 110 determines whether or not the identification was for the last base of the sample. At this time, it is possible to determine whether the base is the last base based on the set number of bases to be analyzed and the number of times the identification is performed.
  • step S9 When it is determined in step S9 that the base is not the last base (No), the control unit 110 drives the liquid feeding mechanism to introduce the cleaning liquid into the sample substrate 101 and remove the fluorescent dye and the protecting group. After that, the stage 109 is driven to arrange the next imaging position of the sample substrate 101 on the optical axis of the objective lens 103, and the process returns to step S1.
  • step S9 If it is determined to be the last base in step S9 (Yes), the control unit 110 ends the operation.
  • the combination of fluorescence of the two colors in steps S3 and S6 is set as follows, for example. That is, the in-focus positions of the fluorescence of the two colors imaged using the same filter unit are close to each other, and are far from the in-focus position of the fluorescence imaged by using another filter unit.
  • a lens has a problem of defocusing due to axial chromatic aberration in which the focal length differs for each wavelength. This axial chromatic aberration can be canceled by complicating the lens system, but the device becomes large as the price increases and the optical system increases.
  • the analyzer of the present embodiment uses two filter units and two two-dimensional sensors to focus and image between the focusing positions of the two colors of fluorescence.
  • defocus due to axial chromatic aberration can be compensated and a high-quality image can be obtained.
  • the number of colors that can be observed with high quality fluorescence by the analyzer of the present embodiment is not limited to four colors.
  • a method of binding a three-color fluorescent dye to a nucleic acid to be analyzed (such as single-stranded DNA) and analyzing its base sequence will be described.
  • fluorescently labeled nucleotides labeled with three color fluorescent dyes (first fluorescent dye to third fluorescent dye) that emit the first fluorescence to the third fluorescence, respectively, are used.
  • nucleic acid bases Since there are four types of nucleic acid bases, one type of base cannot be detected by fluorescence of three colors. Therefore, three of the four nucleotides are each labeled with one of the first fluorescent dye to the third fluorescent dye, and the remaining one nucleotide is, for example, of the first fluorescent dye and the second fluorescent dye. It shall be labeled by two. Thereby, when both the first fluorescence and the second fluorescence are detected, that is, when two of the three colors of fluorescence are detected at the same time, it can be determined to be the fourth type of base.
  • the first fluorescence and the second fluorescence are separated by the filter unit 106A, and the third fluorescence is separated by the filter unit 106B.
  • This method is almost the same as the analysis method (FIG. 2) in the case of the above-mentioned four-color labeling, but in step S6, the objective lens so that the objective lens 103 is focused only on the focus position of the third fluorescence. The position of 103 (second position) is adjusted. Since the other steps are the same as the analysis method in the case of the four-color label, the description thereof will be omitted.
  • the first fluorescence may be separated by the filter unit 106A, and the second fluorescence and the third fluorescence may be separated by the filter unit 106B.
  • the objective lens 103 is focused on the in-focus position of the first fluorescence for imaging, and when the filter unit 106B is used, the in-focus position of the second fluorescence and the third fluorescence are taken. Focus on the in-focus position of the image. As described above, if the in-focus positions of the two colors of fluorescence imaged using the same filter unit are close to each other, and the in-focus positions of the fluorescence imaged by another filter unit are far from each other. Good.
  • a fourth type of base can be detected by combining fluorescence detection with a method other than fluorescence detection such as an electrochemical luminescence method.
  • the analyzer of the present embodiment focuses between the in-focus positions of the two fluorescences and simultaneously performs imaging with the two two-dimensional sensors, and then switches the filter unit to perform the same imaging.
  • defocus due to axial chromatic aberration can be compensated, so that a high-quality fluorescence image can be obtained. Therefore, the base can be identified with high accuracy.
  • the imaging time can be shortened by imaging the fluorescence of two colors at once, the decrease in throughput can be suppressed.
  • FIG. 3 is a schematic view showing a partial configuration of the analyzer according to the second embodiment. Since the configuration of the analyzer of the second embodiment is different from that of the optical system 100 of the first embodiment only in the optical system 200, the configurations other than the optical system 200 and the sample substrate 101 are shown in FIG. It is omitted.
  • the imaging lens 209A is provided in front of the two-dimensional sensor 104A, and the imaging lens 209B is provided in front of the two-dimensional sensor 104B.
  • the imaging lens is composed of two pasted lenses, and chromatic aberration of near wavelength is compensated.
  • the imaging lenses 209A and 209B compensate for the chromatic aberration of only the first fluorescence, the second fluorescence, and the third fluorescence.
  • the objective lens 103 is driven in consideration of the composite lens of 2).
  • the incident side main plane 213 of the first composite lens and the second composite lens is located above the objective lens 103.
  • the emission side main plane 214A (first emission side main plane) of the first synthetic lens is located above the imaging lens 209A, and the emission side main plane 214B (second emission side main plane) of the second composite lens. ) Is located on the right side of the imaging lens 209B.
  • the distance between the incident side main plane 213 and the surface of the sample substrate 101 is z
  • the distance between the emission side main planes 214A and 214B and the fluorescence imaging position is s
  • the combined focal distance is f
  • the emission side main planes 214A and 2 Let dT be the distance from the dimensional sensor 109A, and dR be the distance between the injection side main plane 214B and the two-dimensional sensor 109B.
  • FIG. 3 illustrates the distances z, dT and dR.
  • the focus value when the objective lens 103 is moved in the optical axis direction to change the distance z (sometimes simply referred to as “distance z”) between the incident side main plane 213 and the surface of the sample substrate 101 will be described.
  • a lens has a defocus distance called a depth of focus, which does not change the quality of an image, and this is referred to as a "defocus tolerance" in the present specification. That is, if the image is taken so as to be within the defocus allowable range, a high-quality fluorescent image can be obtained.
  • FIG. 4 is a graph plotting focus values when the distance z is changed to explain a general imaging method.
  • the curve 401A is a curve plotting the focus value of the first synthetic lens when the filter unit 106A is used.
  • the curve 402A is a curve plotting the focus value of the second synthetic lens when the filter unit 106A is used.
  • the curves 401B and 402B are a curve plotting the focus value of the first composite lens and a curve plotting the focus value of the second composite lens when the filter unit 106B is used, respectively.
  • the objective lens driving device 108 is driven to adjust the position of the objective lens 103 so as to satisfy the following equations (5) and (6).
  • s1 ⁇ dT ⁇ s2 when s1> s2 s1 ⁇ dT ⁇ s2 when s1 ⁇ s2, s3 ⁇ dR ⁇ s4 when s3> s4, and s3 ⁇ s4.
  • the position of the objective lens 103 when taking an image using the filter unit 106A and the position of the objective lens 103 when taking an image using the filter unit 106B are adjusted so that s3 ⁇ dR ⁇ s4.
  • the distance z between the incident side main plane 113 and the sample substrate 101 is offset by driving the objective lens 103 so as to satisfy the following equations (7) and (8).
  • FIG. 5 is a graph in which focus values when the distance z is changed are plotted for explaining the imaging method of the present embodiment.
  • the distance z1 is such that the curve 501A plotting the focus value of the first composite lens and the curve 502A plotting the focus value of the second composite lens intersect. Is set, and the image is taken at the position (first position) of the objective lens 103 such that the distance is z1.
  • the distance z2 is such that the curve 501B plotting the focus value of the first composite lens and the curve 502B plotting the focus value of the second composite lens intersect.
  • the image is taken at the position (second position) of the objective lens 103 that is set and has a distance z2.
  • the fluorescence image of each fluorescence is within the defocus range, so that it is possible to capture a fluorescence image in which all the fluorescence is in focus.
  • defocusing is allowed for all fluorescence in consideration of the composite lens system of the objective lens 103 and the imaging lens 209A and the composite lens system of the objective lens 103 and the imaging lens 209B.
  • the position of the objective lens 103 is adjusted so as to be within the range. As a result, a higher quality image can be obtained as compared with the first embodiment, and the base can be identified with high accuracy.
  • the focusing positions of the first fluorescence to the fourth fluorescence are stored in the control unit 110 in advance, and based on this, the objective lens 103 is focused.
  • the third embodiment proposes an example in which the objective lens 103 is focused by autofocus.
  • FIG. 6 is a schematic view showing a partial configuration of the analyzer according to the third embodiment.
  • the configurations other than the optical system 300 and the sample substrate 101 are not shown.
  • the optical system 300 of the present embodiment is provided with an autofocus drive system 311 and a mirror 310 for autofocus.
  • Other configurations are the same as in the second embodiment.
  • the mirror 310 is provided in front of the dichroic mirror 105.
  • the autofocus drive system 311 irradiates the mirror 310 with laser light, whereby the laser light reflected by the mirror 310 is incident on the sample substrate 101. Further, the autofocus drive system 311 detects the laser light reflected from the sample substrate 101.
  • infrared light is used as the laser light of the autofocus drive system 311, and the mirror 310 is used as the mirror 310.
  • a dichroic mirror that reflects infrared light can be used, but is not limited thereto. For example, when a laser beam in a visible light region such as 532 nm is used in the autofocus drive system 311, a mirror 310 that reflects only 532 nm can be used.
  • the irradiation of the laser beam by the autofocus drive system 311 is controlled by the control unit 110. Further, the autofocus drive system 311 outputs a detection signal of the reflected light to the control unit 110. The control unit 110 drives the objective lens driving device 108 based on the detection signal of the autofocus driving system 311.
  • the autofocus drive system 311 outputs a detection signal proportional to the distance z between the objective lens 103 and the sample substrate 101. This detection signal becomes 0 when the fluorescence from the sample is in focus.
  • the control unit 110 sets the detection signal from the autofocus drive system 311 to a value such that the focus value of the first composite lens and the focus value of the second composite lens are equal ( The position of the objective lens 103 is determined so as to be the first target value). That is, the distance between the incident side main plane 213 and the sample substrate 101 is adjusted to the distance z1 shown in FIG. 5, and imaging is performed.
  • the control unit 110 sets the detection signal from the autofocus drive system 311 to a value such that the focus value of the first composite lens and the focus value of the second composite lens are equal (the focus value is equal to that of the second composite lens.
  • the position of the objective lens 103 is determined so as to be the second target value). That is, the distance between the incident side main plane 213 and the sample substrate 101 is adjusted to the distance z2 shown in FIG. 5, and imaging is performed.
  • the first target value and the second target value are different values, and are determined by the focusing positions of the first to fourth fluorescences and the like.
  • the autofocus method is not limited to the above, and other methods can be adopted.
  • the analyzer of the present embodiment uses the autofocus drive system 311 to adjust the position of the objective lens 103 so as to be within the defocus allowable range for all fluorescence, as in the second embodiment. And take an image. Even with such a configuration, a higher quality image can be obtained as compared with the first embodiment, and the base can be identified with high accuracy. Further, when the objective lens 103 is driven to the best focus position obtained in advance (for example, the distance z1 when the curves 501A and 502A shown in FIG. 5 intersect), the actual best focus position changes due to temperature drift or the like. If you do, it will be defocused. On the other hand, by using the autofocus drive system 311 as in the present embodiment, the objective lens 103 can be focused on the actual best focus position, so that a large number of images captured in a short time can be captured with higher accuracy. Can be focused on.
  • the fourth embodiment proposes an optical system further provided with a laser cut filter.
  • FIG. 7 is a schematic view showing a partial configuration of the analyzer according to the fourth embodiment.
  • the configurations other than the optical system 400 and the sample substrate 101 are not shown.
  • the optical system 400 of the present embodiment further includes a laser cut filter 412 between the dichroic mirror 105 and the mirror 310.
  • Other configurations are the same as those in the third embodiment.
  • the autofocus drive system 311 irradiates a laser of infrared light, for example, by using a filter that cuts not only the wavelength of the laser but also the infrared light, the laser light is not prevented from fluorescence from the sample. Only can be removed.
  • the analyzer of the present embodiment has a laser cut filter 412 that cuts the laser light emitted by the autofocus drive system 311. With such a configuration, it is possible to avoid reflection of the laser beam on the fluorescence image, and it is possible to acquire the fluorescence image with stable quality.
  • a nucleic acid analysis apparatus for analyzing a base sequence by labeling a nucleic acid to be analyzed with fluorescence of three or four colors.
  • each embodiment can be applied not only to a nucleic acid analyzer but also to an analyzer that detects a plurality of colors.
  • an additional filter unit is used (three filter units in total), and the focus position of the fifth fluorescence and the focus position of the sixth fluorescence are used. Focus on the focus position and take an image. As a result, it is possible to acquire a high-quality image in which the fluorescence of the six colors is in focus.
  • n filter units in this way, it is possible to acquire a fluorescence image of a maximum of 2n colors with high quality.
  • Optical system 101 ... Sample substrate 102 ... Light source 103 ... Objective lens 104A, 104B ... Two-dimensional sensor 105 ... Dycroic mirror 106A, 106B ... Filter unit 107 ... Filter unit switching mechanism 108 ... Objective lens driving device 109A, 109B ... Imaging lens 110 ... Control unit 111A, 111B ... Transmission filter 112A, 112B ... Dycroic mirror 113A, 113B ... Fluorescent filter 209A, 209B ... Imaging lens 213 ... Incident side main plane 214A, 214B ... Ejection side main plane 310 ... Mirror for autofocus 311 ... Autofocus drive system 412 ... Laser cut filter

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Abstract

This analysis device comprises: a stage having placed thereon a sample substrate that emits first fluorescence, second fluorescence, and third fluorescence; an objective lens; a first filter unit for separating the first fluorescence and second fluorescence; a second filter unit for separating the third fluorescence; a first two-dimensional sensor and second two-dimensional sensor for imaging the first to third fluorescences from the sample substrate; an optical element for separating the first to third fluorescences; a first drive device for switching between the first filter unit and second filter unit; a second drive device for changing the relative distance between the objective lens and sample substrate; and a control unit for at least controlling the first drive device and second drive device. The control unit controls the first drive device and second drive device so as to adjust the distance between the objective lens and sample substrate according to whether imaging is to be carried out using the first filter unit or second filter unit.

Description

分析装置及び分析方法Analytical device and analytical method
　本開示は、分析装置及び分析方法に関する。 This disclosure relates to an analyzer and an analysis method.
　次世代シーケンサは、ＤＮＡなどの核酸を解析する装置として広く用いられている。次世代シーケンサによる計測は、多数の微小反応場が固定されたフローセル（サンプル基板）を用いて行われる。次世代シーケンサは、フローセル上の反応場に対し対物レンズを介して励起光を照射し、ＣＣＤカメラやＣＭＯＳカメラなどの２次元センサにより反応場からの蛍光を検出する。これにより塩基情報を蛍光画像として入手することができる。このように、フローセルに固定された微小反応場上にて化学反応を行い、蛍光観察を行うことにより、目的とするＤＮＡの塩基配列の解析が可能になる。 Next-generation sequencers are widely used as devices for analyzing nucleic acids such as DNA. Measurement by the next-generation sequencer is performed using a flow cell (sample substrate) in which a large number of minute reaction fields are fixed. The next-generation sequencer irradiates the reaction field on the flow cell with excitation light via an objective lens, and detects fluorescence from the reaction field by a two-dimensional sensor such as a CCD camera or a CMOS camera. As a result, the base information can be obtained as a fluorescence image. In this way, by performing a chemical reaction on a microreaction field fixed to a flow cell and observing fluorescence, it is possible to analyze the base sequence of the target DNA.
　次世代シーケンサにおいて重要視される指標の一つとしてスループットがある。スループットとは単位時間当たりに解析できる総塩基数であり、これを増加させるための技術開発が進んでいる。次世代シーケンサによる解析では、各塩基に対応した４種類の蛍光色素が用いられ、それぞれの色素に対応した励起光の照射、蛍光の分離が必要である。従来、各色素に対応した４種類のフィルタセットを用いて検出が行われることが一般的である。この方式においては、４種類の励起光、励起光と蛍光を分離するためのダイクロイックミラー及び蛍光用のフィルタを有するため、少なくとも１２種類の光学素子が用いられる上に、各フィルタセットを切り替えるための機構も必要である。 Throughput is one of the important indicators in next-generation sequencers. Throughput is the total number of bases that can be analyzed per unit time, and technological development is underway to increase this. In the analysis by the next-generation sequencer, four types of fluorescent dyes corresponding to each base are used, and it is necessary to irradiate the excitation light corresponding to each dye and separate the fluorescence. Conventionally, detection is generally performed using four types of filter sets corresponding to each dye. In this method, since it has four types of excitation light, a dichroic mirror for separating the excitation light and fluorescence, and a filter for fluorescence, at least 12 types of optical elements are used and each filter set is switched. A mechanism is also needed.
　蛍光観察の時間に関して、例えば、１色の撮影時間を２００ｍｓ、測定するパネル数を１４０パネルとすると、蛍光撮影だけで約２分の時間を要する。スループットの向上には、このような撮影時間をより短縮することが必要である。 Regarding the fluorescence observation time, for example, assuming that the imaging time for one color is 200 ms and the number of panels to be measured is 140 panels, it takes about 2 minutes for fluorescence imaging alone. In order to improve the throughput, it is necessary to further shorten such shooting time.
　特許文献１には、複数枚のダイクロイックミラーにて蛍光を分離し、２台の２次元センサによって撮影することによって、一度の撮影で２色の色素を同時に読み取ることが記載されている。特許文献１の方式を用いることで、フィルタセットの切替を行う機構を単純化するとともに、蛍光撮影に要する時間を約半分まで短縮することができる。 Patent Document 1 describes that the fluorescence is separated by a plurality of dichroic mirrors and photographed by two two-dimensional sensors to simultaneously read two color dyes in one photographing. By using the method of Patent Document 1, the mechanism for switching the filter set can be simplified and the time required for fluorescence imaging can be reduced to about half.
　特許文献２には、蛍光を通過させる第１のダイクロイックミラーと、蛍光を色素ごとに分離する第２のダイクロイックミラーとを光軸に対して同じ方向に傾けて配置し、光学フィルタの切り替えと２台の２次元センサにより、複数の蛍光色素を同時に観察することが記載されている。 In Patent Document 2, a first dichroic mirror that allows fluorescence to pass through and a second dichroic mirror that separates fluorescence for each dye are arranged at an angle in the same direction with respect to the optical axis, and the optical filter is switched and 2 It is described that a plurality of fluorescent dyes are observed simultaneously by a two-dimensional sensor.
米国特許出願公開第２０１２／０２７０３０５号明細書U.S. Patent Application Publication No. 2012/0270305 特開２０１６－２４１３７号公報Japanese Unexamined Patent Publication No. 2016-24137
　しかしながら、特許文献１においては、２台の２次元センサのフォーカス位置については言及されていない。したがって、対物レンズの色収差等によりピントがずれてしまう可能性があり、高品質の蛍光画像が得られず塩基同定の精度が落ちてしまう。 However, Patent Document 1 does not mention the focus positions of the two two-dimensional sensors. Therefore, there is a possibility that the focus may be out of focus due to chromatic aberration of the objective lens or the like, a high-quality fluorescent image cannot be obtained, and the accuracy of base identification is lowered.
　また、特許文献２においても２台の２次元センサのフォーカス位置については言及されておらず、光学フィルタを切り替えて４枚の画像を撮像する際に、対物レンズの色収差等によりピントがずれてしまう可能性がある。 Further, Patent Document 2 does not mention the focus positions of the two two-dimensional sensors, and when the optical filters are switched to capture four images, the focus shifts due to chromatic aberration of the objective lens or the like. there is a possibility.
　そこで、本開示は、複数色の画像のピントを合わせる技術を提供する。 Therefore, the present disclosure provides a technique for focusing images of a plurality of colors.
　上記課題を解決するために、本開示の分析装置は、少なくとも第１の蛍光、第２の蛍光及び第３の蛍光を発するサンプル基板が載置されるステージと、対物レンズと、前記第１の蛍光及び前記第２の蛍光を分離する第１のフィルタユニットと、前記第３の蛍光を分離する第２のフィルタユニットと、前記サンプル基板からの前記第１の蛍光、前記第２の蛍光及び前記第３の蛍光を撮像する第１の２次元センサ及び第２の２次元センサと、前記第１の２次元センサ及び前記第２の２次元センサに前記第１の蛍光、前記第２の蛍光及び前記第３の蛍光をそれぞれ分離する光学素子と、前記第１のフィルタユニット及び前記第２のフィルタユニットのいずれか一方を用いて前記撮像が行われるようにこれらを切り替える第１の駆動装置と、前記対物レンズと前記サンプル基板との距離を相対的に変化させる第２の駆動装置と、前記第１の２次元センサ、前記第２の２次元センサ、前記第１の駆動装置及び前記第２の駆動装置を少なくとも制御する制御部と、を備え、前記制御部は、前記第１のフィルタユニットを用いて前記撮像を行う場合と前記第２のフィルタユニットを用いて前記撮像を行う場合とで、それぞれ前記対物レンズと前記サンプル基板との距離を調整するように、前記第１の駆動装置及び前記第２の駆動装置を制御することを特徴とする。 In order to solve the above problems, the analyzer of the present disclosure includes a stage on which a sample substrate that emits at least the first fluorescence, the second fluorescence, and the third fluorescence is placed, an objective lens, and the first. A first filter unit that separates the fluorescence and the second fluorescence, a second filter unit that separates the third fluorescence, the first fluorescence from the sample substrate, the second fluorescence, and the said. The first two-dimensional sensor and the second two-dimensional sensor that image the third fluorescence, and the first two-dimensional sensor and the second two-dimensional sensor have the first fluorescence, the second fluorescence, and the second fluorescence. An optical element that separates the third fluorescence, and a first driving device that switches between the first filter unit and the second filter unit so that the imaging can be performed. A second driving device that relatively changes the distance between the objective lens and the sample substrate, the first two-dimensional sensor, the second two-dimensional sensor, the first driving device, and the second driving device. A control unit that controls at least a drive device is provided, and the control unit includes a case where the image pickup is performed using the first filter unit and a case where the image pickup is performed using the second filter unit. It is characterized in that the first drive device and the second drive device are controlled so as to adjust the distance between the objective lens and the sample substrate, respectively.
　本開示に関連する更なる特徴は、本明細書の記述、添付図面から明らかになるものである。また、本開示の態様は、要素及び多様な要素の組み合わせ及び以降の詳細な記述と添付される請求の範囲の様態により達成され実現される。
　本明細書の記述は典型的な例示に過ぎず、本開示の請求の範囲又は適用例を如何なる意味に於いても限定するものではない。 Further features relating to this disclosure will become apparent from the description herein and the accompanying drawings. In addition, the aspects of the present disclosure are achieved and realized by the combination of elements and various elements, the detailed description below, and the aspects of the appended claims.
The description herein is merely a exemplary example, and is not intended to limit the scope or application of the claims of the present disclosure in any way.
　本開示の分析装置によれば、複数色の画像のピントを合わせることができる。
　上記以外の課題、構成及び効果は、以下の実施の形態の説明により明らかにされる。 According to the analyzer of the present disclosure, it is possible to focus an image of a plurality of colors.
Issues, configurations and effects other than the above will be clarified by the following description of the embodiments.
第１の実施形態に係る分析装置を示す概略図。The schematic diagram which shows the analyzer which concerns on 1st Embodiment. 第１の実施形態に係る分析方法を示すフローチャート。The flowchart which shows the analysis method which concerns on 1st Embodiment. 第２の実施形態に係る分析装置の一部の構成を示す概略図。The schematic diagram which shows the structure of a part of the analyzer which concerns on 2nd Embodiment. 合成レンズ系の入射側主平面とサンプル基板との距離ｚを変化させた場合のフォーカス値をプロットしたグラフ。The graph which plotted the focus value when the distance z between the incident side main plane of a synthetic lens system and a sample substrate was changed. 合成レンズ系の入射側主平面とサンプル基板との距離ｚを変化させた場合のフォーカス値をプロットしたグラフ。The graph which plotted the focus value when the distance z between the incident side main plane of a synthetic lens system and a sample substrate was changed. 第３の実施形態に係る分析装置の一部の構成を示す概略図。The schematic diagram which shows the structure of a part of the analyzer which concerns on 3rd Embodiment. 第４の実施形態に係る分析装置の一部の構成を示す概略図。The schematic diagram which shows the structure of a part of the analyzer which concerns on 4th Embodiment.
［第１の実施形態］
＜分析装置の構成＞
　図１は、第１の実施形態に係る分析装置を示す概略図である。図１において、紙面上下方向を鉛直方向とする。本実施形態の分析装置は、例えば核酸の塩基配列を解析する核酸解析装置であり、図１に示すように光学系１００、サンプル基板１０１が載置されるステージ１０９及び分析装置の全体を制御する制御部１１０を備える。 [First Embodiment]
<Analyzer configuration>
FIG. 1 is a schematic view showing an analyzer according to the first embodiment. In FIG. 1, the vertical direction of the paper surface is the vertical direction. The analyzer of this embodiment is, for example, a nucleic acid analyzer that analyzes a nucleic acid base sequence, and controls the entire optical system 100, the stage 109 on which the sample substrate 101 is placed, and the analyzer as shown in FIG. A control unit 110 is provided.
　サンプル基板１０１は例えばフローセルであり、反応液の流路を有している。流路の本数は１本であってもよいし、複数本であってもよい。流路には、一本鎖ＤＮＡなどの読み取り対象の核酸（単に「サンプル」という場合がある）が固定されており、図示しない送液機構により反応液（試薬）が導入される。反応液には、異なる蛍光色素でそれぞれ標識された複数種の蛍光標識ヌクレオチド（ｄＮＴＰ）及びポリメラーゼが含まれる。蛍光標識ヌクレオチドには、次の塩基の伸長を阻害する可逆的ターミネータ（保護基）が結合しており、これにより読み取り対象の核酸に相補的な蛍光標識ヌクレオチドが１塩基だけ取り込まれる。１塩基が取り込まれた後、浮遊する蛍光標識ヌクレオチドが洗浄により除去される。その後、光学系１００により蛍光観察を行い、蛍光色素及び保護基を除去する。以上の工程を１サイクルとして、これを繰り返すことにより蛍光色の配列が決定され、塩基配列が決定される。 The sample substrate 101 is, for example, a flow cell and has a flow path for the reaction solution. The number of flow paths may be one or a plurality. A nucleic acid to be read (sometimes simply referred to as a "sample") such as single-strand DNA is fixed in the flow path, and a reaction solution (reagent) is introduced by a liquid feeding mechanism (not shown). The reaction solution contains a plurality of types of fluorescently labeled nucleotides (dNTPs) and polymerases, each labeled with a different fluorescent dye. A reversible terminator (protecting group) that inhibits the elongation of the next base is bound to the fluorescently labeled nucleotide, whereby only one base of the fluorescently labeled nucleotide that is complementary to the nucleic acid to be read is taken up. After one base is taken up, the floating fluorescently labeled nucleotides are removed by washing. After that, fluorescence observation is performed by the optical system 100 to remove the fluorescent dye and the protecting group. By repeating the above steps as one cycle, the fluorescent color sequence is determined and the base sequence is determined.
　ステージ１０９は、サンプル基板１０１が対物レンズ１０３の光軸と直交するように、サンプル基板１０１を支持する。ステージ１０９は、図示しない駆動装置により少なくとも水平方向に移動可能に構成される。ステージ１０９は、サンプル基板１０１に接する箇所にヒートブロック等の温調機構を有していてもよく、必要に応じてサンプル基板１０１を加熱及び冷却することにより、伸長反応を促進することができる。また、ステージ１０９は、複数のサンプル基板１０１を同時に載置可能に構成されていてもよい。 The stage 109 supports the sample substrate 101 so that the sample substrate 101 is orthogonal to the optical axis of the objective lens 103. The stage 109 is configured to be movable at least in the horizontal direction by a drive device (not shown). The stage 109 may have a temperature control mechanism such as a heat block at a position in contact with the sample substrate 101, and the elongation reaction can be promoted by heating and cooling the sample substrate 101 as needed. Further, the stage 109 may be configured so that a plurality of sample substrates 101 can be mounted at the same time.
　光学系１００は、光源１０２、対物レンズ１０３、２次元センサ１０４Ａ（第１の２次元センサ）、２次元センサ１０４Ｂ（第２の２次元センサ）、ダイクロイックミラー１０５（光学素子）、フィルタユニット１０６Ａ（第１のフィルタユニット）、フィルタユニット１０６Ｂ（第２のフィルタユニット）、フィルタユニット切替機構１０７（第１の駆動装置）及び対物レンズ駆動装置１０８（第２の駆動装置）を有する。 The optical system 100 includes a light source 102, an objective lens 103, a two-dimensional sensor 104A (first two-dimensional sensor), a two-dimensional sensor 104B (second two-dimensional sensor), a dichroic mirror 105 (optical element), and a filter unit 106A ( It has a first filter unit), a filter unit 106B (second filter unit), a filter unit switching mechanism 107 (first drive device), and an objective lens drive device 108 (second drive device).
　光源１０２は、サンプルに結合された蛍光色素を励起可能な波長の励起光を含む光を出射する。光源１０２としては例えばＸｅランプや白色ＬＥＤを用いることができ、１種類の光源だけでなく、例えば複数種類のＬＥＤを組み合わせた光源であってもよい。 The light source 102 emits light including excitation light having a wavelength capable of exciting the fluorescent dye bound to the sample. As the light source 102, for example, an Xe lamp or a white LED can be used, and not only one type of light source but also, for example, a light source in which a plurality of types of LEDs are combined may be used.
　フィルタユニット１０６Ａは、光源１０２からの励起光を透過する透過フィルタ１１１Ａ、励起光を反射してサンプル基板１０１に入射させ、サンプルからの蛍光を透過するダイクロイックミラー１１２Ａ、サンプルからの蛍光のみを透過する蛍光フィルタ１１３Ａを有する。透過フィルタ１１１Ａ及び蛍光フィルタ１１３Ａにより、サンプルを効率よく励起するとともにサンプルから発生した蛍光以外の成分を除去することができ、これにより、得られる蛍光画像のコントラストが上昇する。フィルタユニット１０６Ｂについても同様に、透過フィルタ１１１Ｂ、ダイクロイックミラー１１２Ｂ及び蛍光フィルタ１１３Ｂを有する。フィルタユニット１０６Ａ及び１０６Ｂは、対象とする蛍光体の種類が異なっており、透過させる蛍光の波長及び遮断する波長が異なっている。また、対象とする蛍光体の蛍光を透過させる性能（能力）及び当該蛍光以外の光を遮断する性能については差異が無い。 The filter unit 106A is a transmission filter 111A that transmits the excitation light from the light source 102, a dichroic mirror 112A that reflects the excitation light and is incident on the sample substrate 101, and transmits only the fluorescence from the sample. It has a fluorescent filter 113A. The transmission filter 111A and the fluorescence filter 113A can efficiently excite the sample and remove components other than fluorescence generated from the sample, thereby increasing the contrast of the obtained fluorescence image. Similarly, the filter unit 106B has a transmission filter 111B, a dichroic mirror 112B, and a fluorescence filter 113B. The filter units 106A and 106B have different types of target phosphors, and have different wavelengths of fluorescence to be transmitted and wavelengths to be blocked. In addition, there is no difference in the performance (ability) of transmitting the fluorescence of the target phosphor and the performance of blocking light other than the fluorescence.
　フィルタユニット切替機構１０７は、フィルタユニット１０６Ａ及び１０６Ｂの位置を変更可能に構成され、これらのいずれか一方を対物レンズ１０３の光軸上に配置する。フィルタユニット切替機構１０７の駆動機構としては、例えばモータ又はソレノイドなどを用いることができる。 The filter unit switching mechanism 107 is configured so that the positions of the filter units 106A and 106B can be changed, and one of these is arranged on the optical axis of the objective lens 103. As the drive mechanism of the filter unit switching mechanism 107, for example, a motor or a solenoid can be used.
　ダイクロイックミラー１０５は、対物レンズ１０３の光軸上に配置され、フィルタユニット１０６Ａ又は１０６Ｂを通過した蛍光を２次元センサ１０４Ａ及び１０４Ｂへ入射させる。具体的には、ダイクロイックミラー１０５を透過した蛍光は２次元センサ１０４Ａに結像され、ダイクロイックミラー１０５により反射された蛍光は、２次元センサ１０４Ｂに結像される。ダイクロイックミラー１０５は、対物レンズ１０３の光軸に対して例えば４５°の角度で配置される。なお、ダイクロイックミラー１０５の代わりに、ハーフミラー、ビームスプリッタ、コールドミラーなどの他の光学素子を用いてもよい。 The dichroic mirror 105 is arranged on the optical axis of the objective lens 103, and the fluorescence that has passed through the filter unit 106A or 106B is incident on the two- dimensional sensors 104A and 104B. Specifically, the fluorescence transmitted through the dichroic mirror 105 is imaged on the two-dimensional sensor 104A, and the fluorescence reflected by the dichroic mirror 105 is imaged on the two-dimensional sensor 104B. The dichroic mirror 105 is arranged at an angle of, for example, 45 ° with respect to the optical axis of the objective lens 103. Instead of the dichroic mirror 105, other optical elements such as a half mirror, a beam splitter, and a cold mirror may be used.
　２次元センサ１０４Ａ及び１０４Ｂは、センサ面に入射した蛍光の画像を取得し、該蛍光画像を制御部１１０に送信する。２次元センサ１０４Ａ及び１０４Ｂとしては、例えばＣＣＤカメラやＣＭＯＳカメラを用いることができる。 The two- dimensional sensors 104A and 104B acquire an image of fluorescence incident on the sensor surface and transmit the fluorescence image to the control unit 110. As the two- dimensional sensors 104A and 104B, for example, a CCD camera or a CMOS camera can be used.
　対物レンズ駆動装置１０８は対物レンズ１０３に接続されており、対物レンズ１０３を鉛直方向に移動可能に構成される。これにより、対物レンズ１０３とサンプル基板１０１との距離を調節することが可能である。対物レンズ駆動装置１０８は、例えばステッピングモータ、対物レンズ１０３に固定されるステージ及びパルス発振器等を有する。なお、対物レンズ駆動装置１０８を設ける代わりに、ステージ１０９を水平方向だけでなく鉛直方向にも駆動可能な駆動装置を用いてもよい。 The objective lens driving device 108 is connected to the objective lens 103, and the objective lens 103 is configured to be movable in the vertical direction. This makes it possible to adjust the distance between the objective lens 103 and the sample substrate 101. The objective lens driving device 108 includes, for example, a stepping motor, a stage fixed to the objective lens 103, a pulse oscillator, and the like. Instead of providing the objective lens driving device 108, a driving device capable of driving the stage 109 not only in the horizontal direction but also in the vertical direction may be used.
　制御部１１０は、光源１０２による光の照射、フィルタユニット切替機構１０７による切り替え、２次元センサ１０４Ａ及び１０４Ｂによる撮像、対物レンズ駆動装置１０８の駆動、ステージ１０９の駆動装置（不図示）の駆動、反応液の送液機構（不図示）の駆動などを制御する。また、制御部１１０は、２次元センサ１０４Ａ及び１０４Ｂが取得した蛍光画像に基づいて、核酸の塩基配列を解析する処理を実行する。図示は省略しているが、制御部１１０は、分析装置の各構成要素の駆動や解析処理を実行するためのプログラム及び各種データを格納する記憶部、該プログラム及び各種データを読み出して上記動作を実行するプロセッサ、ユーザがデータや指示を入力するための入力部等を有する。 The control unit 110 irradiates light by the light source 102, switches by the filter unit switching mechanism 107, captures images by the two- dimensional sensors 104A and 104B, drives the objective lens driving device 108, drives the driving device (not shown) of the stage 109, and reacts. Controls the drive of the liquid feeding mechanism (not shown). In addition, the control unit 110 executes a process of analyzing the base sequence of nucleic acid based on the fluorescence images acquired by the two- dimensional sensors 104A and 104B. Although not shown, the control unit 110 reads a program for driving each component of the analyzer and executing analysis processing and a storage unit for storing various data, and reads the program and various data to perform the above operation. It has a processor to execute, an input unit for the user to input data and instructions, and the like.
＜分析方法の一例＞
　本実施形態の分析装置を用いて分析対象の核酸の塩基配列を解析する方法について説明する。本分析方法においては、第１の蛍光～第４の蛍光をそれぞれ発する４色の蛍光色素（第１の蛍光色素～第４の蛍光色素）で標識された蛍光標識ヌクレオチドを１塩基ずつサンプルに結合させて撮像し、塩基配列を解析する。本実施形態において、フィルタユニット１０６Ａは第１の蛍光及び第２の蛍光を分離し、フィルタユニット１０６Ｂは第３の蛍光及び第４の蛍光を分離することとする。 <Example of analysis method>
A method of analyzing the base sequence of the nucleic acid to be analyzed using the analyzer of the present embodiment will be described. In this analysis method, fluorescently labeled nucleotides labeled with four color fluorescent dyes (first fluorescent dye to fourth fluorescent dye) that emit the first fluorescence to the fourth fluorescence are bound to the sample one base at a time. The image is taken and the base sequence is analyzed. In the present embodiment, the filter unit 106A separates the first fluorescence and the second fluorescence, and the filter unit 106B separates the third fluorescence and the fourth fluorescence.
　図２は、本実施形態に係る分析方法を示すフローチャートである。分析装置の動作を開始する前に、ユーザは、予めサンプル基板１０１に対し分析対象の核酸（サンプル）を固定し、蛍光標識ヌクレオチドやポリメラーゼなどの試薬を送液機構に収容しておく。また、制御部１１０の記憶部には、第１の蛍光色素～第４の蛍光色素の種類、第１の蛍光～第４の蛍光の合焦位置、配列の解析対象となる塩基数が記憶されている。 FIG. 2 is a flowchart showing an analysis method according to the present embodiment. Before starting the operation of the analyzer, the user immobilizes the nucleic acid (sample) to be analyzed on the sample substrate 101 in advance, and stores reagents such as fluorescently labeled nucleotides and polymerases in the liquid feeding mechanism. Further, in the storage unit of the control unit 110, the types of the first fluorescent dye to the fourth fluorescent dye, the focusing positions of the first fluorescence to the fourth fluorescence, and the number of bases to be analyzed in the sequence are stored. ing.
　ステップＳ１において、制御部１１０は、サンプル基板１０１をステージ１０９上に載置して、対物レンズ１０３の光軸上にサンプル基板１０１の観察位置が位置するようにステージ１０９を移動させる。その後、制御部１１０は、送液機構を駆動してサンプル基板１０１に反応液を導入し、サンプルに対し蛍光標識ヌクレオチドを１塩基だけ合成させる。 In step S1, the control unit 110 places the sample substrate 101 on the stage 109 and moves the stage 109 so that the observation position of the sample substrate 101 is located on the optical axis of the objective lens 103. After that, the control unit 110 drives the liquid feeding mechanism to introduce the reaction liquid into the sample substrate 101, and causes the sample to synthesize only one base of the fluorescently labeled nucleotide.
　ステップＳ２において、制御部１１０は、フィルタユニット切替機構１０７を駆動して、フィルタユニット１０６Ａを対物レンズ１０３の光軸上に配置する。 In step S2, the control unit 110 drives the filter unit switching mechanism 107 to arrange the filter unit 106A on the optical axis of the objective lens 103.
　ステップＳ３において、制御部１１０は、対物レンズ駆動装置１０８を駆動して、第１の蛍光の合焦位置と第２の蛍光の合焦位置との間に対物レンズ１０３の焦点が合うように、対物レンズ１０３の位置（第１の位置）を調整する。 In step S3, the control unit 110 drives the objective lens driving device 108 so that the objective lens 103 is focused between the focusing position of the first fluorescence and the focusing position of the second fluorescence. The position (first position) of the objective lens 103 is adjusted.
　ステップＳ４において、制御部１１０は、２次元センサ１０４Ａ及び１０４Ｂに撮像の指示を送信し、２次元センサ１０４Ａ及び１０４Ｂは、サンプル基板１０１の蛍光画像を撮像して、撮像した蛍光画像を制御部１１０に送信する。２次元センサ１０４Ａ及び１０４Ｂが同時に撮像することで撮像時間を短縮することができるが、別々に撮像してもよい。 In step S4, the control unit 110 transmits an imaging instruction to the two- dimensional sensors 104A and 104B, the two- dimensional sensors 104A and 104B image the fluorescence image of the sample substrate 101, and the captured fluorescence image is obtained by the control unit 110. Send to. The imaging time can be shortened by simultaneously imaging the two- dimensional sensors 104A and 104B, but they may be imaged separately.
　ステップＳ５において、制御部１１０は、フィルタユニット切替機構１０７を駆動して、フィルタユニット１０６Ｂを対物レンズ１０３の光軸上に配置する。なお、本実施形態においては先にフィルタユニット１０６Ａを用いて撮像を行ったが、フィルタユニット１０６Ａ及び１０６Ｂを用いる順番に限定はない。 In step S5, the control unit 110 drives the filter unit switching mechanism 107 to arrange the filter unit 106B on the optical axis of the objective lens 103. In the present embodiment, imaging is performed using the filter unit 106A first, but the order in which the filter units 106A and 106B are used is not limited.
　ステップＳ６において、制御部１１０は、対物レンズ駆動装置１０８を駆動して、第３の蛍光の合焦位置と第４の蛍光の合焦位置との間に対物レンズ１０３の焦点が合うように、対物レンズ１０３の位置（第２の位置）を調整する。 In step S6, the control unit 110 drives the objective lens driving device 108 so that the objective lens 103 is focused between the in-focus position of the third fluorescence and the in-focus position of the fourth fluorescence. The position (second position) of the objective lens 103 is adjusted.
　ステップＳ７において、２次元センサ１０４Ａ及び１０４Ｂは、サンプル基板１０１の蛍光画像を撮像し、撮像した蛍光画像を制御部１１０に送信する。 In step S7, the two- dimensional sensors 104A and 104B capture a fluorescence image of the sample substrate 101 and transmit the captured fluorescence image to the control unit 110.
　ステップＳ８において、制御部１１０は、ステップＳ４及びＳ７において撮像された４枚の蛍光画像に基づいて、第１の蛍光色素～第４の蛍光色素のいずれが結合されたかを判断し、塩基を同定する。塩基の同定は、例えば４色の蛍光のシグナル強度に基づいて行われる。また、制御部１１０は同定結果を記憶部に記憶する。 In step S8, the control unit 110 determines which of the first fluorescent dye to the fourth fluorescent dye is bound based on the four fluorescent images captured in steps S4 and S7, and identifies the base. To do. Base identification is performed, for example, based on the signal intensity of fluorescence of four colors. Further, the control unit 110 stores the identification result in the storage unit.
　ステップＳ９において、制御部１１０は、サンプルの最後の塩基についての同定であったかどうかを判断する。このとき、設定された解析対象の塩基数及び同定を実行した数に基づいて最後の塩基であるかを判断することができる。 In step S9, the control unit 110 determines whether or not the identification was for the last base of the sample. At this time, it is possible to determine whether the base is the last base based on the set number of bases to be analyzed and the number of times the identification is performed.
　ステップＳ９において最後の塩基ではないと判断された場合（Ｎｏ）、制御部１１０は、送液機構を駆動して洗浄液をサンプル基板１０１に導入し、蛍光色素と保護基を除去する。その後、ステージ１０９を駆動して、サンプル基板１０１の次の撮像位置を対物レンズ１０３の光軸上に配置し、ステップＳ１に戻る。 When it is determined in step S9 that the base is not the last base (No), the control unit 110 drives the liquid feeding mechanism to introduce the cleaning liquid into the sample substrate 101 and remove the fluorescent dye and the protecting group. After that, the stage 109 is driven to arrange the next imaging position of the sample substrate 101 on the optical axis of the objective lens 103, and the process returns to step S1.
　ステップＳ９において最後の塩基であると判断された場合（Ｙｅｓ）、制御部１１０は、動作を終了する。 If it is determined to be the last base in step S9 (Yes), the control unit 110 ends the operation.
　なお、ステップＳ３及びＳ６における２色の蛍光の組み合わせは、例えば以下のように設定される。すなわち、同じフィルタユニットを用いて撮像される２色の蛍光の合焦位置は近接しており、かつ、別のフィルタユニットを用いて撮像される蛍光の合焦位置とは離れている。 The combination of fluorescence of the two colors in steps S3 and S6 is set as follows, for example. That is, the in-focus positions of the fluorescence of the two colors imaged using the same filter unit are close to each other, and are far from the in-focus position of the fluorescence imaged by using another filter unit.
　一般的にレンズは、波長ごとに焦点距離が異なる軸上色収差によるデフォーカスが問題となる。この軸上色収差は、レンズ系を複雑にすることでキャンセルすることができるが、価格の増加や光学系の増大に伴い装置が大型化してしまう。 Generally, a lens has a problem of defocusing due to axial chromatic aberration in which the focal length differs for each wavelength. This axial chromatic aberration can be canceled by complicating the lens system, but the device becomes large as the price increases and the optical system increases.
　これに対し、本実施形態の分析装置は、２つのフィルタユニット及び２つの２次元センサを用い、２色の蛍光の合焦位置の間に焦点を合わせて撮像を行う。これにより、簡単な光学系においても軸上色収差によるデフォーカスを補償し、品質の良い画像を得ることができる。 On the other hand, the analyzer of the present embodiment uses two filter units and two two-dimensional sensors to focus and image between the focusing positions of the two colors of fluorescence. As a result, even in a simple optical system, defocus due to axial chromatic aberration can be compensated and a high-quality image can be obtained.
＜分析方法の他の例＞
　本実施形態の分析装置により高品質に蛍光観察できる色の数は４色に限定されない。以下においては、３色の蛍光色素を分析対象の核酸（一本鎖ＤＮＡなど）に結合させてその塩基配列を解析する方法について説明する。この場合、第１の蛍光～第３の蛍光をそれぞれ発する３色の蛍光色素（第１の蛍光色素～第３の蛍光色素）で標識された蛍光標識ヌクレオチドを用いる。 <Other examples of analysis methods>
The number of colors that can be observed with high quality fluorescence by the analyzer of the present embodiment is not limited to four colors. In the following, a method of binding a three-color fluorescent dye to a nucleic acid to be analyzed (such as single-stranded DNA) and analyzing its base sequence will be described. In this case, fluorescently labeled nucleotides labeled with three color fluorescent dyes (first fluorescent dye to third fluorescent dye) that emit the first fluorescence to the third fluorescence, respectively, are used.
　なお、核酸の塩基は４種類あるため、３色の蛍光では１種類の塩基が検出できない。そこで、４つのヌクレオチドのうち３つはそれぞれ第１の蛍光色素～第３の蛍光色素のいずれかにより標識されており、残りの１つのヌクレオチドは例えば第１の蛍光色素及び第２の蛍光色素の２つにより標識されていることとする。これにより、第１の蛍光及び第２の蛍光の両方が検出された場合、すなわち３色の蛍光のうち２色が同時に検出された場合に、４種類目の塩基と判定することができる。 Since there are four types of nucleic acid bases, one type of base cannot be detected by fluorescence of three colors. Therefore, three of the four nucleotides are each labeled with one of the first fluorescent dye to the third fluorescent dye, and the remaining one nucleotide is, for example, of the first fluorescent dye and the second fluorescent dye. It shall be labeled by two. Thereby, when both the first fluorescence and the second fluorescence are detected, that is, when two of the three colors of fluorescence are detected at the same time, it can be determined to be the fourth type of base.
　本分析方法においては、フィルタユニット１０６Ａにより第１の蛍光及び第２の蛍光を分離し、フィルタユニット１０６Ｂにより第３の蛍光を分離することとする。本方法は、上述の４色標識の場合の分析方法（図２）とほぼ同様であるが、ステップＳ６において、第３の蛍光の合焦位置のみに対物レンズ１０３の焦点が合うように対物レンズ１０３の位置（第２の位置）を調整する。その他のステップについては、４色標識の場合の分析方法と同じであるため説明を省略する。 In this analysis method, the first fluorescence and the second fluorescence are separated by the filter unit 106A, and the third fluorescence is separated by the filter unit 106B. This method is almost the same as the analysis method (FIG. 2) in the case of the above-mentioned four-color labeling, but in step S6, the objective lens so that the objective lens 103 is focused only on the focus position of the third fluorescence. The position of 103 (second position) is adjusted. Since the other steps are the same as the analysis method in the case of the four-color label, the description thereof will be omitted.
　なお、フィルタユニット１０６Ａにより第１の蛍光を分離し、フィルタユニット１０６Ｂにより第２の蛍光及び第３の蛍光を分離するようにしてもよい。この場合、フィルタユニット１０６Ａを用いる際に第１の蛍光の合焦位置に対物レンズ１０３の焦点を合わせて撮像し、フィルタユニット１０６Ｂを用いる際に第２の蛍光の合焦位置と第３の蛍光の合焦位置との間に焦点を合わせて撮像する。上述のように、同じフィルタユニットを用いて撮像される２色の蛍光の合焦位置が近接しており、かつ、別のフィルタユニットを用いて撮像される蛍光の合焦位置が離れていればよい。 The first fluorescence may be separated by the filter unit 106A, and the second fluorescence and the third fluorescence may be separated by the filter unit 106B. In this case, when the filter unit 106A is used, the objective lens 103 is focused on the in-focus position of the first fluorescence for imaging, and when the filter unit 106B is used, the in-focus position of the second fluorescence and the third fluorescence are taken. Focus on the in-focus position of the image. As described above, if the in-focus positions of the two colors of fluorescence imaged using the same filter unit are close to each other, and the in-focus positions of the fluorescence imaged by another filter unit are far from each other. Good.
　以上、３色の蛍光色素を用いて、４色目の塩基には２つの蛍光色素を結合させることにより当該塩基を検出する例について説明したが、４色目の塩基を検出する手法はこれに限定されない。例えば、蛍光の検出と、電気化学発光法などの蛍光検出以外の方法とを組み合わせることによって、４種類目の塩基を検出することもできる。 The example of detecting the base by binding two fluorescent dyes to the base of the fourth color using the fluorescent dyes of three colors has been described above, but the method of detecting the base of the fourth color is not limited to this. .. For example, a fourth type of base can be detected by combining fluorescence detection with a method other than fluorescence detection such as an electrochemical luminescence method.
＜技術的効果＞
　以上のように、本実施形態の分析装置は、２つの蛍光の合焦位置の間に焦点を合わせて２つの２次元センサで同時に撮像を行い、その後フィルタユニットを切り替えて同様に撮像を行う。このような構成により、軸上色収差によるデフォーカスを補償できるので、品質の良い蛍光画像を得ることができる。したがって、高精度で塩基を同定することができる。 <Technical effect>
As described above, the analyzer of the present embodiment focuses between the in-focus positions of the two fluorescences and simultaneously performs imaging with the two two-dimensional sensors, and then switches the filter unit to perform the same imaging. With such a configuration, defocus due to axial chromatic aberration can be compensated, so that a high-quality fluorescence image can be obtained. Therefore, the base can be identified with high accuracy.
　また、２色の蛍光を一度に撮像することで撮像時間を短縮できるので、スループットの低下を抑制することができる。 Further, since the imaging time can be shortened by imaging the fluorescence of two colors at once, the decrease in throughput can be suppressed.
［第２の実施形態］
　上述の第１の実施形態の分析装置においては、光学系１００に含まれるレンズが対物レンズ１０３のみである例について説明した。そこで第２の実施形態は、より高品質の蛍光画像を取得するために、光学系に結像レンズをさらに設け、対物レンズ１０３と結像レンズとの合成レンズ系を考慮した焦点合わせを行う分析装置を提案する。 [Second Embodiment]
In the analyzer of the first embodiment described above, an example in which the lens included in the optical system 100 is only the objective lens 103 has been described. Therefore, in the second embodiment, in order to acquire a higher quality fluorescence image, an imaging lens is further provided in the optical system, and focusing is performed in consideration of the composite lens system of the objective lens 103 and the imaging lens. Propose a device.
＜分析装置の構成＞
　図３は、第２の実施形態に係る分析装置の一部の構成を示す概略図である。第２の実施形態の分析装置の構成は、光学系２００が第１の実施形態の光学系１００と異なっているのみであるため、図３において光学系２００及びサンプル基板１０１以外の構成の図示を省略している。 <Analyzer configuration>
FIG. 3 is a schematic view showing a partial configuration of the analyzer according to the second embodiment. Since the configuration of the analyzer of the second embodiment is different from that of the optical system 100 of the first embodiment only in the optical system 200, the configurations other than the optical system 200 and the sample substrate 101 are shown in FIG. It is omitted.
　図３に示すように、本実施形態の光学系２００は、２次元センサ１０４Ａの手前に結像レンズ２０９Ａが設けられ、２次元センサ１０４Ｂの手前に結像レンズ２０９Ｂが設けられている。 As shown in FIG. 3, in the optical system 200 of the present embodiment, the imaging lens 209A is provided in front of the two-dimensional sensor 104A, and the imaging lens 209B is provided in front of the two-dimensional sensor 104B.
　結像レンズ２０９Ａ及び２０９Ｂとして同一のレンズを使用することでコストを低減できるが、これに限定されない。一般的に、結像レンズは２枚の貼り付けレンズで構成されており、近い波長の色収差が補償される。本実施形態では、結像レンズ２０９Ａ及び２０９Ｂにより第１の蛍光、第２の蛍光及び第３の蛍光のみの色収差が補償されることとする。 Cost can be reduced by using the same lens as the imaging lenses 209A and 209B, but the cost is not limited to this. Generally, the imaging lens is composed of two pasted lenses, and chromatic aberration of near wavelength is compensated. In the present embodiment, the imaging lenses 209A and 209B compensate for the chromatic aberration of only the first fluorescence, the second fluorescence, and the third fluorescence.
＜分析方法＞
　本実施形態の分析装置を用いた分析方法においては、対物レンズ１０３と結像レンズ２０９Ａとの合成レンズ系（第１の合成レンズ）、対物レンズ１０３と結像レンズ２０９Ｂとの合成レンズ系（第２の合成レンズ）を考慮して対物レンズ１０３の駆動が行われる。図３に示すように、第１の合成レンズ及び第２の合成レンズの入射側主平面２１３は、対物レンズ１０３の上方に位置する。第１の合成レンズの射出側主平面２１４Ａ（第１の射出側主平面）は結像レンズ２０９Ａの上方に位置し、第２の合成レンズの射出側主平面２１４Ｂ（第２の射出側主平面）は結像レンズ２０９Ｂの右側に位置する。 <Analysis method>
In the analysis method using the analyzer of the present embodiment, the composite lens system of the objective lens 103 and the imaging lens 209A (first composite lens) and the composite lens system of the objective lens 103 and the imaging lens 209B (first composite lens system). The objective lens 103 is driven in consideration of the composite lens of 2). As shown in FIG. 3, the incident side main plane 213 of the first composite lens and the second composite lens is located above the objective lens 103. The emission side main plane 214A (first emission side main plane) of the first synthetic lens is located above the imaging lens 209A, and the emission side main plane 214B (second emission side main plane) of the second composite lens. ) Is located on the right side of the imaging lens 209B.
　入射側主平面２１３とサンプル基板１０１の表面との距離をｚ、射出側主平面２１４Ａ及び２１４Ｂと蛍光の結像位置との距離をそれぞれｓ、合成焦点距離をｆ、射出側主平面２１４Ａと２次元センサ１０９Ａとの距離をｄＴ、射出側主平面２１４Ｂと２次元センサ１０９Ｂとの距離をｄＲとする。図３には、距離ｚ、ｄＴ及びｄＲを図示している。 The distance between the incident side main plane 213 and the surface of the sample substrate 101 is z, the distance between the emission side main planes 214A and 214B and the fluorescence imaging position is s, the combined focal distance is f, and the emission side main planes 214A and 2 Let dT be the distance from the dimensional sensor 109A, and dR be the distance between the injection side main plane 214B and the two-dimensional sensor 109B. FIG. 3 illustrates the distances z, dT and dR.
　第１の蛍光～第４の蛍光の色収差を考慮した合成焦点距離をそれぞれｆ１～ｆ４とし、、第１の蛍光～第４の蛍光の結像位置をそれぞれｓ１～ｓ４とすると、これらの関係は一般的にそれぞれ下記式（１）～（４）で表される。
　1/z + 1/s1 = 1/f1　…(1)
　1/z + 1/s2 = 1/f3　…(2)
　1/z + 1/s3 = 1/f2　…(3)
　1/z + 1/s4 = 1/f4　…(4) Assuming that the combined focal lengths considering the chromatic aberrations of the first fluorescence to the fourth fluorescence are f1 to f4, respectively, and the imaging positions of the first fluorescence to the fourth fluorescence are s1 to s4, respectively, these relationships are established. Generally, they are represented by the following equations (1) to (4), respectively.
1 / z + 1 / s1 = 1 / f1… (1)
1 / z + 1 / s2 = 1 / f3… (2)
1 / z + 1 / s3 = 1 / f2… (3)
1 / z + 1 / s4 = 1 / f4… (4)
　結像レンズ２０９Ａ及び２０９Ｂにより第１の蛍光、第２の蛍光及び第３の蛍光のみ色収差が補償されていることから、ｆ１≒ｆ２≒ｆ３ないし、ｓ１≒ｓ２≒ｓ３となる。 Since the chromatic aberration is compensated only for the first fluorescence, the second fluorescence, and the third fluorescence by the imaging lenses 209A and 209B, f1≈f2≈f3 or s1≈s2≈s3.
　ここで、対物レンズ１０３を光軸方向に移動させ、入射側主平面２１３とサンプル基板１０１の表面との距離ｚ（単に「距離ｚ」という場合がある）を変化させた場合のフォーカス値について説明する。一般的にレンズには、焦点深度と呼ばれる、画像の品質が変化しないデフォーカス距離があり、これを本明細書においては「デフォーカス許容範囲」と呼ぶ。つまり、デフォーカス許容範囲となるように撮像すれば、品質の良い蛍光画像が得られる。 Here, the focus value when the objective lens 103 is moved in the optical axis direction to change the distance z (sometimes simply referred to as “distance z”) between the incident side main plane 213 and the surface of the sample substrate 101 will be described. To do. Generally, a lens has a defocus distance called a depth of focus, which does not change the quality of an image, and this is referred to as a "defocus tolerance" in the present specification. That is, if the image is taken so as to be within the defocus allowable range, a high-quality fluorescent image can be obtained.
　図４は、一般的な撮像方法を説明するための、距離ｚを変化させた場合のフォーカス値をプロットしたグラフである。曲線４０１Ａは、フィルタユニット１０６Ａを用いた場合における、第１の合成レンズのフォーカス値をプロットした曲線である。曲線４０２Ａは、フィルタユニット１０６Ａを用いた場合における、第２の合成レンズのフォーカス値をプロットした曲線である。同様に、曲線４０１Ｂ及び４０２Ｂは、それぞれフィルタユニット１０６Ｂを用いた場合における、第１の合成レンズのフォーカス値をプロットした曲線、第２の合成レンズのフォーカス値をプロットした曲線である。 FIG. 4 is a graph plotting focus values when the distance z is changed to explain a general imaging method. The curve 401A is a curve plotting the focus value of the first synthetic lens when the filter unit 106A is used. The curve 402A is a curve plotting the focus value of the second synthetic lens when the filter unit 106A is used. Similarly, the curves 401B and 402B are a curve plotting the focus value of the first composite lens and a curve plotting the focus value of the second composite lens when the filter unit 106B is used, respectively.
　対物レンズ１０３とサンプル基板１０１との距離ｚは、一般的には単一波長（例えば第１の蛍光）に合わせてｄＴ＝ｄＲ＝ｓ１になるよう設定され、フィルタユニット１０６Ａを用いる場合とフィルタユニット１０６Ｂを用いる場合のいずれにおいても同じ距離ｚで撮像が行われる。換言すれば、図４に示すように、曲線４０１Ａが頂点となるような距離ｚで、全ての蛍光画像の撮像が行われる。この場合、第１の蛍光、第２の蛍光及び第３の蛍光のみ色収差が補償されていることから、第１～第３の蛍光についてはフォーカス値がデフォーカス許容範囲内となるので、蛍光画像の品質が良い（曲線４０１Ａ、４０２Ａ及び４０１Ｂ）。しかしながら、第４の蛍光の蛍光画像についてはフォーカス値がデフォーカス許容範囲未満となるので、品質の良い画質が得られない（曲線４０２Ｂ）。 The distance z between the objective lens 103 and the sample substrate 101 is generally set so that dT = dR = s1 according to a single wavelength (for example, the first fluorescence), and the case where the filter unit 106A is used and the case where the filter unit 106A is used and the filter unit In any case where 106B is used, imaging is performed at the same distance z. In other words, as shown in FIG. 4, all the fluorescence images are imaged at a distance z such that the curve 401A is the apex. In this case, since the chromatic aberration is compensated only for the first fluorescence, the second fluorescence, and the third fluorescence, the focus value for the first to third fluorescence is within the defocus allowable range, and thus the fluorescence image. Good quality (curves 401A, 402A and 401B). However, since the focus value of the fourth fluorescence image is less than the allowable defocus range, good image quality cannot be obtained (curve 402B).
　そこで本実施形態では、下記式（５）及び（６）を満たすように、対物レンズ駆動装置１０８を駆動して対物レンズ１０３の位置を調整する。
　min(s1, s2)≦dT≦max(s1, s2)　…(5)
　min(s3, s4)≦dR≦max(s3, s4)　…(6) Therefore, in the present embodiment, the objective lens driving device 108 is driven to adjust the position of the objective lens 103 so as to satisfy the following equations (5) and (6).
min (s1, s2) ≤ dT ≤ max (s1, s2)… (5)
min (s3, s4) ≤ dR ≤ max (s3, s4)… (6)
　換言すれば、ｓ１＞ｓ２の場合にはｓ１≧ｄＴ≧ｓ２、ｓ１＜ｓ２の場合にはｓ１≦ｄＴ≦ｓ２、ｓ３＞ｓ４の場合にはｓ３≧ｄＲ≧ｓ４、ｓ３＜ｓ４の場合にはｓ３≦ｄＲ≦ｓ４となるように、フィルタユニット１０６Ａを用いて撮像する際の対物レンズ１０３の位置と、フィルタユニット１０６Ｂを用いて撮像する際の対物レンズ１０３の位置を調整する。 In other words, s1 ≧ dT ≧ s2 when s1> s2, s1 ≦ dT ≦ s2 when s1 <s2, s3 ≧ dR ≧ s4 when s3> s4, and s3 <s4. The position of the objective lens 103 when taking an image using the filter unit 106A and the position of the objective lens 103 when taking an image using the filter unit 106B are adjusted so that s3 ≦ dR ≦ s4.
　さらに、下記式（７）及び（８）を満たすように、対物レンズ１０３の駆動により入射側主平面１１３とサンプル基板１０１との距離ｚをオフセットする。
　フィルタユニット１０６Ａを用いる場合の距離ｚ１（図２のステップＳ３）：
　1/z1=average(1/f1, 1/f3)-average(1/s1, 1/s3)　…(7)
　フィルタユニット１０６Ｂを用いる場合の距離ｚ２（図２のステップＳ６）：
　1/z2=average(1/f2, 1/f4)-average(1/f2, 1/f4)　…(8) Further, the distance z between the incident side main plane 113 and the sample substrate 101 is offset by driving the objective lens 103 so as to satisfy the following equations (7) and (8).
Distance z1 when the filter unit 106A is used (step S3 in FIG. 2):
1 / z1 = average (1 / f1, 1 / f3)-average (1 / s1, 1 / s3)… (7)
Distance z2 when the filter unit 106B is used (step S6 in FIG. 2):
1 / z2 = average (1 / f2, 1 / f4)-average (1 / f2, 1 / f4)… (8)
　すなわち、本実施形態においては図５のように撮像を行う。図５は、本実施形態の撮像方法を説明するための、距離ｚを変化させた場合のフォーカス値をプロットしたグラフである。図５に示すように、フィルタユニット１０６Ａを用いる場合、第１の合成レンズのフォーカス値をプロットした曲線５０１Ａと、第２の合成レンズのフォーカス値をプロットした曲線５０２Ａとが交差するように距離ｚ１が設定され、距離ｚ１となるような対物レンズ１０３の位置（第１の位置）で撮像される。フィルタユニット１０６Ｂを用いて撮像する場合も同様に、第１の合成レンズのフォーカス値をプロットした曲線５０１Ｂと、第２の合成レンズのフォーカス値をプロットした曲線５０２Ｂとが交差するように距離ｚ２が設定され、距離ｚ２となるような対物レンズ１０３の位置（第２の位置）で撮像される。これにより、各蛍光の蛍光画像がデフォーカス範囲内となるため、全ての蛍光のピントが合った蛍光画像を撮像できる。 That is, in the present embodiment, imaging is performed as shown in FIG. FIG. 5 is a graph in which focus values when the distance z is changed are plotted for explaining the imaging method of the present embodiment. As shown in FIG. 5, when the filter unit 106A is used, the distance z1 is such that the curve 501A plotting the focus value of the first composite lens and the curve 502A plotting the focus value of the second composite lens intersect. Is set, and the image is taken at the position (first position) of the objective lens 103 such that the distance is z1. Similarly, when imaging with the filter unit 106B, the distance z2 is such that the curve 501B plotting the focus value of the first composite lens and the curve 502B plotting the focus value of the second composite lens intersect. The image is taken at the position (second position) of the objective lens 103 that is set and has a distance z2. As a result, the fluorescence image of each fluorescence is within the defocus range, so that it is possible to capture a fluorescence image in which all the fluorescence is in focus.
＜技術的効果＞
　以上のように、本実施形態においては、対物レンズ１０３と結像レンズ２０９Ａとの合成レンズ系、並びに対物レンズ１０３と結像レンズ２０９Ｂとの合成レンズ系を考慮し、すべての蛍光についてデフォーカス許容範囲となるように、対物レンズ１０３の位置が調整される。これにより、第１の実施形態と比較してより高品質の画像が得られ、高精度で塩基を同定することができる。 <Technical effect>
As described above, in the present embodiment, defocusing is allowed for all fluorescence in consideration of the composite lens system of the objective lens 103 and the imaging lens 209A and the composite lens system of the objective lens 103 and the imaging lens 209B. The position of the objective lens 103 is adjusted so as to be within the range. As a result, a higher quality image can be obtained as compared with the first embodiment, and the base can be identified with high accuracy.
［第３の実施形態］
　上述のように、第２の実施形態においては、第１の蛍光～第４の蛍光の合焦位置が予め制御部１１０に記憶されており、これに基づいて対物レンズ１０３の焦点を合わせる例について説明した。これに対し、第３の実施形態は、オートフォーカスにより対物レンズ１０３の焦点を合わせる例について提案する。 [Third Embodiment]
As described above, in the second embodiment, the focusing positions of the first fluorescence to the fourth fluorescence are stored in the control unit 110 in advance, and based on this, the objective lens 103 is focused. explained. On the other hand, the third embodiment proposes an example in which the objective lens 103 is focused by autofocus.
＜分析装置の構成＞
　図６は、第３の実施形態における分析装置の一部の構成を示す概略図である。図６において、光学系３００及びサンプル基板１０１以外の構成については図示を省略している。図６に示すように、本実施形態の光学系３００は、オートフォーカス駆動系３１１及びオートフォーカス用のミラー３１０が設けられている。その他の構成については第２の実施形態と同様である。 <Analyzer configuration>
FIG. 6 is a schematic view showing a partial configuration of the analyzer according to the third embodiment. In FIG. 6, the configurations other than the optical system 300 and the sample substrate 101 are not shown. As shown in FIG. 6, the optical system 300 of the present embodiment is provided with an autofocus drive system 311 and a mirror 310 for autofocus. Other configurations are the same as in the second embodiment.
　ミラー３１０は、ダイクロイックミラー１０５の前方に設けられる。オートフォーカス駆動系３１１はミラー３１０にレーザー光を照射し、これによりミラー３１０により反射したレーザー光がサンプル基板１０１に入射する。また、オートフォーカス駆動系３１１は、サンプル基板１０１から反射したレーザー光を検出する。 The mirror 310 is provided in front of the dichroic mirror 105. The autofocus drive system 311 irradiates the mirror 310 with laser light, whereby the laser light reflected by the mirror 310 is incident on the sample substrate 101. Further, the autofocus drive system 311 detects the laser light reflected from the sample substrate 101.
　一般に蛍光試薬は可視光の波長のものが用いられること、また、赤外光は比較的サンプルに対するダメージが小さいことから、オートフォーカス駆動系３１１のレーザー光として赤外光を用い、ミラー３１０としては赤外光を反射するダイクロイックミラーを用いることができるが、これに限定されない。例えば、５３２ｎｍなどの可視光域のレーザー光をオートフォーカス駆動系３１１で用いた場合には、５３２ｎｍのみを反射するミラー３１０を用いることができる。 Generally, a fluorescent reagent having a wavelength of visible light is used, and infrared light has relatively little damage to the sample. Therefore, infrared light is used as the laser light of the autofocus drive system 311, and the mirror 310 is used as the mirror 310. A dichroic mirror that reflects infrared light can be used, but is not limited thereto. For example, when a laser beam in a visible light region such as 532 nm is used in the autofocus drive system 311, a mirror 310 that reflects only 532 nm can be used.
　オートフォーカス駆動系３１１によるレーザー光の照射は、制御部１１０により制御される。また、オートフォーカス駆動系３１１は、反射光の検出信号を制御部１１０に出力する。制御部１１０は、オートフォーカス駆動系３１１の検出信号に基づいて、対物レンズ駆動装置１０８を駆動する。 The irradiation of the laser beam by the autofocus drive system 311 is controlled by the control unit 110. Further, the autofocus drive system 311 outputs a detection signal of the reflected light to the control unit 110. The control unit 110 drives the objective lens driving device 108 based on the detection signal of the autofocus driving system 311.
　具体的には、オートフォーカス駆動系３１１は、対物レンズ１０３とサンプル基板１０１との距離ｚに比例した検出信号を出力する。この検出信号は、サンプルからの蛍光に対しピントが合っている場合に０となる。フィルタユニット１０６Ａを用いた撮像の場合、制御部１１０は、オートフォーカス駆動系３１１からの検出信号が、第１の合成レンズのフォーカス値と第２の合成レンズのフォーカス値が等しくなるような値（第１の目標値）となるように、対物レンズ１０３の位置を決定する。すなわち、入射側主平面２１３とサンプル基板１０１との距離が図５に示す距離ｚ１に調整され、撮像が行われる。フィルタユニット１０６Ｂを用いる場合も同様に、制御部１１０は、オートフォーカス駆動系３１１からの検出信号が、第１の合成レンズのフォーカス値と第２の合成レンズのフォーカス値が等しくなるような値（第２の目標値）となるように、対物レンズ１０３の位置を決定する。すなわち、入射側主平面２１３とサンプル基板１０１との距離が図５に示す距離ｚ２に調整され、撮像が行われる。第１の目標値及び第２の目標値は異なる値であり、第１～第４の蛍光の合焦位置などによって定まる。 Specifically, the autofocus drive system 311 outputs a detection signal proportional to the distance z between the objective lens 103 and the sample substrate 101. This detection signal becomes 0 when the fluorescence from the sample is in focus. In the case of imaging using the filter unit 106A, the control unit 110 sets the detection signal from the autofocus drive system 311 to a value such that the focus value of the first composite lens and the focus value of the second composite lens are equal ( The position of the objective lens 103 is determined so as to be the first target value). That is, the distance between the incident side main plane 213 and the sample substrate 101 is adjusted to the distance z1 shown in FIG. 5, and imaging is performed. Similarly, when the filter unit 106B is used, the control unit 110 sets the detection signal from the autofocus drive system 311 to a value such that the focus value of the first composite lens and the focus value of the second composite lens are equal (the focus value is equal to that of the second composite lens. The position of the objective lens 103 is determined so as to be the second target value). That is, the distance between the incident side main plane 213 and the sample substrate 101 is adjusted to the distance z2 shown in FIG. 5, and imaging is performed. The first target value and the second target value are different values, and are determined by the focusing positions of the first to fourth fluorescences and the like.
　なお、オートフォーカスの方式は上記のものに限定されず、その他の方式を採用することもできる。 The autofocus method is not limited to the above, and other methods can be adopted.
＜技術的効果＞
　以上のように、本実施形態の分析装置は、オートフォーカス駆動系３１１を用いて、第２の実施形態と同様に、すべての蛍光についてデフォーカス許容範囲となるように対物レンズ１０３の位置を調整して撮像を行う。このような構成によっても、第１の実施形態と比較してより高品質の画像が得られ、高精度で塩基を同定することができる。さらに、予め求められたベストフォーカス位置（例えば、図５に示した曲線５０１Ａ及び５０２Ａが交差するときの距離ｚ１）に対物レンズ１０３を駆動する場合、温度ドリフト等によって実際のベストフォーカス位置が変化してしまうと、デフォーカスしてしまう。一方、本実施形態のようにオートフォーカス駆動系３１１を用いることにより、実際のベストフォーカス位置に対物レンズ１０３の焦点を合わせることができるため、短時間で撮像される大量の画像について、より高精度にピントを合わせることができる。 <Technical effect>
As described above, the analyzer of the present embodiment uses the autofocus drive system 311 to adjust the position of the objective lens 103 so as to be within the defocus allowable range for all fluorescence, as in the second embodiment. And take an image. Even with such a configuration, a higher quality image can be obtained as compared with the first embodiment, and the base can be identified with high accuracy. Further, when the objective lens 103 is driven to the best focus position obtained in advance (for example, the distance z1 when the curves 501A and 502A shown in FIG. 5 intersect), the actual best focus position changes due to temperature drift or the like. If you do, it will be defocused. On the other hand, by using the autofocus drive system 311 as in the present embodiment, the objective lens 103 can be focused on the actual best focus position, so that a large number of images captured in a short time can be captured with higher accuracy. Can be focused on.
［第４の実施形態］
　上述の第３の実施形態においては、オートフォーカス駆動系３１１がレーザー光を射出している状態でサンプル基板１０１を撮像すると、２次元センサ１０４Ａ及び１０４Ｂにの画像にレーザー光が写ってしまう可能性がある。サンプルからの蛍光強度よりもレーザー光の強度のほうがはるかに高いため、レーザーが写った位置ではサンプルからの蛍光を撮影することは不可能である。そこで、第４の実施形態は、レーザーカットフィルタをさらに設けた光学系を提案する。 [Fourth Embodiment]
In the third embodiment described above, if the sample substrate 101 is imaged while the autofocus drive system 311 is emitting laser light, the laser light may be reflected in the images on the two- dimensional sensors 104A and 104B. There is. Since the intensity of the laser beam is much higher than the fluorescence intensity from the sample, it is impossible to capture the fluorescence from the sample at the position where the laser is projected. Therefore, the fourth embodiment proposes an optical system further provided with a laser cut filter.
＜分析装置の構成＞
　図７は、第４の実施形態における分析装置の一部の構成を示す概略図である。図７において、光学系４００及びサンプル基板１０１以外の構成については図示を省略している。図７に示すように、本実施形態の光学系４００は、ダイクロイックミラー１０５とミラー３１０との間にレーザーカットフィルタ４１２をさらに備える。その他の構成については第３の実施形態と同様である。 <Analyzer configuration>
FIG. 7 is a schematic view showing a partial configuration of the analyzer according to the fourth embodiment. In FIG. 7, the configurations other than the optical system 400 and the sample substrate 101 are not shown. As shown in FIG. 7, the optical system 400 of the present embodiment further includes a laser cut filter 412 between the dichroic mirror 105 and the mirror 310. Other configurations are the same as those in the third embodiment.
　レーザーカットフィルタ４１２として、オートフォーカス駆動系３１１が射出するレーザーの波長のみをカットするフィルタを用いることにより、レーザーが画像に映り込むことを防ぐことができる。また、オートフォーカス駆動系３１１が例えば赤外光のレーザーを照射する場合には、レーザーの波長だけでなく赤外光もカットするフィルタを用いることで、サンプルからの蛍光を防ぐことなく、レーザー光のみを除去することが可能となる。 By using a filter that cuts only the wavelength of the laser emitted by the autofocus drive system 311 as the laser cut filter 412, it is possible to prevent the laser from being reflected in the image. Further, when the autofocus drive system 311 irradiates a laser of infrared light, for example, by using a filter that cuts not only the wavelength of the laser but also the infrared light, the laser light is not prevented from fluorescence from the sample. Only can be removed.
＜技術的効果＞
　以上のように、本実施形態の分析装置は、オートフォーカス駆動系３１１が射出するレーザー光をカットするレーザーカットフィルタ４１２を有する。このような構成により、蛍光画像へのレーザー光の映り込みを避けることができ、安定した品質で蛍光画像を取得することができる。 <Technical effect>
As described above, the analyzer of the present embodiment has a laser cut filter 412 that cuts the laser light emitted by the autofocus drive system 311. With such a configuration, it is possible to avoid reflection of the laser beam on the fluorescence image, and it is possible to acquire the fluorescence image with stable quality.
［第５の実施形態］
　第１の実施形態～第４の実施形態においては、分析対象の核酸に３色又は４色の蛍光を標識して塩基配列を解析する核酸解析装置について説明した。しかし、核酸解析装置に限らず、複数色を検出する分析装置に対し各実施形態を適用することができる。例えば、６色の蛍光を検出する装置に本開示の構成を採用する場合は、追加のフィルタユニットを用いて（合計３つのフィルタユニット）、第５の蛍光の合焦位置と第６の蛍光の合焦位置との間に焦点を合わせて撮像する。これにより、６色の蛍光にピントが合った高品質の画像を取得することができる。このように、フィルタユニットをｎ個設けることにより、最大２ｎ色の蛍光画像を高品質で取得することができる。 [Fifth Embodiment]
In the first to fourth embodiments, a nucleic acid analysis apparatus for analyzing a base sequence by labeling a nucleic acid to be analyzed with fluorescence of three or four colors has been described. However, each embodiment can be applied not only to a nucleic acid analyzer but also to an analyzer that detects a plurality of colors. For example, when the configuration of the present disclosure is adopted for a device that detects fluorescence of six colors, an additional filter unit is used (three filter units in total), and the focus position of the fifth fluorescence and the focus position of the sixth fluorescence are used. Focus on the focus position and take an image. As a result, it is possible to acquire a high-quality image in which the fluorescence of the six colors is in focus. By providing n filter units in this way, it is possible to acquire a fluorescence image of a maximum of 2n colors with high quality.
［変形例］
　本開示は、上述した実施形態に限定されるものでなく、様々な変形例を含んでいる。例えば、上述した実施形態は、本開示を分かりやすく説明するために詳細に説明したものであり、必ずしも説明した全ての構成を備える必要はない。また、ある実施形態の一部を他の実施形態の構成に置き換えることができる。また、ある実施形態の構成に他の実施形態の構成を加えることもできる。また、各実施形態の構成の一部について、他の実施形態の構成の一部を追加、削除又は置換することもできる。 [Modification example]
The present disclosure is not limited to the embodiments described above, but includes various modifications. For example, the above-described embodiment has been described in detail in order to explain the present disclosure in an easy-to-understand manner, and does not necessarily have all the configurations described. In addition, a part of one embodiment can be replaced with the configuration of another embodiment. It is also possible to add the configuration of another embodiment to the configuration of one embodiment. It is also possible to add, delete, or replace a part of the configuration of another embodiment with respect to a part of the configuration of each embodiment.
１００、２００、３００、４００…光学系
１０１…サンプル基板
１０２…光源
１０３…対物レンズ
１０４Ａ、１０４Ｂ…２次元センサ
１０５…ダイクロイックミラー
１０６Ａ、１０６Ｂ…フィルタユニット
１０７…フィルタユニット切替機構
１０８…対物レンズ駆動装置
１０９Ａ、１０９Ｂ…結像レンズ
１１０…制御部
１１１Ａ、１１１Ｂ…透過フィルタ
１１２Ａ、１１２Ｂ…ダイクロイックミラー
１１３Ａ、１１３Ｂ…蛍光フィルタ
２０９Ａ、２０９Ｂ…結像レンズ
２１３…入射側主平面
２１４Ａ、２１４Ｂ…射出側主平面
３１０…オートフォーカス用ミラー
３１１…オートフォーカス駆動系
４１２…レーザーカットフィルタ 100, 200, 300, 400 ... Optical system 101 ... Sample substrate 102 ... Light source 103 ... Objective lens 104A, 104B ... Two-dimensional sensor 105 ... Dycroic mirror 106A, 106B ... Filter unit 107 ... Filter unit switching mechanism 108 ... Objective lens driving device 109A, 109B ... Imaging lens 110 ... Control unit 111A, 111B ... Transmission filter 112A, 112B ... Dycroic mirror 113A, 113B ... Fluorescent filter 209A, 209B ... Imaging lens 213 ... Incident side main plane 214A, 214B ... Ejection side main plane 310 ... Mirror for autofocus 311 ... Autofocus drive system 412 ... Laser cut filter

Claims (14)

  1. 　少なくとも第１の蛍光、第２の蛍光及び第３の蛍光を発するサンプル基板が載置されるステージと、
    　対物レンズと、
    　前記第１の蛍光及び前記第２の蛍光を分離する第１のフィルタユニットと、
    　前記第３の蛍光を分離する第２のフィルタユニットと、
    　前記サンプル基板からの前記第１の蛍光、前記第２の蛍光及び前記第３の蛍光を撮像する第１の２次元センサ及び第２の２次元センサと、
    　前記第１の２次元センサ及び前記第２の２次元センサに前記第１の蛍光、前記第２の蛍光及び前記第３の蛍光をそれぞれ分離する光学素子と、
    　前記第１のフィルタユニット及び前記第２のフィルタユニットのいずれか一方を用いて前記撮像が行われるようにこれらを切り替える第１の駆動装置と、
    　前記対物レンズと前記サンプル基板との距離を相対的に変化させる第２の駆動装置と、
    　前記第１の２次元センサ、前記第２の２次元センサ、前記第１の駆動装置及び前記第２の駆動装置を少なくとも制御する制御部と、を備え、
    　前記制御部は、
    　前記第１のフィルタユニットを用いて前記撮像を行う場合と前記第２のフィルタユニットを用いて前記撮像を行う場合とで、それぞれ前記対物レンズと前記サンプル基板との距離を調整するように、前記第１の駆動装置及び前記第２の駆動装置を制御することを特徴とする分析装置。     A stage on which a sample substrate that emits at least the first fluorescence, the second fluorescence, and the third fluorescence is placed, and
    With the objective lens
    A first filter unit that separates the first fluorescence and the second fluorescence,
    With the second filter unit that separates the third fluorescence,
    A first two-dimensional sensor and a second two-dimensional sensor that image the first fluorescence, the second fluorescence, and the third fluorescence from the sample substrate.
    An optical element that separates the first fluorescence, the second fluorescence, and the third fluorescence into the first two-dimensional sensor and the second two-dimensional sensor, respectively.
    A first drive device that switches between the first filter unit and the second filter unit so that the imaging can be performed.
    A second driving device that relatively changes the distance between the objective lens and the sample substrate,
    The first two-dimensional sensor, the second two-dimensional sensor, the first driving device, and a control unit that at least controls the second driving device are provided.
    The control unit
    The distance between the objective lens and the sample substrate is adjusted so as to adjust the distance between the objective lens and the sample substrate in the case where the image pickup is performed using the first filter unit and the case where the image pickup is performed using the second filter unit. An analyzer characterized by controlling a first drive device and the second drive device.
  2. 　請求項１の分析装置であって、
    　前記制御部は、
    　前記第１のフィルタユニットを用いて前記撮像を行う場合に、前記第１の蛍光の合焦位置と前記第２の蛍光の合焦位置との間に前記対物レンズの焦点を合わせ、
    　前記第２のフィルタユニットを用いて前記撮像を行う場合に、前記第３の蛍光の合焦位置に前記対物レンズの焦点を合わせるように、前記第１の駆動装置及び前記第２の駆動装置を制御することを特徴とする分析装置。     The analyzer according to claim 1
    The control unit
    When the imaging is performed using the first filter unit, the objective lens is focused between the focusing position of the first fluorescence and the focusing position of the second fluorescence.
    When the image pickup is performed using the second filter unit, the first drive device and the second drive device are set so as to focus the objective lens on the focus position of the third fluorescence. An analyzer characterized by being controlled.
  3. 　請求項２の分析装置であって、
    　前記第１の蛍光の合焦位置と前記第２の蛍光の合焦位置とが近接しており、前記第１の蛍光の合焦位置及び前記第２の蛍光の合焦位置と、前記第３の蛍光の合焦位置とが離れていることを特徴とする分析装置。     The analyzer according to claim 2
    The in-focus position of the first fluorescence and the in-focus position of the second fluorescence are close to each other, and the in-focus position of the first fluorescence, the in-focus position of the second fluorescence, and the third An analyzer characterized in that it is far from the in-focus position of the fluorescence of.
  4. 　少なくとも第１の蛍光、第２の蛍光、第３の蛍光及び第４の蛍光を発するサンプル基板が載置されるステージと、
    　対物レンズと、
    　前記第１の蛍光及び前記第２の蛍光を分離する第１のフィルタユニットと、
    　前記第３の蛍光及び前記第４の蛍光を分離する第２のフィルタユニットと、
    　前記サンプル基板からの前記第１～第４の蛍光を撮像する第１の２次元センサ及び第２の２次元センサと、
    　前記第１の２次元センサ及び前記第２の２次元センサに前記第１～第４の蛍光をそれぞれ分離する光学素子と、
    　前記第１のフィルタユニット及び第２のフィルタユニットのいずれか一方を用いて前記撮像が行われるようにこれらを切り替える第１の駆動装置と、
    　前記対物レンズと前記サンプル基板との距離を相対的に変化させる第２の駆動装置と、
    　前記第１の２次元センサ、前記第２の２次元センサ、前記第１の駆動装置及び前記第２の駆動装置を少なくとも制御する制御部と、を備え、
    　前記制御部は、
    　前記第１のフィルタユニットを用いて前記撮像を行う場合と前記第２のフィルタユニットを用いて前記撮像を行う場合とで、それぞれ前記対物レンズと前記サンプル基板との距離を調整するように、前記第１の駆動装置及び前記第２の駆動装置を制御することを特徴とする分析装置。     A stage on which a sample substrate that emits at least the first fluorescence, the second fluorescence, the third fluorescence, and the fourth fluorescence is placed, and
    With the objective lens
    A first filter unit that separates the first fluorescence and the second fluorescence,
    A second filter unit that separates the third fluorescence and the fourth fluorescence, and
    A first two-dimensional sensor and a second two-dimensional sensor that image the first to fourth fluorescence from the sample substrate, and
    An optical element that separates the first to fourth fluorescences into the first two-dimensional sensor and the second two-dimensional sensor, respectively.
    A first drive device that switches between the first filter unit and the second filter unit so that the imaging can be performed.
    A second driving device that relatively changes the distance between the objective lens and the sample substrate,
    The first two-dimensional sensor, the second two-dimensional sensor, the first driving device, and a control unit that at least controls the second driving device are provided.
    The control unit
    The distance between the objective lens and the sample substrate is adjusted so as to adjust the distance between the objective lens and the sample substrate in the case where the image pickup is performed using the first filter unit and the case where the image pickup is performed using the second filter unit. An analyzer characterized by controlling a first drive device and the second drive device.
  5. 　請求項４の分析装置であって、
    　前記制御部は、
    　前記第１のフィルタユニットを用いて前記撮像を行う際に、前記第１の蛍光の合焦位置と前記第２の蛍光の合焦位置との間に前記対物レンズの焦点を合わせ、
    　前記第２のフィルタユニットを用いて前記撮像を行う際に、前記第３の蛍光の合焦位置と前記第４の蛍光の合焦位置との間に前記対物レンズの焦点を合わせるように、前記第１の駆動装置及び前記第２の駆動装置を制御することを特徴とする分析装置。     The analyzer according to claim 4
    The control unit
    When performing the imaging using the first filter unit, the objective lens is focused between the focusing position of the first fluorescence and the focusing position of the second fluorescence.
    When performing the imaging using the second filter unit, the objective lens is focused so as to be focused between the in-focus position of the third fluorescence and the in-focus position of the fourth fluorescence. An analyzer characterized by controlling a first drive device and the second drive device.
  6. 　請求項５の分析装置であって、
    　前記第１の蛍光の合焦位置と前記第２の蛍光の合焦位置とが近接しており、前記第３の蛍光の合焦位置と前記第４の蛍光の合焦位置とが近接していることを特徴とする分析装置。     The analyzer according to claim 5.
    The focusing position of the first fluorescence and the focusing position of the second fluorescence are close to each other, and the focusing position of the third fluorescence and the focusing position of the fourth fluorescence are close to each other. An analyzer characterized by being present.
  7. 　請求項４の分析装置であって、
    　前記第１の２次元センサの前段に配置される第１の結像レンズと、
    　前記第２の２次元センサの前段に配置される第２の結像レンズと、をさらに備え、
    　前記対物レンズ及び前記第１の結像レンズで構成される第１の合成レンズの第１の出射側主平面、もしくは前記対物レンズ及び前記第２の結像レンズで構成される第２の合成レンズの第２の出射側主平面と、前記第１～第４の蛍光の結像位置との距離をそれぞれｓ１～ｓ４とし、
    　前記第１の出射側主平面と前記第１の２次元センサとの距離をｄＴとし、前記第２の出射側主平面と前記第２の２次元センサとの距離をｄＲとするとき、
    　前記制御部は、
    　ｓ１＞ｓ２の場合にはｓ１≧ｄＴ≧ｓ２、ｓ１＜ｓ２の場合にはｓ１≦ｄＴ≦ｓ２、ｓ３＞ｓ４の場合にはｓ３≧ｄＲ≧ｓ４、ｓ３＜ｓ４の場合にはｓ３≦ｄＲ≦ｓ４を満たすように、前記第２の駆動装置を制御することを特徴とする分析装置。     The analyzer according to claim 4
    A first imaging lens arranged in front of the first two-dimensional sensor and
    A second imaging lens arranged in front of the second two-dimensional sensor is further provided.
    The first emission side main plane of the first composite lens composed of the objective lens and the first imaging lens, or the second composite lens composed of the objective lens and the second imaging lens. The distance between the second main plane on the exit side and the imaging position of the first to fourth fluorescence is s1 to s4, respectively.
    When the distance between the first emission side main plane and the first two-dimensional sensor is dT, and the distance between the second emission side main plane and the second two-dimensional sensor is dR,
    The control unit
    When s1> s2, s1 ≧ dT ≧ s2, when s1 <s2, s1 ≦ dT ≦ s2, when s3> s4, s3 ≧ dR ≧ s4, and when s3 <s4, s3 ≦ dR ≦ An analyzer characterized in that the second drive device is controlled so as to satisfy s4.
  8. 　請求項４の分析装置であって、
    　前記第１の２次元センサの前段に配置される第１の結像レンズと、
    　前記第２の２次元センサの前段に配置される第２の結像レンズと、をさらに備え、
    　前記対物レンズ及び前記第１の結像レンズで構成される第１の合成レンズの第１の出射側主平面、もしくは前記対物レンズ及び前記第２の結像レンズで構成される第２の合成レンズの第２の出射側主平面と、前記第１～第４の蛍光の結像位置との距離をそれぞれｓ１～ｓ４とし、
    　前記第１の合成レンズ又は前記第２の合成レンズにおける、前記第１～第４の蛍光の波長に対する焦点距離をそれぞれｆ１～ｆ４とし、
    　前記第１のフィルタユニットを用いて前記撮像を行う場合の、前記第１及び第２の合成レンズの入射側主平面と前記サンプル基板との距離をｚ１とし、前記第２のフィルタユニットを用いて前記撮像を行う場合の、前記入射側主平面と前記サンプル基板との距離をｚ２としたとき、
    　前記制御部は、
    　1/z1=1/2(1/f1+1/f3)-1/2(1/s1+1/s3)
    　1/z2=1/2(1/f2+1/f4)-1/2(1/s2+1/s4)
    　となるように前記第２の駆動装置を制御することを特徴とする分析装置。     The analyzer according to claim 4
    A first imaging lens arranged in front of the first two-dimensional sensor and
    A second imaging lens arranged in front of the second two-dimensional sensor is further provided.
    The first emission side main plane of the first composite lens composed of the objective lens and the first imaging lens, or the second composite lens composed of the objective lens and the second imaging lens. The distance between the second main plane on the exit side and the imaging position of the first to fourth fluorescence is s1 to s4, respectively.
    The focal lengths of the first synthetic lens or the second synthetic lens with respect to the wavelengths of the first to fourth fluorescences are set to f1 to f4, respectively.
    When the imaging is performed using the first filter unit, the distance between the incident side main plane of the first and second composite lenses and the sample substrate is z1, and the second filter unit is used. When the distance between the incident side main plane and the sample substrate in the case of performing the imaging is z2,
    The control unit
    1 / z1 = 1/2 (1 / f1 + 1 / f3)-1 / 2 (1 / s1 + 1 / s3)
    1 / z2 = 1/2 (1 / f2 + 1 / f4)-1 / 2 (1 / s2 + 1 / s4)
    An analyzer characterized in that the second driving device is controlled so as to be.
  9. 　請求項４の分析装置であって、
    　前記サンプル基板にレーザー光を照射し、前記レーザー光の反射光を検出するオートフォーカス駆動系をさらに備え、
    　前記制御部は、前記オートフォーカス駆動系の検出信号に基づいて前記第２の駆動装置を制御することを特徴とする分析装置。     The analyzer according to claim 4
    The sample substrate is further provided with an autofocus drive system that irradiates the sample substrate with laser light and detects the reflected light of the laser light.
    The control unit is an analysis device that controls the second drive device based on a detection signal of the autofocus drive system.
  10. 　サンプル基板から少なくとも第１の蛍光、第２の蛍光、第３の蛍光及び第４の蛍光が発せられるように、前記サンプル基板に対し光源から光を照射することと、
    　第１の駆動装置により第１のフィルタユニットを対物レンズの光軸上に配置して、前記第１のフィルタユニットにより前記第１の蛍光及び前記第２の蛍光を分離することと、
    　光学素子により前記第１の蛍光及び前記第２の蛍光を第１の２次元センサ及び第２の２次元センサに分離することと、
    　第２の駆動装置により、前記対物レンズを第１の位置に調整することと、
    　前記第１の２次元センサ及び前記第２の２次元センサにより前記第１の蛍光及び前記第２の蛍光の画像を撮像することと、
    　前記第１の駆動装置により第２のフィルタユニットを前記対物レンズの光軸上に配置して、前記第２のフィルタユニットにより前記第３の蛍光及び前記第４の蛍光を分離することと、
    　前記光学素子により前記第３の蛍光及び前記第４の蛍光を前記第１の２次元センサ及び前記第２の２次元センサに分離することと、
    　前記第２の駆動装置により、前記対物レンズを第２の位置に調整することと、
    　前記第１の２次元センサ及び前記第２の２次元センサにより前記第３の蛍光及び前記第４の蛍光の画像を撮像することと、を含む分析方法。     Irradiating the sample substrate with light from a light source so that at least the first fluorescence, the second fluorescence, the third fluorescence, and the fourth fluorescence are emitted from the sample substrate.
    The first drive device arranges the first filter unit on the optical axis of the objective lens, and the first filter unit separates the first fluorescence and the second fluorescence.
    Separation of the first fluorescence and the second fluorescence into a first two-dimensional sensor and a second two-dimensional sensor by an optical element, and
    Adjusting the objective lens to the first position by the second driving device, and
    Taking images of the first fluorescence and the second fluorescence by the first two-dimensional sensor and the second two-dimensional sensor, and
    A second filter unit is arranged on the optical axis of the objective lens by the first driving device, and the third fluorescence and the fourth fluorescence are separated by the second filter unit.
    The optical element separates the third fluorescence and the fourth fluorescence into the first two-dimensional sensor and the second two-dimensional sensor.
    Adjusting the objective lens to the second position by the second driving device, and
    An analysis method including capturing images of the third fluorescence and the fourth fluorescence by the first two-dimensional sensor and the second two-dimensional sensor.
  11. 　請求項１０の分析方法であって、
    　前記対物レンズを前記第１の位置に調整することは、前記第１の蛍光の合焦位置と前記第２の蛍光の合焦位置との間に前記対物レンズの焦点を合わせることを含み、
    　前記対物レンズを前記第２の位置に調整することは、前記第３の蛍光の合焦位置と前記第４の蛍光の合焦位置との間に前記対物レンズの焦点を合わせることを含むことを特徴とする分析方法。     The analysis method of claim 10.
    Adjusting the objective lens to the first position includes focusing the objective lens between the focus position of the first fluorescence and the focus position of the second fluorescence.
    Adjusting the objective lens to the second position includes focusing the objective lens between the focus position of the third fluorescence and the focus position of the fourth fluorescence. Characteristic analysis method.
  12. 　請求項１１の分析方法であって、
    　前記第１の蛍光の合焦位置と前記第２の蛍光の合焦位置とが近接しており、前記第３の蛍光の合焦位置と前記第４の蛍光の合焦位置とが近接していることを特徴とする分析方法。     The analysis method of claim 11.
    The focusing position of the first fluorescence and the focusing position of the second fluorescence are close to each other, and the focusing position of the third fluorescence and the focusing position of the fourth fluorescence are close to each other. An analysis method characterized by being present.
  13. 　請求項１０の分析方法であって、
    　前記第１の２次元センサの前段に配置される第１の結像レンズと前記対物レンズとで構成される第１の合成レンズの第１の出射側主平面、もしくは前記第２の２次元センサの前段に配置される第２の結像レンズと前記対物レンズとで構成される第２の合成レンズの第２の出射側主平面と、前記第１～第４の蛍光の結像位置との距離をそれぞれｓ１～ｓ４とし、
    　前記第１の出射側主平面と前記第１の２次元センサとの距離をｄＴとし、前記第２の出射側主平面と前記第２の２次元センサとの距離をｄＲとするとき、
    　前記対物レンズを前記第１の位置に調整することは、ｓ１＞ｓ２の場合にはｓ１≧ｄＴ≧ｓ２、ｓ１＜ｓ２の場合にはｓ１≦ｄＴ≦ｓ２を満たし、
    　前記対物レンズを前記第２の位置に調整することは、ｓ３＞ｓ４の場合にはｓ３≧ｄＲ≧ｓ４、ｓ３＜ｓ４の場合にはｓ３≦ｄＲ≦ｓ４を満たすことを特徴とする分析方法。     The analysis method of claim 10.
    The first emission side main plane of the first composite lens composed of the first imaging lens and the objective lens arranged in front of the first two-dimensional sensor, or the second two-dimensional sensor. The second emission-side main plane of the second composite lens composed of the second imaging lens and the objective lens arranged in front of the above, and the imaging positions of the first to fourth fluorescence. The distances are s1 to s4, respectively.
    When the distance between the first emission side main plane and the first two-dimensional sensor is dT, and the distance between the second emission side main plane and the second two-dimensional sensor is dR,
    Adjusting the objective lens to the first position satisfies s1 ≧ dT ≧ s2 when s1> s2 and s1 ≦ dT ≦ s2 when s1 <s2.
    Adjusting the objective lens to the second position is an analysis method characterized in that s3 ≧ dR ≧ s4 in the case of s3> s4 and s3 ≦ dR ≦ s4 in the case of s3 <s4.
  14. 　請求項１０の分析方法であって、
    　前記第１の２次元センサの前段に配置される第１の結像レンズと前記対物レンズとで構成される第１の合成レンズの第１の出射側主平面、もしくは前記第２の２次元センサの前段に配置される第２の結像レンズと前記対物レンズとで構成される第２の合成レンズの第２の出射側主平面と、前記第１～第４の蛍光の結像位置との距離をそれぞれｓ１～ｓ４とし、
    　前記第１の合成レンズ又は前記第２の合成レンズにおける、前記第１～第４の蛍光の波長に対する焦点距離をそれぞれｆ１～ｆ４とし、
    　前記対物レンズを前記第１の位置に調整することは、前記第１及び第２の合成レンズの入射側主平面と前記サンプル基板との距離をｚ１とすることを含み、
    　1/z1=1/2(1/f1+1/f3)-1/2(1/s1+1/s3)を満たし、
    　前記対物レンズを前記第２の位置に調整することは、前記入射側主平面と前記サンプル基板との距離をｚ２とすることを含み、
    　1/z2=1/2(1/f2+1/f4)-1/2(1/s2+1/s4)を満たすことを特徴とする分析方法。     The analysis method of claim 10.
    The first emission side main plane of the first composite lens composed of the first imaging lens and the objective lens arranged in front of the first two-dimensional sensor, or the second two-dimensional sensor. The second emission-side main plane of the second composite lens composed of the second imaging lens and the objective lens arranged in front of the above, and the imaging positions of the first to fourth fluorescence. The distances are s1 to s4, respectively.
    The focal lengths of the first synthetic lens or the second synthetic lens with respect to the wavelengths of the first to fourth fluorescences are set to f1 to f4, respectively.
    Adjusting the objective lens to the first position includes setting the distance between the incident side main plane of the first and second composite lenses and the sample substrate to z1.
    Satisfy 1 / z1 = 1/2 (1 / f1 + 1 / f3) -1 / 2 (1 / s1 + 1 / s3),
    Adjusting the objective lens to the second position includes setting the distance between the incident side main plane and the sample substrate to z2.
    An analysis method characterized by satisfying 1 / z2 = 1/2 (1 / f2 + 1 / f4) -1 / 2 (1 / s2 + 1 / s4).
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