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WO2021070259A1 - Dispositif d'analyse et procédé d'analyse - Google Patents

Dispositif d'analyse et procédé d'analyse 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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WIPO (PCT)
Prior art keywords
fluorescence
objective lens
dimensional sensor
filter unit
lens
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PCT/JP2019/039701
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English (en)
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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Hitachi High Tech Corp
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Hitachi High Tech Corp
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Priority to PCT/JP2019/039701 priority Critical patent/WO2021070259A1/fr
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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

L'invention concerne un dispositif d'analyse qui comprend : un étage sur lequel est placé un substrat d'échantillon qui émet une première fluorescence, une deuxième fluorescence et une troisième fluorescence ; une lentille d'objectif ; une première unité de filtre pour séparer la première fluorescence et la deuxième fluorescence ; une seconde unité de filtre pour séparer la troisième fluorescence ; un premier capteur bidimensionnel et un second capteur bidimensionnel pour imager les première à troisième fluorescences à partir du substrat d'échantillon ; un élément optique pour séparer les première à troisième fluorescences ; un premier dispositif d'entraînement pour commuter entre la première unité de filtre et la seconde unité de filtre ; un second dispositif d'entraînement pour modifier la distance relative entre la lentille d'objectif et le substrat d'échantillon ; et une unité de commande pour au moins commander le premier dispositif d'entraînement et le second dispositif d'entraînement. L'unité de commande commande le premier dispositif d'entraînement et le second dispositif d'entraînement de façon à régler la distance entre la lentille d'objectif et le substrat d'échantillon selon que l'imagerie doit être effectuée à l'aide de la première unité de filtre ou de la seconde unité de filtre.
PCT/JP2019/039701 2019-10-08 2019-10-08 Dispositif d'analyse et procédé d'analyse Ceased WO2021070259A1 (fr)

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JP2003140050A (ja) * 2001-11-02 2003-05-14 Olympus Optical Co Ltd 走査型共焦点顕微鏡
JP2010503847A (ja) * 2006-09-14 2010-02-04 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド 単一分子を撮像する装置
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