WO2021117153A1

WO2021117153A1 - Fluorescence detection device and fluorescence detection method

Info

Publication number: WO2021117153A1
Application number: PCT/JP2019/048398
Authority: WO
Inventors: 洋平花崎; 達也山下; 曽根原　剛志; 貴之野田
Original assignee: 株式会社日立ハイテク
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2021-06-17

Abstract

This disclosure provides a fluorescence detection device capable of automatically carrying out calibration for automatic focusing systems. The fluorescence detection device according to the present disclosure acquires a fluorescent light emitting image of a sample for each fluorescent color while moving an objective lens in the light axis direction, acquires a focus value for each fluorescent color, and when peaks of the focus values are present for the fluorescent colors, carries out calibration for the automatic focusing driving system using a preferential color set in advance among the fluorescent colors (see FIG. 1).

Description

Fluorescence detection device, fluorescence detection method

The present disclosure relates to a fluorescence detection device that detects fluorescence emission generated from a sample.

The next-generation sequencer is widely used as a device for analyzing nucleic acids such as DNA (deoxyribonucleic acid). Measurement by the next-generation sequencer is carried out 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 fluoresces from the reaction field using a two-dimensional sensor such as a CCD (Charge Coupled Device) camera or a CMOS (Complementary Metal Oxide Sensor) camera. Is detected. As a result, the base information can be obtained as a fluorescence image. In this way, by causing a chemical reaction on a microreaction field fixed to the flow cell and observing fluorescence, it is possible to analyze the base sequence of the target DNA.

When acquiring a fluorescent image, it is necessary to focus accurately and automatically. Therefore, various autofocus systems are used. For example, Patent Document 1 below describes an autofocus device using a laser and its algorithm.

Japanese Unexamined Patent Publication No. 2005-10665

In the case of an autofocus system using a laser, it is necessary to detect the position of the best focus using another means and correct the zero point of the autofocus system. As a means for detecting the best focus position, a focus value proportional to the contrast of the fluorescence image from the sample can be used. By correcting the zero point at the point where the focus value becomes the maximum, it is possible to focus correctly.

However, if the temperature changes suddenly, the zero point will shift due to the effect of heat drift. Since the next-generation sequencer takes several hours to several tens of hours for one DNA sequence analysis, it may not be possible to focus correctly due to temperature changes during that time. Therefore, in order to follow the temperature change during DNA sequence analysis, it is necessary to calibrate automatically.

The DNA sequence to be analyzed is basically a random sequence, and the ratios of A, T, G, and C are stochastically almost the same. However, a sequence containing identification information of a sample called a barcode sequence is often added to the DNA sequence. Since the barcode sequence is an artificially designed sequence, the proportions of the four types of bases are not equal and may contain only one to three types of bases.

In order to identify the four types of bases A, T, G, and C, it is necessary to use fluorescent dyes of multiple colors. In the case of normal sequence reading, all fluorescent dyes emit light, so any color can be used to calibrate the autofocus system. However, when reading the barcode array, if the fluorescence color at which fluorescence can be obtained is not determined in advance, the search for the best focus position may fail and the calibration may fail.

Furthermore, among the fluorescent dyes of multiple colors, there are fluorescent dyes in which the DNA sequence reading error is less likely to occur when the focus is given priority. Therefore, it is considered more desirable to calibrate the autofocus system after determining the priority color.

The present disclosure has been made in view of the above problems, and provides a fluorescence detection device capable of automatically calibrating an autofocus system.

The fluorescence detection device according to the present disclosure acquires a fluorescence emission image of a sample for each fluorescence color while moving an objective lens in the optical axis direction, acquires a focus value for each fluorescence color, and obtains a focus value for each of the fluorescence colors in a plurality of the fluorescence colors. If there is a peak of the focus value, the autofocus drive system is calibrated using a predetermined priority color among the fluorescent colors.

According to the fluorescence detection device according to the present disclosure, the autofocus system can be calibrated correctly even when the distribution ratio of each fluorescence color is not equal. In addition, it becomes possible to focus on the fluorescent dye to be preferentially focused. Issues, configurations, and effects other than the above will be clarified by the following description of embodiments.

FIG. 5 is a flowchart illustrating a method of capturing a fluorescence image of a sample using the fluorescence detection device according to the first embodiment. It is a block diagram of the fluorescence detection apparatus 1 which concerns on Embodiment 1. FIG. It is a block diagram of the fluorescence detection apparatus 1 which concerns on Embodiment 2. FIG. It is a schematic diagram which shows the structural example of 2D sensor 204.

<Embodiment 1>
In the first embodiment of the present disclosure, an example in which the fluorescence detection device is configured as a next-generation DNA sequencer will be described. First, a general method for determining a base sequence using a next-generation sequencer will be described, and then embodiments of the present disclosure will be described.

Nucleotides fluorescently labeled with multiple colors are bound to the sample. 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, the autofocus is calibrated and a fluorescence image is taken. When imaging is complete, the fluorescent dye and protecting groups are removed. The base sequence is determined by repeating the above steps as one cycle.

FIG. 1 is a flowchart illustrating a method of capturing a fluorescence image of a sample using the fluorescence detection device according to the first embodiment. This flowchart describes a method from binding a fluorescently labeled nucleotide to a DNA sample to completing autofocus calibration. Each step of this flowchart can be carried out by the control unit 209 described later. Each step of FIG. 1 will be described below.

In step S1, nucleotides fluorescently labeled with a plurality of colors are bound to the sample. Four types of fluorescent dyes can be used, one for each of the four types of bases A, T, G, and C, but the present invention is not limited to this. It is possible to identify four types of bases even with two or three types of fluorescent dyes. For example, when three types of fluorescent dyes are used, if they are the first, second, and third fluorescent dyes, the first fluorescent dye is bound to A, the second fluorescent dye is bound to T, and G is used. By designing so that the third fluorescent dye is bound to C and the first and second fluorescent dyes are bound to C, four kinds of bases can be identified by the three kinds of fluorescent dyes.

In step S2, the fluorescent dye bonded in S1 is made to emit light in all colors. If all colors can be imaged at the same time, all colors may be emitted at the same time. Alternatively, each fluorescent color may be emitted in turn and images may be taken in order.

In step S3, a fluorescent image of all fluorescent colors is captured by a two-dimensional sensor. The two-dimensional sensor may be a separate two-dimensional sensor corresponding to each of a plurality of colors, or may be a sensor capable of color imaging that can image all fluorescent colors at once.

In step S4, the focus value is acquired from all the colors of the captured fluorescence image. The focus value is a value that becomes maximum when an image is taken at the best focus position. As the focus value, for example, a value proportional to the contrast of the image can be used. Other appropriate values may be used. The focus value can also be calculated by a predetermined calculation formula. It is not necessary to capture the focus value after saving the acquired image in a storage medium such as a hard disk, and the focus value may be calculated on the image processing circuit attached to the sensor. Examples of the image processing circuit include circuits such as FPGA (Field Programmable Gate Array). Specific examples will be described later.

In step S5, it is determined whether or not a predetermined number of fluorescent images have been captured. In order to determine whether or not a peak of the focus value exists in step S7, it is necessary to acquire the focus value from a plurality of images. In addition, it is necessary to include the image at the position where the best focus is obtained in the plurality of images. The specified number of images is not limited to, for example, if the image is acquired three times, the peak of the focus value may be obtained. If the predetermined number of images have been captured, the process proceeds to step S7, and if not, the process proceeds to step S6.

In step S6, the objective lens is moved in the optical axis direction by the objective lens driving device 207 described later. If the moving distance of the objective lens by performing this step once is small, it may not be possible to take an image at the position where the best focus is obtained, and if the moving distance is large, the image may be taken after passing the best focus position. There is. If the specified number of sheets is increased and the moving distance by one step is reduced, the possibility of imaging at the best focus position increases, but imaging takes time and the sample is irradiated with an excessive amount of excitation light. This may cause the fluorescent dye to fade. Therefore, the driving distance is preferably about 100 nm to 10 um, but is not limited to this.

In step S7, it is determined whether or not a peak of the focus value exists. For a certain fluorescent color, the ratio of the maximum value to the minimum value among the focus values obtained from a plurality of images is calculated. When the ratio of the maximum value to the minimum value is equal to or greater than the threshold value, it can be determined that the fluorescent color is emitting light. As a method for determining whether or not light is emitted, not only the ratio of the maximum value to the minimum value but also whether or not the maximum value of the focus value of each fluorescent color is equal to or greater than the threshold value can be determined. If there is a peak of the focus value in any of the fluorescent colors, the process proceeds to step S8. If there are no peaks in all fluorescent colors, the process proceeds to the abend step.

The following three possibilities are considered as factors for determining that there is no peak in all fluorescent colors in step S7. Regarding Possibility 1 and Possibility 2, if there is a variation in fluorescence emission for each imaging region, it may be possible to deal with it by changing the imaging region. Possibility 3 may be dealt with by expanding the imaging range in the optical axis direction of the objective lens.

(Possibility 1) The sample does not exist in the imaging region or the number is small. (Possibility 2) The fluorescent dye cannot be bound to the sample or the number is small. (Possibility 3) Within the range in which the objective lens is moved. There is no best focus position in

As the processing after the abnormal end, it can be judged as abnormal and the analysis can be stopped. If the analysis is not stopped, (a) change the imaging area and repeat the same procedure according to this flowchart, (b) expand the imaging range, that is, increase the specified number of images, or increase the driving amount of the objective lens. Alternatively, both are performed and the same procedure is repeated according to this flowchart, or (c) the amount of movement of the objective lens for one time is reduced (that is, the step width is reduced). These may be combined. Even if the analysis is not stopped, it is desirable to set the upper limit of the number of changes in the imaging area and the number of expansions of the imaging range, and if it fails a certain number of times, it is judged as abnormal and the analysis is stopped.

In step S8, it is determined whether or not the peak of the focus value is obtained for a plurality of fluorescent colors. When the peak of the focus value is obtained for a plurality of colors, the process proceeds to step S9, and when the peak of the focus value is obtained for only one color, the process proceeds to step S10.

In step S9, the autofocus is calibrated using the priority color. Among the plurality of fluorescent colors, the priority color is the priority color having the highest priority in order from the one having the lowest fluorescence intensity obtained from the sample. In autofocus calibration, it is necessary to calculate the best focus position by some method and correct the zero point of the autofocus system to the focus peak position. Therefore, if the focus peak is calculated using the priority color and the zero point correction is performed at that position, the objective lens can be driven to the best focus position of the priority color. After that, when all the fluorescent colors are emitted and an image is taken by the two-dimensional sensor, it is possible to acquire a fluorescent image in which the priority color is the best focus.

In step S10, autofocus calibration is performed on the fluorescent color from which the focus peak has been obtained. Fluorescent colors for which no focus peak was obtained mean that they do not contain the corresponding bases or are negligibly low in proportion. Therefore, even if those fluorescent colors are priority colors, it is not necessary to focus them, so that the autofocus calibration may be performed using the color from which the focus peak is obtained.

In S9 and S10, the calibration of the autofocus system can be performed by matching the position of the objective lens, which is the peak among the plurality of focus values, with the zero point of the autofocus system. Specifically, both positions can be matched by moving the position of the objective lens in the optical axis direction. After calibrating the autofocus system, the position where the detection signal output by the autofocus drive system 214, which will be described later, is 0 (or the smallest value in the vicinity of 0) can be regarded as the best focus position.

FIG. 2 is a configuration diagram of the fluorescence detection device 1 according to the first embodiment. In FIG. 2, the vertical direction of the paper surface is the vertical direction. The fluorescence detection device 1 is, for example, a nucleic acid analysis device that analyzes a base sequence of a nucleic acid, and includes an optical system 200, a stage 208, and a control unit 209. The stage 208 mounts the sample substrate 201. The control unit 209 controls the entire fluorescence detection device 1.

The sample substrate 201 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, such as single-stranded DNA, is fixed in the flow path, and a reaction solution (reagent) is introduced by a liquid feeding mechanism (not shown).

A method of capturing a fluorescence image of nucleic acid using the fluorescence detection device 1 will be described. In the first embodiment, it is assumed that A, T, G, and C of the DNA sample are labeled with four different fluorescent colors. The filter unit 205A separates the first fluorescence from the second fluorescence, and the filter unit 205B separates the third fluorescence from the fourth fluorescence.

The stage 208 supports the sample substrate 201 so that the sample substrate 201 is orthogonal to the optical axis of the objective lens 203. The stage 208 is configured to be movable at least horizontally by a drive device (not shown). The stage 208 may have a temperature control mechanism such as a heat block at a position in contact with the sample substrate 201, and the elongation reaction can be promoted by heating and cooling the sample substrate 201 as necessary. The stage 208 may be configured so that a plurality of sample substrates 201 can be mounted at the same time.

The optical system 200 includes a light source 202, an objective lens 203, a two-dimensional sensor 204, a filter unit 205A (first unit), a filter unit 205B (second unit), a filter unit switching mechanism 206, and an objective lens driving device 207.

The light source 202 emits light including excitation light having a wavelength capable of exciting the fluorescent dye bound to the sample. The excitation wavelengths of all the fluorescent dyes must be included so that all the fluorescent dyes of multiple colors emit light. Alternatively, a plurality of light sources that emit light of each fluorescence may be prepared, and each of them may be switched to emit light. As the light source 202, for example, an Xe lamp or a white LED can be used. Not only one type of light source but also a light source in which a plurality of types of LEDs are combined may be used.

The filter unit 205A includes a transmission filter 210A, a dichroic mirror 211A, and a fluorescence filter 212A. The transmission filter 210A transmits the excitation light from the light source 202. The dichroic mirror 211A reflects the excitation light and causes it to enter the sample substrate 201 to transmit the fluorescence from the sample. The fluorescence filter 212A allows only the fluorescence from the sample to pass through. The transmission filter 210A and the fluorescence filter 212A can efficiently excite the sample and remove components other than fluorescence generated from the sample. As a result, the contrast of the obtained fluorescent image is increased. Similarly, the filter unit 205B also has a transmission filter 210B, a dichroic mirror 211B, and a fluorescence filter 212B.

The filter unit switching mechanism 206 is configured so that the positions of the

filter units

205A and 205B can be changed, and one of these is arranged on the optical axis of the objective lens 203. As the drive mechanism of the filter unit switching mechanism 206, for example, a motor or a solenoid can be used.

The two-

dimensional sensors

204A and 204B acquire an image of fluorescence incident on the sensor surface and transmit the fluorescence image to the control unit 209. As the two-

dimensional sensors

204A and 204B, for example, a CCD camera or a CMOS camera can be used.

The objective lens driving device 207 is connected to the objective lens 203, and the objective lens 203 is configured to be movable in the vertical direction. This makes it possible to adjust the distance between the objective lens 203 and the sample substrate 201. The objective lens driving device 207 includes, for example, a stepping motor, a stage fixed to the objective lens 203, a pulse oscillator, and the like. Instead of providing the objective lens driving device 207, a driving device capable of driving the stage 208 not only in the horizontal direction but also in the vertical direction may be used.

The control unit 209 irradiates light by the light source 202, switches by the filter unit switching mechanism 206, captures images by the two-

dimensional sensors

204A and 204B, drives the objective lens driving device 207, drives the driving device (not shown) of the stage 208, and reacts. Controls the drive of the liquid feeding mechanism (not shown). The control unit 209 executes a process of analyzing the base sequence of nucleic acid based on the fluorescence images acquired by the two-

dimensional sensors

204A and 204B.

The mirror 213 is provided to reflect the laser to the autofocus drive system 214. The autofocus drive system 214 irradiates the mirror 213 with a laser beam, whereby the laser beam reflected from the mirror 213 is incident on the sample substrate 201. The autofocus drive system 214 detects the laser beam reflected from the sample substrate 201.

Generally, a fluorescent reagent having a wavelength of visible light is used, and since infrared light has relatively little damage to the sample, infrared light is used as the laser light of the autofocus drive system 214, and infrared light is used as the mirror 213. A dichroic mirror that reflects 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 214, a mirror 213 that reflects only 532 nm can be used.

The irradiation of the laser beam by the autofocus drive system 214 is controlled by the control unit 209. The autofocus drive system 214 outputs the detected signal of the reflected light to the control unit 209. The control unit 209 drives the objective lens driving device 207 based on the detection signal of the autofocus driving system 214. Specifically, the autofocus drive system 214 outputs a detection signal proportional to the distance z between the objective lens 203 and the sample substrate 201. The autofocus drive system 214 uses the detection signal to perform autofocus on the objective lens 203 with reference to the zero point set by calibration. The detection signal after calibration becomes 0 when the fluorescence from the sample is in focus.

When the autofocus drive system 214 is calibrated by the method shown in FIG. 1, when a focus peak is obtained with a plurality of fluorescent colors, the detection signal becomes 0 at the best focus position of the priority color, and only one color is focused. If no peak is obtained, the detection signal becomes 0 at the best focus position of the color.

The dichroic mirror 215 is arranged on the optical axis of the objective lens 203, and the fluorescence that has passed through the

filter unit

205A or 205B is incident on the two-

dimensional sensors

204A and 204B. Specifically, the fluorescence transmitted through the dichroic mirror 215 is imaged on the two-dimensional sensor 204A, and the fluorescence reflected by the dichroic mirror 215 is imaged on the two-dimensional sensor 204B. The dichroic mirror 215 is arranged at an angle of, for example, 45 ° with respect to the optical axis of the objective lens 203. A beam splitter (optical element) such as a half mirror may be used instead of the dichroic mirror 215.

In the first embodiment, the first fluorescence and the second fluorescence are obtained when the filter unit 205A is used, and the third fluorescence and the fourth fluorescence are obtained when the filter unit 205B is used. Therefore, when there is one zero point value that can be stored in the autofocus drive system 214, calibration is performed using one priority color. When there are two zero point values that can be stored in the autofocus drive system 214, when a fluorescent image is imaged using the filter unit 205A, one of the first and second fluorescent colors is the priority color. When the calibration is performed using the filter unit 205B and a fluorescent image is captured using the filter unit 205B, the calibration can be performed using one of the priority colors of the third and fourth fluorescent colors. ..

If there is only one fluorescent color with a peak focus value, or if fluorescence is not obtained in either the

filter unit

205A or 205B, calibrate using the two colors of the filter unit that did not obtain fluorescence. Cannot be implemented. In that case, the zero point stored in advance in the autofocus drive system 214 can be used as the calibration value of the filter unit for which fluorescence was not obtained, instead of abnormal termination. The resulting image may be defocused due to the effects of temperature drift, but the two colors do not provide fluorescence in the first place and do not contain the corresponding bases or are contained in negligibly low proportions. Since there is no such thing, the analysis result of the sequence is not affected.

<Embodiment 1: Another Example of Analytical Method>
The number of colors that can be fluorescently imaged with high quality by the fluorescence detection device 1 of the first 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, a fluorescently labeled nucleotide labeled with a three-color fluorescent dye (first fluorescent dye to third fluorescent dye) that emits first fluorescence to third fluorescence, respectively, is used.

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 labeled with, for example, two of the first fluorescent dye and the second fluorescent dye. It is assumed that it has been done. As a result, 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.

In this analysis method, the first fluorescence and the second fluorescence are separated by the filter unit 205A, and the third fluorescence is separated by the filter unit 205B. Since this method is the same as the analysis method (FIG. 2) in the case of the above-mentioned four-color label, the description of other steps will be omitted. The first fluorescence may be separated by the filter unit 205A, and the second fluorescence and the third fluorescence may be separated by the filter unit 205B.

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.

<Embodiment 1: Summary>
The fluorescence detection device 1 according to the first embodiment acquires a focus value for each fluorescence color, and if a peak of the focus value exists, calibrates the autofocus drive system 214 using a predetermined priority color. Implementation. As a result, even when calibration is performed using the fluorescent color of an artificially created base sequence such as a barcode sequence, this can be performed appropriately and automatically.

When the focus value peaks are present in a plurality of fluorescent colors, the fluorescence detection device 1 according to the first embodiment selects the fluorescent colors as priority colors in ascending order of fluorescence emission intensity from the sample. By performing calibration based on a fluorescent color having a low emission intensity, it is possible to improve the accuracy of calibration for other fluorescent colors as well.

<Embodiment 2>
FIG. 3 is a configuration diagram of the fluorescence detection device 1 according to the second embodiment of the present disclosure. In the second embodiment, there is only one filter unit 205, and the filter unit switching mechanism 206 is not provided. The two-dimensional sensor 204 is a color camera capable of simultaneously capturing a plurality of fluorescent colors. Since it is not necessary to switch the filter unit and only one imaging is required, the structure can be simplified and the time required for imaging can be shortened.

On the surface of the color camera, there are pixels that detect only one fluorescent color among a plurality of fluorescent colors. As a method of arranging the pixels, a plurality of types of pixels may be arranged regularly in a plane, for example, in a Bayer arrangement. Alternatively, the pixels may be arranged in the vertical direction, and the pixels for detecting each fluorescent color may be arranged in multiple layers.

In the second embodiment, the transmission filter 210A is capable of separating excitation light for a plurality of fluorescent colors, the dichroic mirror 211A is capable of separating a plurality of fluorescences from a sample, and the fluorescence filter 212A is capable of separating a plurality of fluorescences from a sample. Only multiple fluorescence from is transmitted. The fluorescence filter 212A can be omitted if the pixels corresponding to each fluorescence are not affected by other fluorescence.

<Embodiment 3>
FIG. 4 is a schematic view showing a configuration example of the two-dimensional sensor 204. The two-dimensional sensor 204 may include an arithmetic circuit 2042 (for example, FPGA) in addition to the sensor element 2041. The sensor element 2041 and the arithmetic circuit 2042 can be mounted on different boards or on the same board. FIG. 4 shows an example of mounting on another substrate. The two-

dimensional sensors

204A and 204B described in the first embodiment can also have the same configuration.

The arithmetic circuit 2042 performs fluorescence imaging in parallel with outputting the fluorescence imaging image from the sensor element 2041 to the control unit 209, or before outputting the fluorescence imaging image from the sensor element 2041 to the control unit 209. Calculate the focus value of the image. As a result, the focus value can be calculated efficiently. On the other hand, when the fluorescence image is output from the sensor element 2041 to the control unit 209 and the focus value is calculated by the control unit 209, in a sample in which a large amount of time is required to transfer the fluorescence image. Will take a long time to perform auto-calibration. Therefore, there is an advantage that the focus value is calculated in the two-dimensional sensor 204 as in the third embodiment.

<About a modified example of the present disclosure>
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.

In the above embodiment, the control unit 209 can be configured by hardware such as a circuit device that implements the function, or an arithmetic unit such as a CPU (Central Processing Unit) executes software that implements the function. It can also be configured by. The control unit 209 further includes a storage unit that stores a program and various data for driving and analyzing each component of the fluorescence detection device 1, a processor that reads the program and various data and executes the above operation, and a user. It may have an input unit for inputting data and instructions, and the like.

In the above embodiments, the DNA sequencer has been described as an example of the fluorescence detection device, but the fluorescence detection device according to the present disclosure is not limited to this. The method according to the present disclosure can also be used in other fluorescence detection devices that acquire a fluorescence emission image of a sample.

200: Optical system 201: Sample substrate 202: Light source 203:

Objective lens

204, 204A, 204B: Two-

dimensional sensor

205A, 205B: Filter unit 206: Filter unit switching mechanism 207: Objective lens drive device 208: Stage 209:

Control unit

210A , 210B:

Transmission filter

211A, 211B:

Dichroic mirror

212A, 212B: Fluorescent filter 213: Mirror 214: Autofocus drive system 215: Dichroic mirror

Claims

A fluorescence detection device that detects fluorescence emission generated from a sample.
An objective lens arranged to face the sample,
An objective lens driving device that moves the objective lens in the optical axis direction,
An autofocus drive system that outputs a focus detection signal indicating whether or not the objective lens is in focus with respect to the sample and also performs autofocus of the objective lens.
A sensor that captures a fluorescence emission image of the sample,
A control unit that adjusts the focus position of the objective lens with respect to the sample by controlling the objective lens driving device.
With
The control unit acquires a fluorescence emission image of the sample for each fluorescence color while moving the objective lens in the optical axis direction.
The control unit acquires the maximum focus value when an image is taken at the best focus position from the fluorescence emission image for each fluorescence color.
When the peak of the focus value exists in each of the plurality of fluorescent colors, the control unit calibrates the autofocus drive system using a predetermined priority color among the fluorescent colors. A fluorescence detection device characterized in that.
When the focus value peak is present only in the single fluorescent color, the control unit calibrates the autofocus drive system using the fluorescent color in which the focus value peak is present. The fluorescence detection device according to claim 1.
When the peak of the focus value does not exist in any of the fluorescent colors, the control unit may use the control unit.
The imaging region imaged by the sensor is changed on the surface of the sample.
When moving the objective lens in the optical axis direction, the movement width moved by one movement is reduced.
Increasing the moving distance of the objective lens in the optical axis direction,
The fluorescence detection device according to claim 1, wherein the focus value is acquired from the fluorescence emission image for each fluorescence color after performing at least one of the above.
The control unit acquires the maximum value and the minimum value from the focus values acquired for the same fluorescent color.
When the ratio of the maximum value to the minimum value is equal to or greater than a threshold value, the control unit determines that a peak of the focus value exists in the fluorescent color obtained from the maximum value and the minimum value. The fluorescence detection device according to claim 1.
The control unit acquires the maximum value from the focus values acquired for the same fluorescent color.
The fluorescence detection device according to claim 1, wherein when the maximum value is equal to or greater than a threshold value, the control unit determines that a peak of the focus value exists in the fluorescence color that has acquired the maximum value.
The fluorescence according to claim 1, wherein the control unit selects as the priority color in ascending order of fluorescence emission intensity from the sample from among the plurality of fluorescence colors in which the peak of the focus value exists. Detection device.
The fluorescence detector further comprises a filter unit for filtering light.
The filter unit is
A transmission filter that allows light from a light source to pass through,
A fluorescent filter that transmits fluorescent light from the sample,
A filter element that reflects light transmitted through the transmission filter toward the objective lens and transmits fluorescence emission from the sample toward the fluorescence filter.
The fluorescence detection device according to claim 1, further comprising.
The sensor includes a first image sensor that captures the first fluorescence and a second image sensor that captures the second fluorescence.
The filter unit includes a first unit that transmits the first fluorescence from the sample toward the first image sensor, and a second unit that transmits the second fluorescence from the sample toward the second image sensor. Equipped with a unit,
7. The seventh aspect of the present invention is characterized in that the fluorescence detection device further includes a filter unit switching mechanism for switching which of the first unit and the second unit is arranged on the optical path of fluorescence from the sample. Fluorescence detector.
The first image sensor captures a third fluorescence in addition to the first fluorescence.
The second image sensor captures a fourth fluorescence in addition to the second fluorescence.
The first unit transmits the third fluorescence from the sample toward the first image sensor.
The fluorescence detection device according to claim 8, wherein the second unit transmits the fourth fluorescence from the sample toward the second image pickup device.
The sensor is configured as a multicolor camera capable of capturing a plurality of fluorescent colors.
The fluorescence detection device according to claim 7, wherein the filter element transmits fluorescence emission of each of the fluorescent colors imaged by the sensor toward the fluorescent filter.
The control unit calibrates the autofocus drive system by setting a zero point of the focus detection signal at a position of the objective lens at which the focus value peaks. 1. The fluorescence detection device according to 1.
The sensor includes an arithmetic circuit for calculating the focus value.
The sensor obtains the focus value of the fluorescence emission image in parallel with outputting the fluorescence emission image to the control unit or before outputting the fluorescence emission image to the control unit. The fluorescence detection device according to claim 1, wherein the fluorescence is calculated by an arithmetic circuit.
By imaging the fluorescence generated from each of the four types of DNA bases of the sample, the sensor captures the fluorescence emission image describing the information about the base composition of the sample.
The fluorescence detection device according to claim 1, wherein the control unit is configured to analyze the base configuration of the sample by analyzing the fluorescence emission image.
A fluorescence detection method for detecting fluorescence emission generated from a sample.
A step of acquiring a fluorescence emission image of the sample for each fluorescence color while moving an objective lens arranged to face the sample in the optical axis direction.
A step of acquiring the maximum focus value when an image is taken at the best focus position from the fluorescence emission image for each fluorescence color.
Steps for calibrating the autofocus drive system for autofocusing the objective lens,
Have,
In the step of performing the calibration, when a peak of the focus value is present in each of the plurality of fluorescent colors, the calibration is performed using a predetermined priority color among the fluorescent colors. A fluorescence detection method characterized by.