JP2003262588A - Apparatus and method for reading microarray of organism-related substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately acquire information on the location or size of a section to be detected to which an organism-related substance is immobilized even in a specimen of a low fluorescent intensity and a microarray of a low arrangement accuracy. <P>SOLUTION: In this apparatus (1) for reading the microarray, fluorescence emitted by a fluorescent substance is read from the microarray (A) in which sections except the section to which the organism-related substance is immobilized are opaquely formed. The apparatus (1) for reading the microarray comprises a light source (2) for transmission for transmitting light through the microarray, a light source (6) for excitation for exciting the fluorescent substance present in the section, an image pickup means (4) for both picking up the image of light shone from the light source for transmission and transmitted through the microarray and picking up the image of the fluorescence emitted by the fluorescent substance excited by the light source for excitation, and a detecting means (5) for detecting the location and/or size of the section arranged on the microarray on the basis of the image of light picked up by the image pickup means and transmitted through the microarray. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bio-related substance microarray in which probes are immobilized and arrayed, a specimen provided with a fluorescent substance is reacted, and excitation light is irradiated to read the amount of excited fluorescence. A material microarray reader and method.

[0002]

2. Description of the Related Art In recent years, genome projects in various organisms have been under way, and many genes including human genes and their nucleotide sequences are rapidly being clarified. The function of the gene whose sequence has been elucidated can be examined by various methods. As one of the effective methods, gene expression analysis using the revealed nucleotide sequence information is known. In this analysis method, for example, a different gene peptide, protein, antibody, for example, in any of the surface of the substrate, the concave portion, the convex portion, or the compartment of the polymer gel-like substance retained in the inner wall portion of the capillary or the hollow portion of the capillary is used. A microarray in which tens to tens of thousands of bio-related substances are immobilized as a probe is used. By utilizing various specific reactions with this probe, the relationship between the substance used as the probe and its biological function expression can be investigated. Such a method enables a collective expression analysis of a large number of genes in order to carry out a comprehensive and systematic analysis of an extremely large number of genes at the individual level, as is being revealed through today's genome projects. Microarray method (DN
It has been developed as a new analytical method or method called the A-chip method.

In the method of detecting a reaction product of a probe and a specimen, first, a fluorescence-labeled specimen and a bio-related substance immobilized as a probe on a microarray are reacted. Then, by detecting the amount of fluorescence with a fluorescence measurement device such as a fluorescence laser scanner or a fluorescence microscope, it is possible to detect with which probe the fluorescence-labeled sample formed a reaction product.

[0004]

However, when the fluorescence intensity of the sample is low or the positional accuracy of the array of the microarray is low, the detected fluorescence and the biological substance immobilized on the microarray as a probe are detected. There is a problem in that it is difficult to accurately recognize the correspondence between the two, and it is difficult to easily and accurately detect with which probe the sample has hybridized.

In order to solve such a problem, Japanese Unexamined Patent Application Publication No. 2004-242242 describes a microarray reading method.
In this method, in a bio-related substance to be immobilized as a probe on a microarray, a fluorescent substance different from the fluorescent substance for labeling the specimen is previously mixed, and the fluorescent substance mixed with the probe is excited. The position of the spot is detected by detecting the obtained fluorescence.

[0006] However, in this method, it is necessary to add a fluorescent substance different from the fluorescent substance given to the sample to the biological substance immobilized on the microarray to form the probe. The fluorescent substance, which is attached to this biological substance, for detecting the position of the probe in advance,
There is a problem that it becomes a noise source when the fluorescent substance attached to the sample, which should be originally detected, is detected. There is also a problem that it is necessary to construct a new optical system such as a filter for detecting a fluorescent substance newly mixed with the biological substance.

According to the present invention, even in a sample having a low fluorescence intensity or an array having a low array accuracy, a bio-related substance is immobilized on a compartment to be detected without mixing a new substance with the bio-related substance used as a probe. It is an object of the present invention to provide a microarray reading apparatus and method capable of accurately obtaining position and / or size information.

[0008]

[Patent Document 1] Japanese Patent Laid-Open No. 2001-27607

[0009]

In order to solve the above-mentioned problems, the present invention detects fluorescence emitted by a fluorescent substance from a bio-related substance microarray including a plurality of compartments in which a specimen to which the fluorescent substance is applied is reacted. A reader for reading, which is a transmissive light source that transmits light to the microarray, an excitation light source that excites the fluorescent substance existing in the compartment, and an image of the light that is emitted from the transmissive light source and that has passed through the microarray, and is excited. Based on the image of the light transmitted through the microarray, which is imaged by the imaging means for imaging the fluorescence emitted from the fluorescent substance excited by the light source, and / or the position of the section arranged on the microarray And a detection means for detecting the size.

Further, it is preferable that the portion other than the section of the microarray is made opaque.

Furthermore, it is characterized in that it has means for moving the section from the position of the section arranged on the microarray into the visual field of the image pickup means.

Next, the specimen to which the fluorescent substance is applied is reacted with the section of the microarray to form a reaction product of the probe and the specimen of the microarray. Next, the microarray is irradiated with light from a light source for transmission, and the transmitted light that has passed through the microarray is imaged by an imaging unit.

The detecting means detects the position and / or size of the section on the microarray from the image of the transmitted light taken. Preferably, from the image of the transmitted light taken,
The position of the entire section on the microarray is detected. Next, the microarray is moved so that the detected center position of the entire section on the microarray coincides with the center position of the visual field of the imaging means. Then, the microarray is irradiated with light from the light source for transmission again, the transmitted light transmitted through the sections of the microarray is imaged by the imaging means, and the position and / or size of the section on the microarray is determined from the captured image of the transmitted light. To detect.

Further, the excitation light source irradiates the microarray with excitation light to cause the fluorescent substance existing on the microarray to fluoresce. This fluorescence is imaged by the imaging means, and the imaged image is analyzed based on the position and / or size of the previously detected section.

With this configuration, it is possible to easily recognize the position and / or size of the section formed on the microarray, and therefore, when using a microarray having a poor positional accuracy for arranging the section, or from the microarray. Even when the emitted fluorescence is weak, the fluorescence can be accurately detected.

It is preferable that the detecting means selects a part of the image of the light transmitted through the section imaged by the imaging means and detects the fluorescence of the selected part.

In the present invention thus constructed,
Image information of a part of the spot, which is an image of the section detected by capturing the transmitted light, is extracted, and analysis is performed based on the image information. According to this configuration, fluorescence emitted from other than the fluorescent substance to be detected on the microarray,
For example, it is possible to eliminate the influence of autofluorescence emitted from the material other than the section of the microarray.

Preferably, the detection means further comprises a correction means for correcting the difference between the optical system for imaging the light transmitted through the microarray and the optical system for imaging the fluorescence emitted from the fluorescent substance.

In the present invention thus constructed,
An optical system for imaging light transmitted through the microarray,
Even when the optical system for capturing the fluorescence emitted from the fluorescent substance is different and the difference in the optical system causes a shift in the image, the fluorescence in the section can be detected without error.

The image pickup means can be composed of a CCD camera.

Further, the light source for transmission and the light source for excitation may be combined.

Further, the light source for transmission or the light source for excitation can be constituted by a light emitting means and an optical fiber bundle for guiding the light emitted by the light emitting means.

In the present invention thus constructed,
The transmission light source or the excitation light source can be arranged at any position, and the optical fiber can prevent the heat rays emitted from the light source from being applied to the microarray.

Further, a transmission light source or an excitation light source is set to L
It can also be configured by an ED light source.

In the present invention thus constructed,
The life of the transmission light source or the excitation light source can be extended.

Further, the reading method of the bio-related substance microarray according to the present invention comprises the steps of preparing a bio-related substance microarray including a plurality of compartments reacted with a specimen to which a fluorescent substance is applied, and irradiating the microarray with light. Imaging the transmitted light transmitted through the microarray, detecting the position and / or size of the compartment, irradiating the microarray with excitation light to excite the fluorescent substance existing in the compartment of the macroarray, and And a step of reading the fluorescence imaged in this step based on the position and / or the size of the section detected based on the transmitted light.

Further, the reading method of the bio-related substance microarray of the present invention comprises the steps of preparing a bio-related substance microarray including a plurality of compartments reacted with a specimen to which a fluorescent substance is applied, and irradiating the microarray with light. , Imaging the transmitted light transmitted through the microarray,
Based on the transmitted light imaged in this step, a step of moving the position of the section to an appropriate position in the field of view for imaging, irradiating the microarray again with light, and imaging the transmitted light transmitted through the microarray, Detecting the position and / or size of the section, irradiating the microarray with excitation light to excite the fluorescent substance existing in the section of the macroarray, and imaging the fluorescence emitted from this fluorescent substance; Reading the fluorescence imaged in step based on the position and / or size of the section detected based on transmitted light;
It is characterized by having.

Furthermore, in the microarray reading method of the present invention, a mask is applied to an image obtained by imaging the fluorescence emitted from the fluorescent substance based on the position and / or size of the section detected based on the transmitted light. It has a feature.

[0029]

Embodiments of the present invention will now be described with reference to the accompanying drawings. FIG. 1 shows the first or second aspect of the present invention.
2 is a schematic configuration diagram of a microarray reader according to the embodiment of FIG.

FIG. 2 is a plan view of the microarray A used in the microarray reader according to this embodiment. As shown in FIG. 1, the microarray reader 1 according to the first embodiment of the present invention irradiates the microarray A with a transmissive light source 2 that radiates transmitted light that transmits the microarray A and light for exciting a fluorescent substance. And a light source 6 for excitation. Further, the microarray reading device 1 includes a CCD camera 4 which is an imaging unit for imaging the transmitted light transmitted through the microarray A and the fluorescence emitted from the fluorescent substance applied to the sample on the microarray A,
The detection means 5 analyzes the sample based on the image captured by the CCD camera 4.

Further, the microarray reader 1 includes a dichroic mirror 8 which transmits or reflects a predetermined light.
The microscope 16 is provided with an optical system including an absorption filter 10, an excitation filter 12, and an objective lens 14 for the microarray A. Further, the microarray reading device 1 includes an XY stage 18 for moving the microarray A to a desired position in a horizontal plane, a transmitted light source 2, a CCD camera 4, an excitation light source 6, and a microarray A imaged on the CCD camera 4. A focus stage (not shown) that moves the XY stage 18 or the objective lens 14 up and down to adjust the focus of the image, and a control device 20 that controls the XY stage 18 and the like.

The transmissive light source 2 is arranged below the XY stage 18 so that the optical axis of the light emitted from the light source and the optical axis of the microscope 16 are aligned with each other. As the transmissive light source 2, for example, a halogen lamp or an LED array light source in which a plurality of LEDs are arranged can be used. In this embodiment, an LED array light source and a halogen lamp are used as the transmissive light source 2.

The transmissive light source 2 does not necessarily have to be arranged below the XY stage 18, and can be connected to the optical axis of the microscope 16 from a position other than the position below the XY stage 18 via a lens, a mirror, etc. as appropriate. It may be configured to irradiate light in the matching direction. Alternatively, light may be guided from a light source to a predetermined position by using an optical fiber bundle. As described above, when the light is guided using the optical fiber bundle, the heat ray of the light beam can be removed by the optical fiber.

By aligning the emitting end of each optical fiber with the position where the section C of the microarray A is arranged, each section C can be efficiently irradiated with light. Furthermore, when an LED is used as the light source, a stable light amount,
The life of the light source can be extended.

The excitation light source 6 is arranged so as to emit light from the side surface of the lens barrel of the microscope 16 into the lens barrel. Preferably, as the excitation light source 6, a light source containing a large amount of light of a wavelength component capable of exciting the fluorescent substance on the microarray A is selected. In this embodiment, a high pressure mercury lamp is used as the excitation light source 6.

The excitation filter 12 transmits only the wavelength component of the light emitted from the excitation light source 6 that can excite the fluorescent substance on the microarray A, and blocks the other wavelength components. Is configured. Also,
The absorption filter 10 is configured to transmit light having a wavelength of fluorescence emitted from the microarray A and not transmit light having a wavelength component that excites the fluorescent substance. The dichroic mirror 8 is transmitted through the excitation filter 12,
It is constructed and arranged so as to reflect light of a wavelength component capable of exciting the fluorescent substance and direct it toward the microarray A.

Further, the dichroic mirror 8 is constructed so as to transmit the light having the wavelength of the fluorescence emitted from the fluorescent substance on the microarray A.

In this embodiment, C is used as the fluorescent substance.
Using y3, the absorption filter 10 has a center wavelength of 61
A bandpass filter having a wavelength of 0 nm and a full width at half maximum of 75 nm is used as the excitation filter 12 with a center wavelength of 545 nm and a full width at half maximum of 3
The bandpass filter of 0 nm is the dichroic mirror 8
As such, a mirror that reflects light having a wavelength of about 570 nm or less and transmits light having a wavelength of more than that is used. The dichroic mirror 8, the absorption filter 10, and the excitation filter 12 are selected and configured according to the excitation wavelength of the fluorescent substance used and the wavelength of the fluorescence emitted by the fluorescent substance, and a plurality of fluorescent substances are used. In some cases, the microscope 16
In the above, it is preferable that the switch can be easily performed.

The optical axis of the CCD camera 4 is the microscope 16
It is attached to the end of the lens barrel of the microscope 16 on the side opposite to the objective lens 14 so as to align with the optical axis of. Preferably, as the CCD camera 4, a cooled CCD camera having a camera cooling function is used so that the weak fluorescence emitted from the microarray A can be imaged with less noise. In this embodiment, 1392 × 10
CC that has 40 effective pixels and is used by cooling to -30 ° C
A D camera is used. Further, in the present embodiment, the microscope 16 is an epi-fluorescence microscope, an objective lens having a magnification of 2 is used, a lens having a magnification of 0.5 is inserted in front of the CCD camera, and an optical system having a magnification of 1 is used. is doing.

The detection means 5 is configured to perform various image processings based on the image data picked up by the CCD camera 4 to detect the fluorescence emitted from the microarray A. The detection means 5 is, for example, the CCD camera 4
Interface for inputting image data from
It can be configured by a personal computer, a computer program for operating the personal computer, and the like.

The XY stage 18 is constructed so that the CCD camera 4 images each part of the microarray A, so that the microarray A can be moved within a horizontal plane. The focus stage (not shown) is C
In order to adjust the focus of the image of the microarray A projected on the CD camera 4, the XY stage 18 or the objective lens 14
Is configured to move up and down. Further, the control device 20 controls the XY stage 18 and the image pickup by the CCD camera 4.
ON / OF of moving and transmitting light source 2 and excitation light source 6
F, the movement of the focus stage, etc. are controlled. Alternatively,
ON / O is frequently used as the transmission light source 2 or the excitation light source 6.
When a type of light source that is not suitable for repeated use of FF is used, a shutter (not shown) for blocking light is provided in front of the transmissive light source 2 or the excitation light source 6, and the shutter is opened and closed. Are controlled by the control device 20. The control device 20 includes, for example, an actuator that operates each device, a personal computer that sends a signal to the actuator, and the like, and the personal computer can also serve as the personal computer used in the detection unit 5.

Next, with reference to FIG. 2, a microarray A used in the microarray reader according to the first or second embodiment of the present invention will be described. The microarray A shown in FIG. 2 is a microarray in which compartments C are formed by through holes, and a plurality of compartments C are arranged in an orderly manner. Such microarrays are described, for example, in WO 00/53.
736, JP-A 2000-60554, and JP-A 2000-78998.

In each compartment, a bio-related substance that can serve as a probe is fixed directly to the inner wall of the compartment or via a gel.

The structure of the base material portion B other than the section is not particularly limited as long as it has a light transmittance different from that of the section C. Further, the base material portion B and the section C only need to have a difference in light transmittance, and it does not matter which is transparent.

Preferably, the base material portion B is made of an opaque material. Here, “opaque” means a property that light is less likely to be transmitted than a section of the microarray A on which the probes are fixed, and does not necessarily mean that the light transmittance is 0%.

In FIG. 2, a hollow circular object is illustrated as the section C, but the outer shape of the section is not limited to a circle. Section C has a structure inner diameter of 170 μm and outer diameter of 220 μm
m.

In FIG. 2, the light transmittance is 1 in the thick portion of the section C and the resin base portion B including it.
The figure illustrates a microarray composed of a material whose content is less than or equal to%. The thick portion of the section C is formed of PMMA or polycarbonate resin mixed with 2% of carbon black. This section C has 5 rows in parallel and 4 rows in parallel.
They are arranged at equal intervals with a pitch of 20 μm, and the surrounding area is
It is included in a urethane resin mixed with 2.5% of carbon black.

Next, the operation of the microarray reader 1 according to the first embodiment of the present invention will be described. Here, a verification experiment for verifying the detection accuracy of the microarray reader 1 according to the present embodiment will be described before actually detecting the fluorescence emitted from the sample. In the verification experiment, a fluorescent substance is directly immobilized at a preset concentration on the gel-like material filled in each section C of the microarray A, and the fluorescence emitted from the fluorescent substance is detected.

First, the fluorescent substance is immobilized at different concentrations on the gel-like material filled in each section C of the microarray A. In this verification experiment, CY3 was used as the fluorescent substance. Table 1 shows the concentration of the fluorescent substance immobilized in each section C.

[0050]

[Table 1] As shown in Table 1, each section C is numbered from 1 to 25, the first row is numbered 1 to 5 in order from the left end, and the left end of the second row is numbered 6 In the same manner, numbers are given up to the number 25 on the right end of the fifth row. The center coordinates of each section C and the concentration of the immobilized fluorescent substance are as shown in Table 1. Also, the center coordinates and the numerical values of the inner diameter of each section C are
This is the design value of the microarray A.

Table 2 shows that the microarray A thus constructed was irradiated with light by a projector,
The results of measuring the position and spot diameter of the spot of the light transmitted through each section are shown. The values in Table 2 are the average values of the five measurements.

[0052]

[Table 2] Next, this microarray A is set on the XY stage 18 of the microarray reader 1 according to the first embodiment of the present invention, the light of the light source 2 for transmission is transmitted, and the transmitted light is imaged by the CCD camera 4. FIG. 3 shows an example of an image of a spot of transmitted light imaged in this way.
Each pixel forming the image of the spot is classified into a binary value by a predetermined threshold value, and a portion brighter than the predetermined threshold value is recognized as a spot portion and a darker portion than the predetermined threshold value is recognized as a portion other than the spot. Then, the spot diameter of the image of the spot shown in FIG. 3 is contracted by the detecting means 5. Here, the diameter of each spot is contracted by about 70 μm. Image-Pro PLUS, which is imaging software manufactured by MEDIA CYBERNETICS, is used for this image processing.
(Registered trademark) was used. That is, the Pr of this software
Morphological which is the Filter function in the ocess menu
In Erode, the diameter of each spot was shrunk by 70 μm using the 11 × 11 Circle option.

In FIG. 4, image processing is performed by the detecting means 5,
An example of the result of contracting the diameter of each spot is shown. However, as the software used for image processing and the image processing method, any appropriate software can be used. For example,
Here, the diameter of each spot is reduced by a fixed amount, but the diameter of each spot may be reduced at a constant rate,
It is also possible to set a circular area having a constant radius from the center position of the spot.

Table 3 shows an example of the results of measuring the position and size of each spot shown in FIG.

[0055]

[Table 3] Comparing Table 2 and Table 3, it can be seen that the center coordinates of each spot are in good agreement, and the spot diameter in Table 3 is smaller than that in Table 2 by about 70 μm. Therefore, Table 3
By measuring the fluorescence of each spot according to the position and size of each spot obtained as described above, the peripheral portion of each section C on the microarray A is excluded, and the measurement is performed on the region near the center of the section C. It will be possible.

Next, in the microarray A set on the XY stage 18 of the microarray reader 1,
Excitation light is emitted from the excitation light source 6, and the fluorescence from the fluorescent substance immobilized on the gel-like material filled in each section C arranged in the microarray A is imaged by the CCD camera 4. FIG. 5 shows an example of an image captured by the CCD camera 4. In addition, FIG. 6 shows the image of FIG.
The result which overlapped the outline of each spot measured from FIG. 4 is shown.

The detection means 5 calculates the average CCD reading value D _AV obtained by averaging the CCD reading values representing the brightness of each pixel in the contour line of FIG. FIG. 7 is a graph showing the relationship between the average CCD read value D _{AV of} each spot thus obtained and the concentration of the fluorescent substance immobilized on the corresponding spot. However, each average CC on the vertical axis
The D read value D _AV is a fluorescent substance concentration of 0 [nmol / ml], that is, a value obtained by averaging the CCD read values of all the spots to which the fluorescent substance is not immobilized, from the average value of the CCD read values of the respective spots. It is the reduced value. The average C on the vertical axis
Both the CD read value D _AV and the fluorescent substance concentration on the horizontal axis are shown on a logarithmic scale.

As shown in FIG. 7, the relationship between the fluorescent substance concentration and the average CCD read value D _AV is almost linear on the log-log graph. Therefore, the concentration of the fluorescent substance immobilized on the gel-like material in each section C can be obtained from the average CCD read value D _AV .

Next, the operation of the microarray reader 1 according to the second embodiment of the present invention will be described. First, the microarray A including the probes in each section C is prepared. On the other hand, a fluorescent substance is added to the sample to be analyzed, and this sample is allowed to act on the microarray A. When a certain sample acts on the probe having a predetermined structure, the probe and the sample react with each other to form a reaction product, so that the fluorescent substance applied to the sample is fixed in the section C of the microarray A. Therefore, the fluorescent substance exists only in the section C in which the probe having a structure capable of forming a hybrid with the analyte to be analyzed is fixed.

Before the reading of the microarray A is started, a uniform light diffusion plate (not shown) is set on the XY stage 18 of the microarray reader 1. Next, the excitation light source 6 irradiates the diffusion plate with light, and the light reflected by the diffusion plate is imaged by the CCD camera 4. Information of the captured diffused image is stored in the detection means 5 in order to correct the light amount unevenness of the excitation light source 6. The image taken here is an image of the irradiation spots of the excitation light in order to correct the irradiation spots of the excitation light on the sample surface in a later step, and is generally in a state where the diffuser plate is out of focus. Take an image with. Further, without using a diffusion plate, nothing is placed on the XY stage 18, that is, light is emitted from the excitation light source 6 in a state where nothing is present on the sample surface, and the CCD is in that state.
The image may be captured by the camera 4.

Next, the microarray A is mounted on the XY stage 1.
Set to 8. Microarray A on the XY stage 18
When the position of the XY stage 18 when setting is different from the position of the XY stage 18 when measuring the microarray A, the XY stage 18 is moved to the measurement position.

Next, the light source 2 for transmission transmits light to the microarray A. The light emitted from the transmission light source 2 and transmitted through the microarray A passes through the objective lens 14 to C
The image is taken by the CD camera 4. The image captured at this time is, for example, due to factors such as the dimensional accuracy of the microarray A, the array accuracy of the sections C of the microarray A, the positional accuracy when the microarray A is set on the XY stage 18, the moving accuracy of the XY stage 18, and the like. As shown in FIG.
The section C of the microarray A may not fit within the field of view of the CCD camera 4. Here, in the field of view of the CCD camera 4 in FIG. 9, in order to make the operation speed of the CCD camera 4 as fast as possible, the range for capturing an image is adjusted according to the arrangement of the section C of the microarray A used.

Next, the position of the entire section C of the microarray A is calculated by the detecting means 5 based on the information such as the position and number of the section C in the image taken by the CCD camera 4, and the section C of the microarray A is calculated. The XY stage 18 is moved by the control device 20 so that the whole can be imaged by the CCD camera 4. Then, the light source 2 for transmission again irradiates the microarray A with light. The light emitted from the transmission light source 2 and transmitted through the microarray A is
An image is taken by the CCD camera 4 through the objective lens 14. If the entire section C of the microarray A does not fit within the entire field of view of the CCD camera 4, the area gathered for each preset number of sections C fits within the field of view of the CCD camera 4, and XY Using the stage 18, the microarray A is sequentially moved to image all the sections C. At this time, the microarray A is sequentially moved in consideration of the movement accuracy of the XY stage 18 and the like, and all the sections C are
Instead of taking the image of
After imaging the section C and before moving the XY stage 18,
It is desirable to perform the fluorescence measurement together.

The spot of the transmitted light imaged by the CCD camera 4 is subjected to image analysis by the detecting means 5 and the position and size of each spot are measured. Further, the measured spot size is contracted by a predetermined amount by the detecting means 5, and the outline of the contracted spot is stored in the detecting means 5. Depending on the application, when capturing the transmitted light, the dichroic mirror 8 and the absorption filter 1 may be used.
0 and the excitation filter 12 may be moved so as to be off the optical axis of the microscope 16.

The rate of contracting the spot can be appropriately selected according to the magnitude of noise due to the influence of the base material portion B around the section C and the wall surface of the section C. For example, if the spots around the spot have large irregularities in shape and the immobilization rate of the probe is different between the center and the periphery of the spot, or if the autofluorescence of the wall surface of the base material portion B or section C is strong, the shrinkage is caused. The ratio is large, that is, only the image near the center of the section C is adopted. Generally, it is preferable to reduce the approximate diameter of the original spot to a size of 30 to 100%. The procedure of contracting the spot diameter by the detecting means 5 is the same as that in the verification experiment described above, and thus the description thereof is omitted.

Further, in a microarray of the type in which the probe is directly immobilized on the inner wall of each section, or in the type of microarray in which a gel-like material is provided only on the inner wall of each section, the probe is It exists only near the inner wall, and becomes hollow near the center. Therefore, in such a microarray, the transmitted light intensity near the center of the section becomes even stronger than the transmitted light intensity near the inner wall of the section. When reading this type of microarray,
Each pixel inside the spot is further classified by a second threshold,
The image of the hollow portion near the center of the spot may be removed for later analysis.

After imaging the spot of the transmitted light, the light of the transmissive light source 2 is blocked, and then the excitation light source 6 is turned on.
Alternatively, the excitation light of the excitation light source 6 is applied to the microarray A by opening the shutter of the excitation light source 6. The light emitted from the excitation light source 6 is incident on the excitation filter 12, and only the light having the wavelength necessary for exciting the fluorescent substance is transmitted. The excitation light transmitted through the excitation filter 12 is reflected by the dichroic mirror 8 and directed toward the objective lens 14 of the microscope 16. The excitation light reflected by the dichroic mirror 8 is condensed by the objective lens 14 and applied to the microarray A.

When the microarray A is irradiated with excitation light,
The fluorescent substance existing on the microarray A is excited by the excitation light and emits fluorescence having a wavelength different from that of the excitation light.
The fluorescence emitted from the microarray A is the objective lens 1
4. Filter 1 for absorption through the dichroic mirror 8
It is incident on 0. Further, the wavelength component of the excitation light reflected by the microarray A and returned to the dichroic mirror 8 is reflected by the dichroic mirror 8 and therefore hardly enters the absorption filter 10. The absorption filter 10 transmits the wavelength component of the fluorescence emitted from the fluorescent substance on the microarray A of the incident light and blocks the light of the wavelength component of the excitation light. The fluorescence that has passed through the absorption filter 10 is captured by the CCD camera 4.

The spot image of the fluorescence emitted from the fluorescent substance on the microarray A, which is picked up by the CCD camera 4, is previously picked up, and the shading correction is performed on the basis of the diffusion image stored in the detecting means 5. . That is, the light amount unevenness of the excitation light with which the microarray A is irradiated is
It is numerically corrected by the detection means 5.

Further, when the transmitted light is imaged, the dichroic mirror 8, the absorption filter 10, and the excitation filter 12 are used.
The presence or absence of an optical system by removing the lens from the optical axis of the microscope 16, or even if these are not removed, the aberration of the optical system used may cause a positional deviation of the image, and this deviation is stored in the detection means 5 in advance. It is preferable to correct the focus position of image capture, the positions of the fluorescence image and the spot image, and the like based on this. Next, the image of the contour of the spot of the transmitted light, which was previously captured and stored in the detection means 5, is superimposed on the shading-corrected fluorescent spot image. The CCD reading values of each pixel inside the outline of each fluorescent spot are averaged by the detecting means 5,
The average CCD reading for that spot is determined. The amount of the sample to be analyzed which has hybridized with each probe can be determined from the obtained average CCD reading value.

Further, in the shading-corrected fluorescent spot image, unnecessary portions of the image can be masked by the contour of the spot of the transmitted light stored in the detecting means 5. An image masked in this way can have its storage capacity reduced by a compression process.

With the microarray reader according to the first or second embodiment of the present invention, it is possible to recognize in advance which position on the microarray the partition is arranged, so that a specimen with low fluorescence intensity or Even in a microarray having low sequence accuracy, it is possible to accurately discriminate which probe the detected fluorescence is emitted from a sample forming a reaction product. Furthermore, the spot of the transmitted light imaged by the CCD camera is contracted,
Since the fluorescence is analyzed by using the image of a part of the spot, it is possible to effectively remove the influence of noise other than the fluorescence emitted by the fluorescent substance, such as autofluorescence of the section itself.

In the above-described first or second embodiment, one type of fluorescent substance is provided to one microarray, but a plurality of fluorescent substances having different fluorescence wavelengths are provided to one microarray. It can be given at the same time. In this case, the set of the dichroic mirror 8, the absorption filter 10, and the excitation filter 12 may be appropriately exchanged by the control device 20 in accordance with the wavelength of fluorescence to be detected. If a slight shift occurs in the image forming position of the spot by exchanging the set of the dichroic mirror 8, the absorption filter 10, and the excitation filter 12, this shift is measured in advance and used. It can be configured to correct the deviation according to a set of filters or the like.

Next, the third or fourth embodiment of the present invention will be described with reference to FIG. The microarray reading device 30 according to the third or fourth embodiment of the present invention differs from the above-described embodiments in that the transmission light source and the excitation light source are shared. Therefore, here, only the points in which the third or fourth embodiment is different from the above-described embodiment will be described,
Description of similar configurations, operations, and effects will be omitted.

FIG. 8 is a schematic block diagram of a microarray reader 30 according to the third or fourth embodiment of the present invention. As shown in FIG. 8, the reading device 30 for a microarray according to the second embodiment of the present invention includes a transmission / excitation light source 32 for irradiating the transmission light that transmits the microarray A and the excitation light that excites the fluorescent substance, and the transmission light source 32.・ Excitation light source 3
An excitation filter 34 for filtering the emitted light of 2;
Have. Further, the reading device 30 of the microarray has a CCD camera 4 for picking up transmitted light and fluorescent light, and a detection means 5 for analyzing the sample.

Further, the reading device 30 of the microarray is
Microscope 16 including an optical system including a dichroic mirror 8, an absorption filter 10, and an objective lens 14.
Have. Further, the microarray reader 30 moves the microarray A to a desired position in the horizontal plane X.
A focus stage (not shown) for moving the XY stage 18 or the objective lens 14 up and down in order to adjust the focus of the Y stage 18, the transmission / excitation light source 32, the CCD camera 4, and the image of the microarray A captured by the CCD camera 4. No.) and a control device 20 for controlling the XY stage 18
Have and.

In the present embodiment, the transmission / excitation light source 32 and the excitation filter 34 are arranged below the XY stage 18. The excitation filter 34 is configured to transmit only light having a wavelength that excites the fluorescent substance existing on the microarray A.

The other constituent elements of the third embodiment of the present invention are the same as those of the above-mentioned embodiment, and therefore their explanations are omitted.

Next, the operation of the microarray reader 30 according to the fourth embodiment of the present invention will be described. First, when irradiating the microarray A with transmitted light, the excitation filter 34 is removed from the optical axis, and the transmission / excitation light source 32 directly irradiates the microarray A with transmission light. The light emitted from the transmission / excitation light source 32 passes through the microarray A and is imaged by the CCD camera 4. Next, when the fluorescent substance on the microarray A is excited from the transmission / excitation light source 32, the excitation filter 34 is arranged on the optical axis, and the excitation light having a wavelength capable of exciting the fluorescent substance is moved downward. The microarray A is irradiated with the light.
The excitation light that has passed through the microarray A is reflected by the dichroic mirror 8 and is deflected to the outside of the optical axis that enters the CCD camera 4. The fluorescence emitted from the fluorescent substance excited by the irradiation of the excitation light passes through the dichroic mirror 8 and the absorption filter 10 and enters the CCD camera 4. The operation other than the above points is the same as that of the above-described embodiment, and thus the description thereof is omitted.

According to this embodiment, it is possible to use both the light source for transmission and the light source for excitation.

Further, since the excitation light is emitted from below the microarray A, all the light incident on the microscope 16 is the light transmitted through the section C of the microarray A. Therefore, unlike the first or second embodiment, the excitation light with which the microarray A is irradiated is reflected by the base portion B of the microarray A, and the reflected light does not enter the microscope 16. Thereby, the S / N of the fluorescence to be detected
The ratio can be improved.

[0082]

According to the microarray reader of the present invention, even in a sample having a low fluorescence intensity or a microarray having a low array accuracy, the probe-immobilized section to be detected without mixing a new substance with the probe. It is possible to accurately obtain the position and / or size information of the.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a microarray reader according to a first embodiment of the present invention.

FIG. 2 is a plan view of a microarray used in the microarray reader according to the first embodiment of the present invention.

FIG. 3 is a diagram showing an example of a spot of light transmitted through a microarray, which is imaged by a CCD camera.

FIG. 4 shows an example of a result of reducing the diameter of the spot of the transmitted light shown in FIG.

FIG. 5 is a diagram showing an example of a spot of fluorescence emitted from a microarray, which is imaged by a CCD camera.

6 is a diagram showing a result of superimposing the outline of the spot of the transmitted light having the reduced diameter shown in FIG. 4 on the fluorescent spot shown in FIG.

FIG. 7 is a graph showing the relationship between the CCD reading value of each fluorescent spot and the concentration of the fluorescent substance immobilized on the corresponding spot.

FIG. 8 is a schematic configuration diagram of a microarray reader according to a second embodiment of the present invention.

FIG. 9 is a diagram showing an example of a case where a spot of light transmitted through a microarray, which is imaged by a CCD camera, is deviated from the field of view of the CCD camera.

[Explanation of symbols]

A microarray B substrate portion C section 1 microarray reader 2 according to the first embodiment of the present invention 2 light source for transmission 4 CCD camera 5 detection means 6 light source for excitation 8 dichroic mirror 10 absorption filter 12 excitation filter 14 objective lens 14 16 microscope 18 XY stage 20 controller 30 microarray reader 32 according to the second embodiment of the present invention 32 transmission / excitation light source 34 excitation filter

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 G01N 33/53 M 37/00 102 37/00 102 (72) Inventor Chiaki Nagahama Yokohama, Kanagawa Prefecture Sanryo Rayon Co., Ltd. 10-1 Daikoku-cho, Tsurumi-ku, Yokohama F-Term (Reference) 2D043 AA01 BA16 CA04 EA01 EA13 FA01 FA02 GA08 GB01 HA01 HA05 HA09 JA03 KA02 KA05 LA03 NA01 NA11 2G054 CA22 CA23 FA19 FA20 FA32 GA04 4B029 AA23 BB15 BB17 BB20 CC03 FA10 FA12 4B063 QA01 QA18 QQ42 QQ53 QQ79 QQ96 QR32 QR48 QR56 QR82 QS33 QS34 QX01 QX10

Claims (12)

[Claims] 1. A reader for reading fluorescence emitted by a fluorescent substance from a bio-related substance microarray containing a plurality of compartments, which is reacted with an analyte to which the fluorescent substance is applied, and is a transmissive light source for transmitting light to the microarray. And an imaging light source that excites the fluorescent substance existing in the compartment and an image of the light that is emitted from the transmissive light source and that has passed through the microarray, and an image that captures the fluorescence emitted from the fluorescent substance that is excited by the excitation light source. Means and detection means for detecting the position and / or the size of the section arranged on the microarray based on the image of the light transmitted through the microarray, which is picked up by the image pickup means. Bio-related material microarray reader. 2. The microarray according to claim 1, wherein a portion other than the partition is opaque. 3. The reading device according to claim 1, further comprising means for moving the section from the position of the section arranged on the microarray into the field of view of the imaging means. 4. The detection means selects a part of an image of light transmitted through the microarray, which is imaged by the imaging means, and detects fluorescence of the selected part.
7. The microarray reader according to any one of 1. 5. The detecting means further comprises a correcting means for correcting a difference between an optical system for imaging the light transmitted through the microarray and an optical system for imaging the fluorescence emitted from the fluorescent substance. The microarray reader according to any one of 1 to 4. 6. The image pickup means is a CCD camera.
6. The microarray reader according to any one of 5 to 5. 7. The microarray reader according to claim 1, wherein the light source for transmission and the light source for excitation are combined. 8. The microarray reader according to claim 1, wherein the light source for transmission or the light source for excitation has a light emitting means and an optical fiber bundle for guiding the light emitted by the light emitting means. . 9. The microarray reader according to claim 1, wherein the light source for transmission or the light source for excitation is an LED light source. 10. A step of preparing a bio-related substance microarray including a plurality of compartments in which a specimen to which a fluorescent substance is applied is reacted, and a step of irradiating the microarray with light and imaging transmitted light transmitted through the microarray. The step of detecting the position and / or size of the section based on the transmitted light imaged in the step, and irradiating the microarray with excitation light to excite the fluorescent substance existing in the section of the microarray, An image of fluorescence emitted from the body, and a step of reading the fluorescence imaged in the step based on the position and / or the size of the section detected based on the transmitted light. Reading method of related substance microarray. 11. A step of preparing a bio-related substance microarray including a plurality of compartments reacted with a specimen to which a fluorescent substance is applied, and a step of irradiating the microarray with light and imaging transmitted light transmitted through the microarray. , Moving the position of the section to an appropriate position within the field of view to be imaged based on the transmitted light imaged in the step, and irradiating the microarray again with light, and imaging the transmitted light transmitted through the microarray A step of detecting the position and / or size of the section, a step of irradiating the microarray with excitation light to excite the fluorescent substance existing in the section of the macroarray, and imaging the fluorescence emitted from this fluorescent substance, The fluorescence imaged in the step is converted into the position and / or size of the section detected based on the transmitted light. A method for reading a microarray for bio-related substances, comprising: 12. The method according to claim 10, further comprising the step of masking an image obtained by capturing the fluorescence emitted from the fluorescent substance based on the position and / or the size of the section detected based on the transmitted light. A method for reading a microarray of a bio-related substance according to [4].