JP2004184379A - Method of reading microarray - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of reading microarray by which the intensity of fluorescence can be measured with accuracy, even from a microarray having a high S/N ratio and high dynamic range and low intensity for fluorescence, even when the wavelength of the fluorescence is close to that of exciting light. <P>SOLUTION: The intensity of the fluorescence can be measured with accuracy, even from the microarray having the high S/N radio and dynamic range and low intensity of fluorescence, by using an image obtained by picking up the image of light obtained by photographing the exciting light, in a state with the microarray not existing, as the background. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microarray reading method in which a sample provided with a fluorescent substance is caused to react with a microarray in which probes are immobilized and arranged, and irradiation with excitation light is performed to read the amount of excited fluorescence.
[0002]
[Prior art]
In recent years, genome projects for various organisms have been promoted, and a large number of genes, including human genes, and their base sequences have been rapidly elucidated. The function of the sequenced gene can be examined by various methods. As one of the promising methods, gene expression analysis using the revealed base sequence information is known. In this analysis method, for example, a different gene peptide, protein, antibody, or the like, in any of the surface of the substrate, the concave portion, the convex portion, the inner wall portion of the capillary, or the section of the polymer gel held in the hollow portion of the capillary. A microarray in which tens to tens of thousands of biologically-related substances such as biomaterials are immobilized is used. By utilizing various specific reactions to this probe, the relationship between the substance used as the probe and its biological function expression can be examined. Such a method is a DNA that enables simultaneous expression analysis of a large number of genes in order to perform comprehensive and systematic analysis of a very large number of genes at the level of one individual, which is being clarified through today's genome project. It has been developed as a new analytical method or methodology called a microarray method (DNA chip method).
[0003]
In the method of detecting a reaction product between a probe and a sample, first, a fluorescently labeled sample is reacted with a biological substance immobilized as a probe on a microarray. Then, by detecting the amount of fluorescence with a fluorescence measuring device such as a fluorescence laser scanner or a fluorescence microscope, it is possible to detect which probe and a reactant the fluorescently labeled sample has formed.
[0004]
[Problems to be solved by the invention]
Since an apparatus such as a fluorescent laser scanner uses a laser as an excitation light source for exciting a fluorescent substance, the wavelength band of the excitation light is narrow and the separation from the wavelength band of the fluorescence is relatively easy. For this reason, a high S / N when fluorescence is detected can be obtained. However, a mechanism for scanning the laser is required, which tends to be relatively expensive, and has a problem in maintainability.
[0005]
In contrast, an apparatus such as a fluorescence microscope uses a white light source to separate excitation light and fluorescence by an optical filter. For this reason, for example, when the fluorescent substance is changed, it is possible to respond by a relatively easy method of changing the combination of the optical filters, and maintenance is easy. However, when the wavelengths of the excitation light and the fluorescence are close to each other, it is difficult to completely separate the excitation light and the fluorescence from the characteristics of the optical filter, which causes a reduction in S / N and dynamic range. .
[0006]
The present invention relates to a microarray capable of accurately measuring fluorescence intensity even in an array having a high S / N and a high dynamic range and a low fluorescence intensity even when the excitation light wavelength and the fluorescence wavelength are close to each other. It is an object to provide a reading method.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have made intensive studies and as a result, have obtained an image obtained by capturing light obtained by irradiating excitation light in the absence of a microarray and obtaining an S / S The present inventors have found that it is possible to accurately measure the fluorescence intensity even in an array having a high N and a dynamic range and a low fluorescence intensity, and have completed the present invention.
[0008]
That is, the present invention provides a microarray reading method for reading fluorescence emitted by a fluorescent substance from a microarray in which a specimen to which a fluorescent substance has been applied is reacted on a section on which a probe is immobilized, wherein the microarray is provided at a predetermined sample holding position. A first step of preparing a microarray, a second step of irradiating the microarray with excitation light to excite a fluorescent substance present in a section of the macroarray, and imaging fluorescence emitted from the fluorescent substance, A third step of capturing light obtained by irradiating the excitation light with the microarray not at the holding position, wherein the image obtained in the third step is used as a background and the image obtained in the second step is used. From a microarray reading method.
[0009]
The present invention also provides a microarray reading method for reading fluorescence emitted from a fluorescent substance from a microarray obtained by reacting a sample to which a fluorescent substance has been applied to a section on which a probe is immobilized, wherein the method comprises the steps of: A first step of preparing a microarray, and a second step of irradiating the microarray with excitation light to excite a fluorescent substance present in a section of the macroarray, and imaging fluorescence emitted from the fluorescent substance a plurality of times. A third step of imaging light obtained by irradiating excitation light with no microarray at the sample holding position,
Using the image obtained in the third step as a background, subtracting each of the plurality of images obtained in the second step, and adding the plurality of images obtained by the subtraction, Reading method.
[0010]
Furthermore, the present invention provides a method for reading a microarray that reads fluorescence emitted from a fluorescent substance from a microarray in which a specimen to which a fluorescent substance has been applied is reacted on a section on which a probe is immobilized, wherein the method includes the steps of: A first step of preparing a microarray, and a second step of irradiating the microarray with excitation light to excite a fluorescent substance existing in a section of the macroarray, and capturing fluorescence emitted from the fluorescent substance at a plurality of exposure times. And a third step of imaging light obtained by irradiating the sample with the excitation light in a state where the microarray is not present at the sample holding position, and using the image obtained in the third step as a background, Each of the images obtained in the step is subtracted from each other, and the obtained images are subtracted by a value proportional to 1 / exposure time. And-normalized, to extract data from the obtained plurality of images, a reading method, the microarray characterized by.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0012]
FIG. 1 is a schematic configuration diagram of an apparatus for reading a microarray using a fluorescence microscope.
[0013]
As shown in FIG. 1, a microarray reader 1 that reads a microarray using a fluorescence microscope 16 includes an excitation light source 6 that irradiates the microarray A with light for exciting a fluorescent substance, and a specimen on the microarray A. A CCD camera 4 that is an imaging unit that captures fluorescence emitted from the fluorescent substance given to the device, and a detection unit 5 that analyzes a sample based on an image captured by the CCD camera 4.
[0014]
The fluorescence microscope 16 has an optical system including a dichroic mirror 8 that transmits or reflects predetermined light, an absorption filter 10, an excitation filter 12, and an objective lens 14 for the microarray A. Further, the microarray reader 1 holds the microarray A and moves it to a desired position in a horizontal plane, and the XY stage 18 adjusts the focus of the image of the microarray A captured on the CCD camera 4. Alternatively, it has a focus stage (not shown) for moving the objective lens 14 up and down, and a control device 20 for controlling the XY stage 18 and the like.
[0015]
Further, the excitation light source 6 is arranged so as to emit light from the side of the lens barrel of the fluorescence microscope 16 into the lens barrel. Preferably, as the excitation light source 6, a light source containing a large amount of light having a wavelength component capable of exciting the fluorescent substance on the microarray A is selected. In the present embodiment, a high-pressure mercury lamp is used as the excitation light source 6.
[0016]
The excitation filter 12 is configured to transmit only wavelength components that can excite the fluorescent substance on the microarray A in the light emitted from the excitation light source 6, and block other wavelength components. ing. In addition, 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 configured and arranged to reflect light having a wavelength component capable of exciting a fluorescent substance, which has passed through the excitation filter 12, and direct the light toward the microarray A. The dichroic mirror 8 is configured to transmit light having a wavelength of fluorescence emitted from a fluorescent substance on the microarray A.
[0017]
In the present embodiment, Cy3 was used as the fluorescent substance. A commercially available filter set for Cy3 is used as the absorption filter 10, the excitation filter 12, and the dichroic mirror 8, and a bandpass filter having a center wavelength of 610 nm and a half-value width of 75 nm is used as the absorption filter 10, respectively. As the filter 12, a band-pass filter having a center wavelength of 545 nm and a half value width of 30 nm was used. As the dichroic mirror 8, a mirror that reflects light having a wavelength of about 570 nm or less and transmits light having a wavelength longer 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 to be used and the wavelength of the fluorescence emitted from the fluorescent substance, and use a plurality of fluorescent substances. In such a case, it is preferable that the fluorescence microscope 16 be configured to be easily switchable.
[0018]
The CCD camera 4 is attached to the end of the lens barrel of the fluorescence microscope 16 opposite to the objective lens 14 so that its optical axis is aligned with the optical axis of the fluorescence microscope 16. Preferably, a cooled CCD camera having a camera cooling function is used as the CCD camera 4 so that weak fluorescent light emitted from the microarray A can be imaged with little noise. In the present embodiment, a CCD camera having 1392 × 1040 effective pixels and used by cooling to −30 ° C. is used. In the present embodiment, the fluorescence microscope 16 is an epi-illumination fluorescence microscope, uses an objective lens with a magnification of 2 ×, inserts a 0.5 × imaging lens in front of the CCD camera, and provides a 1 × optical microscope. Using the system.
[0019]
The XY stage 18 is configured to be able to move the microarray A in a horizontal plane because the CCD camera 4 captures an image of each part of the microarray A. The focus stage (not shown) is configured to move the XY stage 18 or the objective lens 14 up and down in order to adjust the focus of the image of the microarray A captured on the CCD camera 4. Further, the control device 20 controls imaging by the CCD camera 4, movement of the XY stage 18, ON / OFF of the excitation light source 6, movement of the focus stage, and the like. Alternatively, when a type of light source that is not suitable for use that is frequently turned on / off is used as the excitation light source 6, a shutter (not shown) for blocking light before the excitation light source 6 is used. And the opening and closing of the shutter is controlled by the control device 20. The control device 20 can be composed of, for example, an actuator that operates each device, a personal computer that sends a signal to the actuator, and the like.
[0020]
Next, a microarray A used for reading a microarray according to the embodiment of the present invention will be described with reference to FIG.
[0021]
As shown in FIG. 2, the microarray A is a microarray in which the sections C are formed by through holes, and a plurality of the sections C are orderly arranged. Such a microarray is prepared, for example, by the method described in WO 00/53736, JP-A-2000-60554, and JP-A-2000-78998.
[0022]
In each section, a bio-related substance that can serve as a probe is fixed directly on the inner wall of the section or via a gel.
[0023]
The configuration of the substrate portion B other than the section is not particularly limited as long as the section C has a different light transmittance. Further, it is sufficient that there is a difference in light transmittance between the base portion B and the section C, and it does not matter which is transparent.
[0024]
Preferably, the base portion B is made of an opaque material. Here, “opaque” means that the probe of the microarray A transmits light less easily than the fixed section, and does not necessarily mean that the light transmittance is 0%.
[0025]
FIG. 2 illustrates a hollow circular object as the section C, but the outer shape of the section is not limited to a circle. The structural inner diameter of the section C is 170 μm, and the outer diameter is 220 μm.
[0026]
FIG. 2 illustrates a microarray made of a material having a light transmittance of 1% or less in the thick portion of the section C and the resin base material B including the thick section. The thick portion of the section C is formed of PMMA or polycarbonate resin mixed with 2% of carbon black. The sections C are arranged in parallel at regular intervals of 5 rows and 5 rows at a pitch of 420 μm, and the periphery thereof is covered with a urethane resin mixed with 2.5% of carbon black.
[0027]
Next, a method for reading a microarray according to an embodiment of the present invention will be described.
[0028]
First, a microarray A in which each section C is filled with a polymer gel containing a probe is prepared. Further, depending on the application, the probe can be directly fixed to the inner wall of each section C. On the other hand, a fluorescent substance is provided to the sample to be analyzed, and this sample is caused 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 reactant, so that the fluorescent substance given to the sample is fixed in the section C of the microarray A. Therefore, the fluorescent substance is present only in the section C where the probe having a structure capable of forming a hybrid with the sample to be analyzed is fixed.
[0029]
Before starting reading the microarray A, nothing is placed on the XY stage 18 of the microarray reader 1 and light is emitted from the excitation light source 6, and the light detected in this state is emitted by the CCD camera 4. Take an image. At this time, it is desirable that a portion of the XY stage 18 to which the excitation light is irradiated from the objective lens 14 has a structure in which the excitation light is not blocked but transmitted. The information of the captured image is information representing the unevenness of the light amount of the excitation light source 6 and the noise captured by the CCD camera 4 as light other than the fluorescence emitted from the microarray A when performing the fluorescence measurement of the microarray A. The information becomes light representing light, and is stored in the detection unit 5 as the correction image 100. In order to remove noise due to dark current or the like of the CCD camera 4, the excitation image light source 6 is turned off or the shutter is closed so that excitation light is not emitted as the correction image 100. It is desirable to use an image obtained by irradiating light and exposing for the same exposure time as when the image was captured by the CCD camera 4 and subtracting the image captured by the CCD camera 4. FIG. 3 shows an example of the correction image 100 captured by the CCD camera 4.
[0030]
Next, the microarray A is set on the XY stage 18. When the position of the XY stage 18 when setting the microarray A on the XY stage 18 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.
[0031]
Next, the microarray A set on the XY stage 18 is irradiated with excitation light from the light source 6 for excitation, and the fluorescence immobilized on the gel material filled in each section C arranged in the microarray A The fluorescence from the substance is imaged by the CCD camera 4. The captured image is stored in the detection unit 5 as the fluorescence image 101. In addition, in order to remove noise due to dark current or the like of the CCD camera 4, the excitation light is used as the fluorescence image 101 in a state where the excitation light source 6 is turned off or the shutter is closed so that the excitation light is not irradiated. It is desirable to use an image obtained by irradiating and exposing for the same exposure time as when imaging with the CCD camera 4 and subtracting the image captured with the CCD camera 4. FIG. 4 shows an example of a fluorescence image 101 captured by the CCD camera 4.
[0032]
The fluorescence image 101 includes light spots of the excitation light source 6 and stray light due to reflections from the lenses and the like included in the fluorescence microscope 16, especially when the excitation light wavelength and the fluorescence wavelength are close to each other. Due to the characteristics of the filter 12, the dichroic mirror 8, and the absorption filter 10, it is difficult to completely separate the excitation light and the fluorescent light, and the background increases due to the stray light, resulting in a poor S / N. FIG. 5 shows a line profile of a straight line D on the image of FIG.
[0033]
Here, although the reflected light of the excitation light by the lens group constituting the objective lens 14 is reduced by the characteristics of the dichroic mirror 8 and the absorption filter 10, the stray light cannot be completely removed, but the stray light cannot be completely removed. Incident light is considered to be the main factor, and its intensity distribution is almost proportional to the correction image 100. The correction image 100 is also substantially proportional to the intensity distribution of the excitation light emitted from the objective lens 14 to the microarray A.
[0034]
Next, an image obtained by multiplying the correction image 100 by a predetermined value is subtracted from the fluorescent image 101 and stored in the detecting unit 5 as a stray light corrected fluorescent image 102. With this operation, the portion corresponding to the stray light is removed from the image.
[0035]
Next, in order to correct the fluorescence emission spot due to the spot of the excitation light, the stray light correction fluorescence image 102 is divided by the correction image 100 and further multiplied by the average value of the correction image 100 to obtain a stray light and excitation light spot correction fluorescence image 103. It is stored in the detecting means 5. It is desirable that the calculation of the images be performed as images in a floating-point format. In the above method, the average value of the correction image 100 is multiplied, but the present invention is not limited to this. For example, the maximum value, the minimum value, and a constant value can be set. When the constant value is set, even when the intensity of the excitation light source 6 decreases with the passage of time, the same level of excitation light is irradiated. Value can be corrected.
[0036]
In FIG. 6, the correction image of FIG. 3 is multiplied by a value corresponding to [(exposure time of the fluorescence image of FIG. 4) / (exposure time of the correction image of FIG. 3)] from the fluorescence image of FIG. FIG. 4 shows a stray light and excitation light spot corrected fluorescent image in which the image has been subtracted, then divided by the corrected image of FIG. 3, and further multiplied by the average value of the image of FIG. FIG. 7 shows a line profile of a straight line D on the image of FIG. Here, the correction image in FIG. 3 is multiplied by a value corresponding to [(exposure time of the fluorescent image in FIG. 4) / (exposure time of the correction image in FIG. 3)]. Is not limited to this, and can be set based on the relationship between the correction image 100 and the stray light intensity.
[0037]
Through the above operation, the effects of the stray light and the excitation light irradiation spots can be removed, and an image with improved S / N can be obtained. However, when the level of stray light is large, a sufficient exposure time cannot be obtained due to saturation of the CCD camera 4, and it may be difficult to measure a section having a weak fluorescence intensity. Therefore, a plurality of n fluorescence images 101-1, 101-2,..., 101-n are imaged with an exposure time in a range where the CCD camera 4 is not saturated, and the stray light and the excitation light irradiation spot are corrected for each of the images. Further, by adding the n pieces of the stray light and the excitation light spot corrected fluorescence images, the apparent exposure time can be extended, thereby enabling measurement of a section having a weak fluorescence intensity. The image obtained by adding the n pieces of the stray light and the excitation light spot corrected fluorescence image is stored in the detection unit 5 as the n-piece added stray light and the excitation light spot corrected fluorescence image 104-n. In FIG. 8, when n = 5, five fluorescent images are captured under the same conditions as the conditions under which the fluorescent image of FIG. 4 is captured, and the processing using the above-described method is performed. The image is shown in. FIG. 9 shows a line profile of a straight line D on the image of FIG. Here, in the above-described method, a plurality of fluorescent images are captured with an exposure time in a range where the CCD camera 4 does not saturate. However, the saturation of the CCD camera 4 is allowed for a section having a high fluorescence intensity (measurement values are ignored). ), When it is desired to accurately measure a section with a low fluorescence intensity, the exposure time should be extended within a range where saturation of a section with a high fluorescence intensity does not affect other sections to be measured, and a plurality of fluorescence images should be captured. You can also.
[0038]
Further, fluorescent images 101-1 (X), 101-2 (X),..., 101-n (X) captured in sections having a high fluorescent intensity with an exposure time (X) in a range where the CCD camera 4 is not saturated, .., 101-n (Y) are captured at the exposure time (Y) for accurately measuring a section having weak fluorescence intensity. Correction of the stray light and the excitation light irradiation spot and addition processing of the n pieces of the stray light and the excitation light spot correction fluorescence image are performed for each of them, and a plurality of n exposure light and excitation light spot correction fluorescence images 104-n (X), 104 for which n exposure times are added. −n (Y) is stored in the detection means 5.
[0039]
The reading of the microarray A is performed for each section of the microarray A. The image to be read is a stray light and excitation light spot corrected fluorescent image 103, or an n-number added stray light and excitation light spot corrected fluorescent image 104-n, or a multi-exposure time n-number added stray light and excited light spot corrected fluorescent image 104-n. (X), 104-n (Y), and the exposure time (Y) is used to read the stray light and the excitation light spot corrected fluorescent images 104-n (X), 104-n (Y) for the n exposure times for a plurality of exposure times. If the captured fluorescent image 101-m (Y), m = 1, 2,..., N is not saturated in the section to be read, n-multiple exposure time added stray light and excitation light spot corrected fluorescent image 104-n (Y) If the image is saturated in the section to be read, and the image is saturated in the section to be read, the stray light and the excitation light spot corrected fluorescence image 104-n (X) are added for a plurality of exposure times, and the CCD camera 4 shows a linear characteristic with respect to the exposure time. thing Et al., Its value Y / X times the readings. Here, the case of two exposure times X and Y has been described as a plurality of exposure times, but it is also possible to divide the exposure time into more exposure times.
[0040]
【The invention's effect】
According to the method for reading a microarray of the present invention, even in a microarray using a specimen having a low fluorescence intensity, it is possible to accurately and accurately obtain information on a section to be detected in which a probe is immobilized.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram showing an example of a microarray reader using a fluorescence microscope.
FIG. 2 is a plan view showing an example of a microarray used in a microarray reader.
FIG. 3 is an image showing an example of a correction image captured by a CCD camera.
FIG. 4 is an image showing an example of a fluorescence image captured by a CCD camera.
FIG. 5 is a graph showing a line profile of a straight line D on the image of FIG. 4;
FIG. 6 is an image showing a stray light and excitation light spot corrected fluorescent image obtained by applying the method of the present invention to the images of FIGS. 3 and 4;
FIG. 7 is a graph showing a line profile of a straight line D on the image of FIG. 6;
FIG. 8 is an image showing an example of a five-image addition stray light and excitation light spot corrected fluorescence image to which the method of the present invention is applied.
FIG. 9 is a graph showing a line profile of a straight line D on the image of FIG. 8;
[Explanation of symbols]
Reference Signs List A Microarray B Substrate portion C Section D Straight line displaying line profile on image 1 Microarray reader 4 CCD camera 5 Detecting means 6 Excitation light source 8 Dichroic mirror 10 Absorption filter 12 Excitation filter 14 Objective lens 16 Fluorescence Microscope 18 XY stage 20 Controller

Claims (3)

A method of reading a microarray that reads fluorescence emitted by a fluorescent substance from a microarray in which a specimen to which a fluorescent substance has been applied is reacted with a section in which a probe is immobilized,
A first step of preparing the microarray at a predetermined sample holding position;
A second step of irradiating the microarray with excitation light to excite a fluorescent substance present in the section of the macroarray, and capturing an image of fluorescence emitted from the fluorescent substance;
A third step of imaging light obtained by irradiating excitation light with no microarray at the sample holding position,
A method for reading a microarray, comprising subtracting an image obtained in the second step from an image obtained in the third step as a background. A method of reading a microarray that reads fluorescence emitted by a fluorescent substance from a microarray in which a specimen to which a fluorescent substance has been applied is reacted with a section in which a probe is immobilized,
A first step of preparing the microarray at a predetermined sample holding position;
A second step of irradiating the microarray with excitation light to excite a fluorescent substance present in a section of the macroarray, and capturing fluorescence emitted from the fluorescent substance a plurality of times;
A third step of imaging light obtained by irradiating excitation light with no microarray at the sample holding position,
Using the image obtained in the third step as a background, subtracting each of the plurality of images obtained in the second step, and adding the plurality of images obtained by the subtraction, Reading method. A method of reading a microarray that reads fluorescence emitted by a fluorescent substance from a microarray in which a specimen to which a fluorescent substance has been applied is reacted with a section in which a probe is immobilized,
A first step of preparing the microarray at a predetermined sample holding position;
A second step of irradiating the microarray with excitation light to excite a fluorescent substance present in a section of the macroarray, and capturing fluorescence emitted from the fluorescent substance at a plurality of exposure times;
A third step of imaging light obtained by irradiating excitation light with no microarray at the sample holding position,
Using the image obtained in the third step as a background, each is subtracted from the plurality of images obtained in the second step, and the plurality of images obtained by the subtraction are calculated with a value proportional to 1 / exposure time. A method for reading a microarray, which comprises normalizing and extracting data from a plurality of obtained images.

Images