JP2004317366A - Image processing method and image processor in microarray observation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an image processing method and an image processor in microarray observation which can extend a dynamic range and can acquire data having high luminance through low luminance. <P>SOLUTION: The image processing method in microarray observation is provided with a light detection means (1) which observes a microarray substrate, control means (2, 3) which control a laser output or PMT (photomultiplier) sensitivity when making observations by the light detection means (1), and image forming means (4) which forms an microarray image on the basis of data obtained by changing step-by-step the laser output or PMT sensitivity for the microarray substrate by the control means. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an image processing method and an image processing apparatus for microarray observation.
[0002]
[Prior art]
In recent years, microarray technology has been used as a technique for analyzing gene expression frequency and SNPs on a large scale. The microarray technology is a very useful technology with high throughput because it is possible to analyze a large number of genes at once.
[0003]
In this microarray technology, generally, a large number of probe DNA spots are arranged on a microarray substrate using a certain standard slide glass (approximately 7.5 cm × 2.5 cm). Detect using. Then, the obtained image data is digitized, and the expression frequency is quantified based on the numerical result. For fluorescence observation, confocal fluorescence detection scanners and the like are widely used, and it is possible to perform high-resolution, high-precision fluorescence detection by reducing background noise from the substrate itself. .
[0004]
[Problems to be solved by the invention]
As described above, a large number of probe DNA spots are immobilized on the microarray substrate, and each of them exhibits a different fluorescence intensity value, so that high- and low-luminance spots are mixed.
[0005]
However, at present, the fluorescence intensity range that can be analyzed on the microarray substrate, that is, the dynamic range, is larger than that of the fluorescence detection device. For this reason, data from all spots cannot be acquired due to the limitation of the apparatus performance.
[0006]
FIGS. 4A and 4B show microarray observation images according to a conventional example. The observation condition of (a) is a laser intensity of 40% and a PMT (photomultiplier tube) sensitivity of 100%, and the observation condition of (b) is a laser intensity of 60% and a PMT sensitivity of 100%.
[0007]
As shown in FIG. 4A, when trying to observe the spot with the highest luminance (white spot) among a large number of spots, little signal is obtained from other spots. Also, as shown in FIG. 4B, when the laser output of the fluorescence detection device is increased to observe a low-brightness spot, the number of spots at which the signal is saturated increases, and analysis may be performed. Can not.
[0008]
Further, conventionally, in order to increase the depth of focus while maintaining the brightness and the high resolution of the images, it has been considered to add a plurality of images having different in-focus positions and to perform appropriate processing on these images. As an example, Japanese Patent Application Laid-Open No. 4-83478 discloses that the distance and brightness of a partial image to each subject are automatically measured, input and addition conditions are set, and these input images are added together. A method is disclosed. This process is performed in order to eliminate the gradation jump and the defocusing of the image that occur during the observation with the microscope.
[0009]
On the other hand, it has been considered that not only an image with a large depth of focus is obtained but also acquired images are added to form one data for the purpose of observing samples of various luminances. As an example, in Japanese Patent Application Laid-Open No. H10-243289, a series of data is obtained by taking an observation sample of a microscope with a television camera capable of controlling the exposure time and performing addition and recovery processing.
[0010]
However, JP-A-10-243289 does not strictly reflect the relationship between the laser intensity or PMT sensitivity and the output signal during fluorescence observation because the brightness conversion is performed using only the length of the exposure time as an index. . In actual measurements, the laser intensity or PMT sensitivity output signal is not proportional and varies logarithmically. Therefore, data cannot be corrected using a uniform conversion rate using the length of the exposure time as an index.
[0011]
As described above, with the conventional method, it is not possible to obtain numerical values from all spots based on the microarray observation image obtained under one observation condition.
[0012]
An object of the present invention is to provide an image processing method and an image processing apparatus in microarray observation that can expand a dynamic range and acquire data from high luminance to low luminance.
[0013]
[Means for Solving the Problems]
In order to solve the problem and achieve the object, an image processing method and an image processing apparatus in microarray observation according to the present invention are configured as follows.
[0014]
(1) The image processing method for microarray observation according to the present invention controls laser output or PMT sensitivity when observing a microarray substrate, and changes the laser output or PMT sensitivity for the microarray substrate stepwise by this control. A microarray image is formed on the basis of the data obtained.
[0015]
(2) The image processing method for microarray observation of the present invention is the method described in (1) above, and forms a microarray image by synthesizing the data.
[0016]
(3) The image processing method for microarray observation according to the present invention is the method described in (1) above, and the relationship between the fluorescent dye concentration and the laser intensity or PMT sensitivity is checked in advance, and observation is possible based on the data. Set the appropriate conditions.
[0017]
(4) The image processing method for microarray observation according to the present invention is the method described in (1) above, and the position of a spot is determined by arranging a position marker on the microarray substrate, and a large number of spots are gridded. It is divided into shapes.
[0018]
(5) The image processing method in microarray observation of the present invention is the method described in (1) above, and recognizes the position of a spot outside the observation range in the first observation, and observes only that portion under different conditions. I do.
[0019]
(6) The image processing method for microarray observation of the present invention is the method described in (1) above, and uses a plurality of fluorescent dyes for the microarray substrate.
[0020]
(7) The image processing method for microarray observation of the present invention is the method described in (1) above, and uses a microarray substrate equipped with a dye dilution series whose dye concentration is known.
[0021]
(8) The image processing method for microarray observation of the present invention is the method described in (1) above, and the microarray substrate uses nucleic acids, antibodies, and proteins as probe DNA.
[0022]
(9) The image processing apparatus for microarray observation according to the present invention comprises: a light detection unit for observing the microarray substrate; a control unit for controlling laser output or PMT sensitivity when observing with the light detection unit; Image forming means for forming a microarray image based on data obtained by changing the laser output or the PMT sensitivity of the microarray substrate in a stepwise manner.
[0023]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0024]
(First Embodiment)
FIG. 1 is a block diagram illustrating a schematic configuration of the image processing apparatus according to the first embodiment of the present invention. In FIG. 1, a laser output control unit 2 and a PMT sensitivity control unit 3 are connected to a fluorescence detection device 1. The image forming unit 4 is connected to the fluorescence detection device 1. The fluorescence detection device 1 includes a PMT and a laser light source.
[0025]
In the first embodiment, when observing a microarray substrate including spots from high luminance to low luminance, the microarray is used for each fluorescent dye contained in the spot in advance (when a plurality of fluorescent dyes are used). The laser intensity on the substrate and the PMT sensitivity to the fluorescence from the microarray substrate are changed (modulated) stepwise, and the relationship between the fluorescent dye concentration and the PMT sensitivity or the laser intensity is examined. Using the measured data as a reference, image data is converted. In this case, since the characteristics are different depending on the fluorescent dye to be used, it is possible to reduce the difference between the dyes during the competition reaction. Note that the microarray substrate of the present embodiment uses nucleic acids, antibodies, and proteins as probe DNA.
[0026]
FIGS. 2A and 2B are diagrams showing the values of the fluorescence intensity associated with changes in PMT sensitivity or laser intensity. FIGS. 2A and 2B show data representing the relationship between PMT sensitivity or laser intensity and output value (fluorescent intensity) for a flat substrate to which one type of fluorescent dye Cy3 is applied.
[0027]
FIG. 2A shows a state in which the laser intensity of the laser light source of the fluorescence detection device 1 is fixed by the laser output control unit 2 and the PMT sensitivity control unit 3 obtains the PMT sensitivity while changing the PMT sensitivity from 50% to 100% every 5%. The fluorescence detection values obtained are graphed. Specifically, data on a glass microarray substrate Atlas Human 3.8 (Becton Dickinson) equipped with a dye dilution series prepared by diluting Cy3 dye (Amersham Pharmacia) at ^10-6 M in two-fold dilutions is summarized. As described above, the logarithmic change is observed between the fluorescent dye concentration and the PMT sensitivity, and when the same experiment is performed for various dye concentrations, data of the optimal observation conditions can be represented as one surface.
[0028]
In this graph, spots having a fluorescence intensity value exceeding 255 have saturated values, and data cannot be interpreted. When the fluorescence intensity value is low, the value is not stable due to various errors at the time of detection. Therefore, it is desirable to use the acquired data under the condition that a certain fluorescence intensity value is held for the analysis.
Further, as shown in FIG. 2B, a similar tendency is observed for data in which the laser intensity is changed. As described above, in the fluorescence observation, either the PMT sensitivity or the laser intensity is fixed, and once data is obtained by rough scanning, based on the data, each region can be observed under optimal observation conditions. Data is acquired by an intensity modulation method or a time width (pulse width) modulation method.
[0029]
FIG. 3 is a diagram illustrating a configuration of a microarray substrate with a marker. As shown in FIG. 3, the positions of the spots on the microarray substrate are determined by arranging some position markers so that the spots can be recognized, and a large number of spots are divided (divided) in a grid-like region. This makes it possible to recognize the position of the spot that was outside the observation range and could not be optimally observed in the first observation, and it is also possible to re-acquire the data of only that spot under further different conditions. It is considered that the observation time is reduced and the influence on the fading of the fluorescent dye or the like is reduced by randomly scanning the target spot instead of the fixed operation.
[0030]
When an absolute density is required, a known density spot for correction may be placed on the microarray substrate side.
[0031]
Through these steps, continuous data with a wide dynamic range can be acquired by reflecting conversion rates such as PMT sensitivity and laser intensity in observation data. Then, based on the acquired data, the image forming unit 4 can form a microarray image.
[0032]
(Second embodiment)
In the second embodiment, for a microarray substrate including spots from high luminance to low luminance, either the condition of PMT sensitivity or the laser intensity is fixed, and the conditions other than the fixed are stepwise changed. To obtain multiple data. Further, a plurality of control spots whose concentrations are known in a stepwise manner are placed on the microarray substrate, and data obtained by changing PMT sensitivity or laser intensity with respect to these control spots is used as correction data.
[0033]
Then, the image forming unit 4 adds (combines) each correction data to the acquired data of the microarray substrate to obtain a series of data, and combines the series of data to form a microarray image. I do. Note that the summing method can be realized by a generally used method.
[0034]
As described above, according to the present embodiment, it is possible to acquire data of a wide dynamic range from all spots of the microarray substrate including the probe DNA from high luminance to low luminance.
[0035]
Note that the present invention is not limited to the above embodiments, and can be appropriately modified and implemented without changing the gist.
[0036]
【The invention's effect】
According to the present invention, it is possible to provide an image processing method and an image processing apparatus in microarray observation that can expand a dynamic range and acquire data from high luminance to low luminance.
[Brief description of the drawings]
FIG. 1 is a block diagram showing a schematic configuration of an image processing apparatus according to an embodiment of the present invention.
FIG. 2 is a diagram showing a fluorescence intensity value accompanying a change in PMT sensitivity or laser intensity according to the embodiment of the present invention.
FIG. 3 is a diagram showing a configuration of a microarray substrate with a marker according to the embodiment of the present invention.
FIG. 4 is a microarray observation image according to a conventional example.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Fluorescence detection device 2 ... Laser output control part 3 ... PMT sensitivity control part 4 ... Image forming part

Claims (9)

Control the laser output or PMT sensitivity when observing the microarray substrate,
An image processing method in microarray observation, wherein a microarray image is formed based on data obtained by stepwise changing the laser output or the PMT sensitivity for the microarray substrate by this control. 2. The image processing method according to claim 1, wherein a microarray image is formed by combining the data. 2. The image processing method for microarray observation according to claim 1, wherein the relationship between the fluorescent dye concentration and the laser intensity or the PMT sensitivity is checked in advance, and observable conditions are set based on the data. 2. The image processing method for microarray observation according to claim 1, wherein the position of the spot is determined by arranging a position marker on the microarray substrate, and a large number of spots are divided into a grid. 2. The image processing method for microarray observation according to claim 1, wherein the position of the spot that was outside the observation range in the first observation is recognized, and only that part is observed under different conditions. 2. The image processing method for microarray observation according to claim 1, wherein a plurality of fluorescent dyes are used for the microarray substrate. 2. The image processing method for microarray observation according to claim 1, wherein a microarray substrate equipped with a dye dilution series having a known dye concentration is used. 2. The image processing method according to claim 1, wherein the microarray substrate uses a nucleic acid, an antibody, and a protein as probe DNA. Light detection means for observing the microarray substrate,
Control means for controlling laser output or PMT sensitivity when performing observation with the light detection means;
Image forming means for forming a microarray image based on data obtained by stepwise changing the laser output or PMT sensitivity to the microarray substrate by the control means;
An image processing apparatus for microarray observation, comprising:

Images