JP2000180362A - Image data reading method and apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enhance the detection sensitivity of fluorescence without using a high power exciting light source high in cost in an image data reading apparatus. SOLUTION: A control unit 95 selects a laser beam suitable for exciting the fluorescent reagents (A1, A2, A3) of a sample 10 and another laser beam lower than this suitable laser beam in exciting efficiency but capable of exciting the fluorescent reagents among laser beams L1, L2, L3 mutually different in wavelength zone corresponding to the fluorescent reagents (A1, A2, A3) of the sample 10 placed on a sample stand 20 to irradiate the sample 10 with two selected laser beams at the same time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an image information reading method and an image information reading apparatus, and more particularly, to an improvement in a method of irradiating excitation light for irradiating a sample from which image information is to be read.

[0002]

2. Description of the Related Art Conventionally, in the field of biochemistry and molecular biology, a fluorescence detection system using a fluorescent reagent as a labeling substance has been known. According to this system, the gene sequence, the expression level of the gene, and the amount of the administered substance in the experimental mouse are obtained by photoelectrically reading the fluorescence image information on the sample such as the gel in which the biological substance labeled with the fluorescent reagent is distributed. Metabolism, absorption and excretion pathways
The state, the separation and identification of proteins, or the evaluation of molecular weight and characteristics can be performed.

[0003] For example, by electrophoresis in which a living cell in a suspension or a biological compound (protein, etc.) in a solution is moved by an electric charge to an anode or a cathode, a solution containing a plurality of DNA fragments is obtained. After adding the fluorescent reagent, a plurality of DNA fragments are electrophoresed on a gel support, or a plurality of DNA fragments are electrophoresed on a gel support containing a fluorescent reagent, or a plurality of DNA fragments are gel-supported. After electrophoresis on the body, the gel support is immersed in a solution containing a fluorescent reagent to obtain a gel support on which the fluorescently labeled DNA fragments are distributed. By irradiating with excitation light to excite the reagent, the fluorescence emitted on the gel support is photoelectrically read, thereby obtaining image information representing the distribution of the DNA fragment labeled with the fluorescence. By displaying a visible image (the distribution image (fluorescent image)) on the display unit such as a CRT based on image information, it is possible to evaluate, for example, the molecular weight of DNA fragments.

An image information reading apparatus for irradiating the gel support with excitation light, photoelectrically reading fluorescence to obtain image information, and displaying a fluorescent image on a display device based on the obtained image information, Widely used in biochemistry and molecular biology.

There are various types of fluorescent reagents for labeling biological substances, which are suitable for each type of biological substance to be detected. The wavelength of the excitation light for efficiently emitting each fluorescent reagent is described. The bands do not always match. For this reason, in order to be able to obtain distribution images of various biological substances by the above-described image information reading device, it is necessary to prepare so as to be able to emit excitation light in an emission wavelength band corresponding to each of a plurality of types of fluorescent reagents. There is a need to.

[0006] In order to meet such a demand, an image information reading apparatus equipped with a plurality of excitation light sources each emitting a plurality of excitation lights having different emission wavelength bands has been developed and has been put into practical use.

[0007]

By the way, the fluorescence to be read by the image information reading device is very weak, and if the detection sensitivity is to be further increased, a larger excitation energy is applied to reduce the emission intensity of the fluorescence. It is conceivable to increase.

However, since an excitation light source capable of providing high-power excitation energy is expensive, if such an excitation light source is provided in an image information reading apparatus, the manufacturing cost increases and the product price rises. become.

The present invention has been made in view of the above circumstances, and provides an image information reading method and an image information reading apparatus capable of increasing the fluorescence detection sensitivity without using a high-cost, high-power excitation light source. The purpose is to provide.

[0010]

According to the present invention, there is provided an image information reading method and an image information reading apparatus for simultaneously irradiating a sample with a plurality of types of excitation light prepared to excite a plurality of types of fluorescent reagents. Thus, the excitation energy applied to the fluorescent reagent is improved without increasing the amount of single excitation light.

That is, the image information reading method of the present invention is prepared as a method suitable for exciting each of a plurality of types of fluorescent reagents in a sample in which a biological substance labeled with a predetermined fluorescent reagent is distributed. Of the two or more excitation lights having different emission wavelength bands, the excitation light suitable for exciting the predetermined fluorescent reagent and the excitation efficiency lower than the suitable excitation light are used to excite the predetermined fluorescent reagent. Another one
Selecting the above excitation light, irradiating the selected two or more excitation lights simultaneously, and receiving the irradiation of the selected two or more excitation lights, and reading the fluorescence emitted from the fluorescent reagent photoelectrically. Reading image information representing a distribution image of the biological substance in the sample.

Here, the two or more excitation lights having different emission wavelength bands prepared as suitable for exciting each of a plurality of types of fluorescent reagents include, for example, excitation lights having different emission wavelength bands. When A1, A2, and A3 are prepared, the excitation light A1 is prepared to excite the fluorescent reagent B1 with high excitation efficiency, and the excitation light A2 is used to excite the fluorescent reagent B2 with high excitation light efficiency. Means that the excitation light A3 is prepared to excite the fluorescent reagent B3 with high excitation efficiency. Conventionally, the biological substance labeled with the fluorescent reagent B2 is distributed. When the sample to be read is to be read, the excitation light A2 prepared as suitable for exciting the fluorescent reagent B2 is selected, and only the excitation light A2 is irradiated on the sample. In the image information reading method of the present invention, not only the excitation light A2 having high excitation efficiency for the fluorescent reagent B2 but also other excitation light A1 as long as the fluorescent reagent B2 can be excited even if the excitation efficiency is lower than the excitation light A2. A3
Is also irradiated with the excitation light A2, so that the fluorescent reagent B2
Can be improved.

When photoelectrically reading the fluorescence emitted from the fluorescent reagent in the sample, it is desirable not to read the two or more excitation lights described above together with the fluorescence. It is only necessary to use a filter or the like whose band is limited so as to cut and pass the fluorescence.

An image information reading apparatus according to the present invention is an apparatus for carrying out the image information reading method according to the present invention, and includes a sample table on which a sample in which a biological substance labeled with a predetermined fluorescent reagent is distributed is placed. And two or more excitation light sources, each of which emits two or more excitation lights having different emission wavelength bands, each of which is suitable for exciting each of a plurality of types of fluorescent reagents,
Among the above excitation light sources, excitation light suitable for exciting the predetermined fluorescent reagent and one or more other excitation lights which have lower excitation efficiency than the suitable excitation light but can excite the predetermined fluorescent reagent A light source control means for controlling the light source so as to simultaneously irradiate the selected two or more excitation lights to the sample; and receiving the two or more excitation lights to emit the fluorescent reagent. Photoelectric reading means for reading image information representing a distribution image of the biological substance in the sample by photoelectrically reading fluorescence emitted from the sample.

It is preferable that a filter is further provided between the sample and the photoelectric reading means, the band being set so as to block the passage of the two or more excitation lights and allow the passage of the fluorescence. .

The irradiation of the excitation light in the image information reading method and the image information reading apparatus of the present invention may be performed by simultaneously irradiating the entire surface of the sample, or may be performed by applying a narrow beam of excitation light to the main scanning and the sub-scanning. A method of sequentially irradiating the entire surface of the sample by performing a combination with scanning may be employed.

[0017]

According to the image information reading method and the image information reading apparatus of the present invention, by simultaneously irradiating a sample with a plurality of types of excitation light prepared to excite a plurality of types of fluorescent reagents, The excitation energy applied to the fluorescent reagent can be improved without increasing the amount of single excitation light.

That is, among two or more excitation lights prepared for exciting each of a plurality of types of fluorescent reagents and having different emission wavelength bands, the method is suitable for exciting a specific fluorescent reagent to be read. Simultaneously irradiating the sample with excitation light and other excitation light that is not necessarily suitable for exciting that particular fluorescent reagent, but which can cause the fluorescent reagent to have a single excitation light Irradiating the sample with a larger amount of excitation energy than that of the sample, thereby increasing the amount of fluorescent light emitted from the sample. In addition, the increase in the excitation energy applied to the sample is not achieved by newly providing a single excitation light having an increased excitation energy, but rather as a suitable one for exciting other fluorescent reagents. Since only those prepared are diverted, it can be realized without incurring a substantial increase in cost.

By simultaneously irradiating a plurality of excitation lights of the same emission wavelength band suitable for exciting a specific fluorescent reagent, the excitation efficiency can be further increased and a single excitation light having a high excitation energy is used. Although it is possible to reduce costs, it is necessary to prepare multiple excitation lights for excitation lights of other emission wavelength bands in order to excite other fluorescent reagents. However, since the image information reading method and the image information reading apparatus of the present invention effectively use existing components, such a problem does not occur. Rather, it is useful in this regard.

[0020]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, specific embodiments of an image information reading method and an image information reading apparatus according to the present invention will be described in detail with reference to the drawings.

FIG. 1 is a diagram showing the configuration of an embodiment of the image information reading apparatus of the present invention. Image information reading device shown
Reference numeral 100 denotes a transparent sample table 20 on which a gel support sample 10 on which a biological substance labeled with a fluorescent reagent is distributed and which is disposed at a predetermined position, and a light emission suitable for exciting the fluorescent reagent A1. A gas laser 31 that emits a laser beam L1 having a wavelength, and a laser beam L2 having an emission wavelength suitable for exciting the fluorescent reagent A2.
And a first SHG laser 32 that emits a laser beam L3 having an emission wavelength suitable for exciting the fluorescent reagent A3.
An HG laser 33, a first dichroic mirror 41 transmitting the laser light L1 and reflecting the laser light L2, and a laser light L
A second dichroic mirror 42 for transmitting the laser beam L3 while passing through the first and L2, a photomultiplier (hereinafter referred to as PMT) 90 for photoelectrically detecting the fluorescence emitted when the fluorescent reagent of the sample 10 is excited, The laser light emitted from each of the lasers 31, 32, and 33 is applied to the sample 10 placed on the sample stage 20.
And an optical head 50 for guiding the fluorescence emitted from the sample 10 by the irradiation to the PMT 90, and two types of band-pass filters 81 and 82 disposed on the optical path from the optical head 50 to the PMT 90. Switchable filter set 80, main scanning means 60 for moving optical head 50 at a constant speed in the direction of arrow X, lasers 31, 32, 33, optical head
50, filter set 80 and PMT 90 are integrated with arrow Y
Sub-scanning means 70 for moving in the direction (the direction orthogonal to the arrow X direction), an amplifier 91 for logarithmically amplifying the detection signal detected by the PMT 90, and A / D conversion for A / D converting the amplified detection signal. Device 92 and the sample placed on sample stage 20
Laser light L according to 10 fluorescent reagents A1, A2, A3
At least two laser beams to be emitted are selected from among L1, L2, and L3, and the laser (light source) that emits the selected laser beam is controlled to emit the laser beam, and the selected laser beam is emitted. Filter set 80 according to the laser beam
And a control unit (Cont. Unit) 95 for selecting one of the band-pass filters 81 and 82 and controlling the selected band-pass filter to be disposed on the optical path.

Here, the gas laser 31 has a wavelength of, for example, 633 nm.
The first SHG laser 32 emits a laser beam L2 having a wavelength of 532 nm, and the first SHG laser 32 emits a laser beam L2 having a wavelength of 532 nm.
The second SHG laser 33 emits a laser beam L3 having a wavelength of 473 nm. The laser beam L1 having a wavelength of 633 nm emitted from the gas laser 31 is suitable for exciting a fluorescent reagent A1 called, for example, Cy5, and the wavelength 532 emitted from the first SHG laser 32.
The laser light L2 of nm is suitable for exciting a fluorescent reagent A2, for example, Cy3, and the laser light L3 of 473 nm emitted from the second SHG laser 33 is suitable for exciting, for example, a fluorescent reagent A3 of Florescein. I have.

FIG. 2A is a graph showing the emission characteristics of the fluorescent reagent A1 (Cy5 (Syfive): trade name of Amersham Pharmacia Biotech Co., Ltd.). Illumination efficiency is shown, and II shows the distribution of the spectrum contained in the emitted fluorescence K1. According to this figure, in the range of the wavelength band in which the fluorescent reagent A1 can be detected separately from the excitation light, it is efficient to excite with the excitation light L1 having the wavelength of 633 nm (luminous efficiency a
1) It can be seen that other wavelengths, for example, wavelength 532
When excited by the excitation light L2 of nm, the emission efficiency is inferior to the excitation light L1 (emission efficiency a2), but the emission of the fluorescence K1 is observed. Similarly, FIG. 2B is a graph showing the emission characteristics of the fluorescent reagent A2 (Cy3 (Sciety): trade name of Amersham Pharmacia Biotech Co., Ltd.). And II shows the distribution of the spectrum contained in the emitted fluorescence K2. According to this figure, in the range of the wavelength band in which the fluorescent reagent A2 can be detected separately from the excitation light, the excitation efficiency is high when the excitation light L2 having the wavelength of 532 nm is excited (the emission efficiency b
1) It can be seen that other wavelengths, for example, wavelength 473
When excited by the excitation light L3 of nm, the emission efficiency is inferior to the excitation light L2 (emission efficiency b2), but the emission of the fluorescence K2 is observed.

Therefore, when the reagent in the sample 10 to be read inputted from the host computer or the like is A1, the control unit 95 emits the laser beams L1 and L2 from the gas laser 31 and the first SHG laser 32, respectively. When the input reagent is A2, the lasers 31, 32, and 33 are controlled such that the first SHG laser 32 and the second SHG laser 33 emit laser beams L2 and L3, respectively.

The filter 81 of the two band-pass filters 81 and 82 of the filter set 80 has a band set so as to block transmission of light having a wavelength of 633 nm or less and allow transmission of light having a wavelength of 633 nm or more. Are arranged on the optical path when the laser light L1 is emitted, and the filter 82 has a wavelength of 532 nm.
The band is set so as to block transmission of the following light and allow transmission of light exceeding 532 nm.
Is controlled by the control unit so that the light is emitted and arranged on the optical path. Even when the laser light L2 is emitted, when the laser light L1 is emitted at the same time, the filter 82 is compared with the optical path and the filter 81 is arranged on the optical path.

The optical head 50 includes a plane mirror 51 for reflecting the laser beams L1, L2, and L3 traveling in the direction of the arrow X in a direction perpendicular to the sample 10 (upward in the drawing), and the reflected laser beam. Small hole 52a large enough to allow passage
Is formed, and a perforated mirror 52 that reflects most of the fluorescence emitted downward from the laser beam irradiation surface (the lower surface in the drawing) of the sample 10 in the direction of arrow X and makes it enter the PMT 90.
And a lens 53 that converts the fluorescent light emitted from the lower surface of the sample 10 into a substantially parallel beam.

Next, the operation of the image information reading apparatus 100 according to this embodiment will be described.

First, a gel support sample 10 (hereinafter, referred to as a gel support sample) on which a specific DNA labeled with the fluorescent reagent A1 is distributed on a sample stage 20.
A sample 10) is placed, and the type A of the fluorescent reagent
1 is input to the control unit 95 from a host computer (not shown). The control unit 95
The gas laser 31 and the first SHG laser 32 are controlled so as to emit the first laser light L1 and the second laser light L2 based on the input type A1 of the fluorescent reagent. The first laser light L1 is emitted, and the second laser light L2 is emitted from the first SHG laser 32. Meanwhile, control unit 95
Controls the filter set 80 such that the first filter 81 is arranged on the optical path between the optical head 50 and the PMT 90. The filter set 80 arranges the first filter 81 on the optical path. I do.

The first laser light L1 emitted from the gas laser 31 is applied to the first and second dichroic mirrors 41, 4
2 and travels in the direction of arrow X, and enters the plane mirror 51 of the optical head 50. On the other hand, the second laser beam L2 emitted from the first SHG laser 32 is reflected by the first dichroic mirror 41, travels in the direction of arrow X, passes through the second dichroic mirror 42, and passes through the first laser beam. Similarly to the light L1, the light enters the plane mirror 51 of the optical head 50.

Each of the laser beams L1 and L2 incident on the plane mirror 51 of the optical head 50 is reflected upward in the drawing, passes through the small hole 52a of the perforated mirror 52, and enters the lens 53.
A small area of the sample 10 placed on the sample stage 20 is irradiated through the lens 53.

At this time, the optical head 50 is moved at high speed and at a constant speed in the arrow X direction by the main scanning means 60, and the laser beams L1 and L2 scan the sample 10 in the arrow X direction in the main direction. During the main scanning, if the DNA labeled with the fluorescent reagent A1 exists in the minute area irradiated with the laser beams L1 and L2, the fluorescent reagent A1 is excited by the irradiated laser beams L1 and L2. If the DNA emits the fluorescence K1 and the DNA does not exist in the region, the fluorescence K1 is not emitted.

The amount of fluorescence K1 emitted when DNA labeled with the fluorescent reagent A1 is present is the amount of fluorescence emitted when excited only by the first laser beam L1 (the amount of light depending on the efficiency a1). ), The second laser light L
The amount of light is increased by the amount of light that is excited and emitted by 2 (the amount of light that depends on the efficiency a2).

The emitted fluorescent light K1 spreads out from the lower surface of the sample 10 and is emitted. The emitted fluorescent light K1 is turned into a substantially parallel downward beam in the figure by the lens 53 of the optical head 50. It is incident on 52. Of the fluorescent light K1 incident on the apertured mirror 52, a part of the fluorescent light incident on the small hole 52a proceeds downward as it is, but the diameter of the small hole 52a is minute compared to the beam diameter of the fluorescent light K1. Therefore, most of the fluorescence K1 is reflected by the reflection surface of the perforated mirror 52,
It proceeds in the direction along the arrow X direction. The fluorescence K1 that has traveled in the direction of the arrow X passes through the first bandpass filter 81 and enters the PMT 90.

The laser beams L1 and L irradiating the sample 10
A part of the sample 2 is scattered / reflected by the sample 10 or the sample stage 20 and travels in the direction of the PMT 90 similarly to the fluorescence K1, but is cut by the first band-pass filter 81 arranged on the optical path. Therefore, it does not enter the PMT 90.

The fluorescence K1 incident on the PMT 90 is
The signal is amplified and detected by photoelectric conversion, is read as a corresponding electric signal, is amplified by a logarithmic amplifier 91, is converted into a digital signal by an A / D converter 82, and is output to an external image processing device, analysis device, or the like. Used for processing and quantitative analysis.

When the reading of the image information by one main scanning is completed in this way, the optical head 50 is returned to the original position by the main scanning means 60, while the sub-scanning means 70 is in the meantime.
However, the lasers 31, 32, 33, the dichroic mirrors 41, 42, the optical head 50, the filter set 80, and the PMT 90 are integrally sub-scanned in the arrow Y direction. By repeating the main scanning and the sub-scanning described above, laser light L1 and L2 are irradiated over the entire surface of the sample 10, and digital image signals corresponding to each position of the sample 10 are obtained.
Image information representing the distribution of the DNA labeled with the fluorescent reagent A1 can be obtained.

It is needless to say that the sub-scanning does not necessarily have to be performed after the main scanning has been completed for one cycle, but may proceed simultaneously with the main scanning. The sub-scanning means 70 includes lasers 31, 32, 33, dichroic mirrors 41, 42, an optical head 50, and a filter set 80.
And the PMT 90 are shown as being integrally sub-scanned in the arrow Y direction.
The reference numeral 70 may cause the sample stage 20 to sub-scan in the direction opposite to the arrow Y direction.

As described above, according to the image information reading apparatus 100 of the present embodiment, the amount of fluorescence emitted by another laser beam is smaller than the amount of fluorescence emitted when excited by only a single laser beam. High intensity fluorescent light can be emitted by the amount of emitted light, and the S / N of the detection signal is improved.
Detection sensitivity can be increased.

Although not described, other fluorescent reagents A
When the sample 10 in which the DNA or the like labeled with 2 is distributed is to be read, the control unit 95 sets the second laser beam L2 and the second laser beam L2 based on the type A2 of the fluorescent reagent input from a host computer (not shown). Third laser beam L
The first SHG laser 32 and the second SHG laser 33 are controlled so as to emit the third laser light 3, whereby the second laser light L2 is emitted from the first SHG laser 32 and
The third laser beam L3 is emitted from the second SHG laser 33, while the control unit 95 operates the second filter
The filter set 80 controls the filter set 80 so that the second filter 82 is arranged on the optical path between the optical head 50 and the PMT 90, and the filter set 80 arranges the second filter 82 on the optical path. In the same manner as the
When the fluorescent reagent A2 in the medium is excited only by the single second laser beam L2,
High-intensity fluorescent light can be emitted by the amount of light that is excited and emitted by the laser light L3.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of an embodiment of an image information reading apparatus of the present invention.

FIG. 2 is a graph showing emission characteristics of a fluorescent reagent.

[Explanation of symbols]

 10 Sample 20 Sample stage 31 Gas laser 32,33 SHG laser 41,42 Dichroic mirror 50 Optical head 51 Flat mirror 52 Perforated mirror 53 Lens 60 Main scanning means 70 Sub scanning means 80 Filter set 81,82 Band pass filter 90 Photomultiplier 91 Amplifier 92 A / D converter 95 Control unit 100 Image information reading device L1, L2, L3 Laser beam K1 Fluorescence

 ──────────────────────────────────────────────────続 き Continued on front page F term (reference) 2G043 AA04 BA16 CA03 DA02 DA06 EA01 EA19 FA01 FA06 GA02 GA04 GB18 GB19 HA02 HA09 JA03 KA05 KA09 LA02 NA04 NA05 NA13 2G054 AA03 AA08 BB03 CA22 CE02 EA03 EB01 EB02 FA12 FA19 FA19 FA03 GB02 JA02

Claims (3)

[Claims] 1. A sample in which a biological substance labeled with a predetermined fluorescent reagent is distributed is prepared so as to be suitable for exciting each of a plurality of types of fluorescent reagents and has different emission wavelength bands from each other. Among the above-mentioned excitation lights, excitation light suitable for exciting the predetermined fluorescent reagent and one or more other excitation lights which have lower excitation efficiency than the suitable excitation light but can excite the predetermined fluorescent reagent And simultaneously irradiating the selected two or more excitation lights, and receiving the irradiation of the selected two or more excitation lights and emitting fluorescence from the fluorescent reagent, photoelectrically reading the sample, thereby obtaining the sample. An image information reading method which reads image information representing a distribution image of the biological substance in the inside. 2. A sample table on which a sample on which a biological substance labeled with a predetermined fluorescent reagent is distributed, and an emission wavelength band suitable for exciting each of a plurality of types of fluorescent reagents are different from each other. Two or more excitation light sources for separately emitting two or more excitation lights, of the two or more excitation light sources, excitation light suitable for exciting the predetermined fluorescent reagent, and excitation efficiency higher than the suitable excitation light Selecting means for selecting one or more other excitation lights that are low but capable of exciting the predetermined fluorescent reagent, and controlling the light source so as to simultaneously irradiate the sample with the selected two or more excitation lights. A light source control unit, and photoelectrically reads fluorescence emitted from the fluorescent reagent in response to the irradiation of the two or more excitation lights, thereby reading image information representing a distribution image of the biological substance in the sample. Reading Image information reading apparatus characterized by comprising a stage. 3. Between the sample and the photoelectric reading means,
3. The image information reading apparatus according to claim 2, further comprising a filter whose band is set so as to block passage of the two or more excitation lights and allow passage of the fluorescence.