JP2002168784A - Pinhole position adjustment/positioning method and device of confocal scanner - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pinhole position adjustment/positioning method and a device of a confocal scanner capable of adjusting and positioning the position of a pinhole as required simply in a short time relative to a scanner using a confocal optical system. SOLUTION: A diffusion plate 51 is set on a sample stage 20, and the diffusion plate is irradiated with a laser beam, and the laser beam diffused by the diffusion plate and converged by the confocal optical system is received by a position sensor 50 capable of receiving light two-dimensionally, to thereby generate light-receiving position data. After calculating the optimum center position data corresponding to the optimum position where the center of the pinhole is to be positioned, the pinhole is arranged on the front of the position sensor, and a light source for pinhole position adjustment is positioned on an optical path of fluorescence of the confocal optical system, and light from the light source is received by the position sensor through the pinhole, to thereby generate pinhole position data, and the pinhole is moved so that the center of the pinhole agrees with the optimum position where the center of the pinhole is to be positioned.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for adjusting and positioning a pinhole position of a confocal scanner. More specifically, the present invention relates to a scanner using a confocal optical system, which is simple and short. The present invention relates to a method and an apparatus for adjusting and positioning a pinhole position of a confocal scanner capable of adjusting and positioning a pinhole position as desired in a time, and in particular, in a scanner using a confocal optical system, a plurality of scanners. Even if you have a light source of
The present invention relates to a pinhole position adjustment method and apparatus for a confocal scanner that can easily and quickly adjust and position a pinhole position as desired.

[0002]

2. Description of the Related Art When irradiated with radiation, the energy of the radiation is absorbed, stored, recorded, and then excited using electromagnetic waves in a specific wavelength range. A stimulable phosphor having a characteristic of emitting a stimulating amount of radiated light is used as a radiation detecting material, and a substance provided with a radioactive label is administered to an organism, and then the organism or a tissue of the organism is treated. Partly as a sample,
This sample is superimposed on a stimulable phosphor sheet provided with a stimulable phosphor layer for a certain period of time, whereby radiation energy is accumulated and recorded on the stimulable phosphor, and thereafter, the stimulable phosphor is stimulated by electromagnetic waves. Scans the stimulable phosphor layer to excite the stimulable phosphor, photoelectrically detects the stimulable light emitted from the stimulable phosphor, generates a digital image signal, and performs image processing. An autoradiography detection system configured to reproduce an image on display means such as a CRT or a recording material such as a photographic film is known (for example, Japanese Patent Publication No. 1-60784, Japanese Patent Publication No. No. 60782, Japanese Patent Publication No. 4-3952).

An autoradiography detection system using a stimulable phosphor sheet as a material for detecting an image, unlike the case of using a photographic film, not only does not require a chemical treatment called a development process, but also obtains an obtained image. By performing image processing on image data, there is an advantage that an image can be reproduced or quantitative analysis can be performed by a computer as desired.

[0004] On the other hand, a fluorescence detection system using a fluorescent substance as a labeling substance instead of a radioactive labeling substance in an autoradiography system is known. According to this system, by reading a fluorescent image, gene sequence, gene expression level, separation and identification of protein, or evaluation of molecular weight and characteristics can be performed. After adding a fluorescent dye to the solution containing the fragments,
A plurality of DNA fragments are electrophoresed on a gel support, or a plurality of DNA fragments are placed on a gel support containing a fluorescent dye.
After the NA fragment is subjected to electrophoresis, or a plurality of DNA fragments are subjected to electrophoresis on a gel support, the gel support is immersed in a solution containing a fluorescent dye, etc. By labeling, exciting a fluorescent dye with excitation light, and detecting the generated fluorescence, an image is generated, and the distribution of DNA on the gel support is detected. After electrophoresis on a support, the DNA is denaturated, and then at least a part of the denatured DNA fragment is transferred onto a transfer support such as nitrocellulose by Southern blotting to obtain the desired DNA. A probe prepared by labeling DNA or RNA complementary to the DNA to be labeled with a fluorescent dye and a denatured DNA fragment is hybridized, and the probe DNA
Alternatively, only the DNA fragment complementary to the probe RNA is selectively labeled, and the excitation light excites the fluorescent dye,
By detecting the generated fluorescence, an image can be generated and the distribution of the target DNA on the transfer support can be detected. Further, a DNA probe complementary to the DNA containing the target gene labeled with the labeling substance is prepared, hybridized with the DNA on the transcription support, and the enzyme is reacted with the complementary DNA labeled with the labeling substance. After binding, it is brought into contact with a fluorescent substrate to convert the fluorescent substrate into a fluorescent substance that emits fluorescence, and the excitation light excites the generated fluorescent substance and detects the generated fluorescence to generate an image. However, the distribution of the target DNA on the transcription support can also be detected. This fluorescence detection system has an advantage that a gene sequence or the like can be easily detected without using a radioactive substance.

[0005] In recent years, hormones, tumor markers, enzymes, antibodies, antigens, and the like have been placed at different positions on the surface of a carrier such as a slide glass plate, a membrane filter, or a gel support.
Abzyme, other proteins, nucleic acids, cDNA, D
NA, RNA, etc., can be specifically bound to a substance derived from a living body, and a specific binding substance whose base sequence, base length, composition, etc. are known is dropped using a spotter device,
Form a number of independent spots, followed by hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, mRNA
Such as, extraction, isolation, etc., collected from a living body, or, furthermore, a chemical substance, a biologically-derived substance that has been subjected to a treatment such as chemical modification, and is labeled with a labeling substance such as a fluorescent substance or a dye. Microarrays that irradiate excitation light to microarrays that have hybridized substances that have been hybridized, and that photoelectrically detect light such as fluorescence emitted from fluorescent substances, dyes, and other labeled substances, and analyze biologically derived substances Detection systems have been developed. According to this microarray detection system, a large number of specific binding substance spots are formed at different positions on a carrier surface such as a slide glass plate or a membrane filter at a high density, and a biological substance labeled with a labeling substance is formed. Is advantageous in that a substance derived from a living body can be analyzed in a short time.

[0006] In addition, hormones, tumor markers, enzymes, and the like can be located at different positions on the surface of a carrier such as a membrane filter.
Antibodies, antigens, abzymes, other proteins, nucleic acids,
Using a spotter, a specific binding substance that can specifically bind to a substance derived from a living body, such as cDNA, DNA, or RNA, and has a known base sequence, base length, or composition, is dropped using a spotter device. Form a number of independent spots, followed by hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNAs, DNs
A, mRNA, etc., a substance derived from a living body that has been collected from a living body by extraction, isolation, or the like, or that has been further subjected to a chemical treatment, a chemical modification, or the like, and that has been labeled with a radioactive labeling substance. The macroarray hybridized with the above is brought into close contact with the stimulable phosphor sheet on which the stimulable phosphor layer containing the stimulable phosphor is formed, and the stimulable phosphor layer is exposed, and then the luminescent phosphor layer is exposed. A macroarray detection system using a radioactive labeling substance that irradiates the stimulable phosphor layer with excitation light, photoelectrically detects the stimulable phosphor emitted from the stimulable phosphor layer, and analyzes substances derived from living organisms Has also been developed.

[0007]

An image reading device and a data generating device for generating data for biochemical analysis such as light emission amount data used in these systems include a scanner using a scanner and a two-dimensional sensor. Although there is a case where a scanner is used, there is an advantage that data can be generated with high resolution when a scanner is used, as compared with the case where a two-dimensional sensor is used.

Among scanners used as image reading devices used in these systems and data generating devices for generating data for biochemical analysis such as light emission amount data, scanners for a fluorescence detection system include gel support. Since it is intended to detect the fluorescent light emitted from the fluorescent substance by exciting the fluorescent substance contained in the body or transfer support, it is necessary to have an optical system with a deep depth of focus. Even in a scanner for an autoradiography detection system using a phosphor sheet, stimulable phosphor in the stimulable phosphor layer formed on the surface of the stimulable phosphor sheet emits stimulable phosphor. Therefore, while it is required to have an optical system with a certain depth of focus, a scanner for a microarray detection system requires a slide glass. The S / N ratio is improved by photoelectrically detecting the fluorescence from a fluorescent substance labeling a biological substance that has hybridized with a specific binding substance on the surface of a plate or a membrane filter. For the purpose, it is necessary to employ a confocal optical system and photoelectrically detect fluorescence through a pinhole.

In such a scanner for a microarray detection system, it is indispensable to adjust the position of the pinhole so that the fluorescence is focused in the pinhole.

Therefore, conventionally, a sample that emits fluorescence when excited by excitation light is set on a sample stage of a scanner for a microarray detection system, and the position of the pinhole is changed while the excitation light is actually changed. Illuminate the sample, and detect the fluorescence emitted from the sample photoelectrically by a photodetector, generate a signal, and position the pinhole at a position where the most signal is obtained. The position of was adjusted.

However, such a method has a problem that it is extremely complicated and requires a long time for adjustment.

In particular, since the wavelength of the excitation light that can be efficiently excited differs depending on the fluorescent substance that labels the sample,
As a scanner for a microarray detection system, one provided with a plurality of excitation light sources that emit excitation lights having different wavelengths has been proposed.However, when a confocal optical system is employed, the wavelength of fluorescence emitted from a fluorescent substance is reduced. Depending on the position, the focus of the fluorescence is two-dimensionally different, and the excitation light excites the fluorescent substance and the wavelength of the emitted fluorescence is longer than the wavelength of the excitation light. In a scanner for a microarray detection system having a plurality of excitation light sources that emit excitation lights having different wavelengths, the position of the pinhole is determined so that the fluorescence is focused in the pinhole with any of the excitation light sources. It will be essential to adjust.

Therefore, in the case of the conventional method, the sample which is excited by the excitation light and emits the fluorescence,
While setting it on the sample stage of the scanner for the microarray detection system and changing the position of the pinhole,
Actually, the operation of irradiating the excitation light and detecting the fluorescence emitted from the sample photoelectrically by the photodetector, generating the signal, and positioning the pinhole at the position where the most signal is obtained. Must be repeated for each excitation light source, and there is a problem that it takes an enormous amount of time to adjust the position of the pinhole.

In a scanner employing a confocal optical system as well as a scanner for a microarray detection system, similarly, it is extremely complicated to position a pinhole at a desired position, and a lot of time is required for adjustment. There is a problem that requires a confocal optical system, and in a scanner having a plurality of light sources, similarly, a scanner using a confocal optical system and, like a scanner having a plurality of excitation light sources,
There has been a problem that it takes an enormous amount of time to adjust the position of the pinhole.

Accordingly, the present invention relates to a scanner using a confocal optical system, which can easily and quickly adjust the position of a pinhole as desired and position it. It is an object of the present invention to provide a pinhole position adjustment / positioning method and apparatus.

Another object of the present invention is to provide a scanner using a confocal optical system, in which the position of a pinhole can be easily and quickly set as desired even if the scanner has a plurality of light sources. It is an object of the present invention to provide a method and an apparatus for adjusting and positioning a pinhole position of a confocal scanner capable of adjusting and positioning.

[0017]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
At least one laser excitation light source, a sample stage for setting a sample including a sample labeled with a fluorescent substance, and a laser beam emitted from the at least one laser excitation light source, on the sample set on the sample stage. By scanning, by the laser light, a scanning mechanism that excites a fluorescent substance contained in the sample, and a photodetector that is excited by the laser light and photoelectrically detects light emitted from the sample. A confocal optical system for guiding light emitted from the sample to the photodetector, and a pinhole forming member having a pinhole formed between the confocal optical system and the photodetector. A method of adjusting and positioning the pinhole position of a confocal scanner for adjusting and positioning the pinhole position of a confocal scanner to be used. Te, on the sample stage,
A diffusion plate is set, and a laser beam emitted from the at least one laser excitation light source is applied to the diffusion plate, and the laser beam diffused by the diffusion plate and collected by the confocal optical system is converted into light. Is received photoelectrically by a position sensor capable of receiving light two-dimensionally, light receiving position data corresponding to the light receiving position of the laser light is generated, and the light receiving position data is digitized to digital light receiving position data. After calculating the optimal center position data corresponding to the optimal position where the center of the pinhole should be located, the pinhole forming member is positioned in front of the position sensor, and the downstream side of the confocal optical system. A light source for adjusting a pinhole position is located in the optical path of the fluorescence, and light emitted from the light source for adjusting the pinhole position is transmitted to the pinhole forming unit. Digital pinhole position data obtained by photoelectrically receiving, by the position sensor, generating pinhole position data corresponding to the position of the pinhole and digitizing the pinhole position through the pinhole formed in And moving the pinhole forming member so that the center of the pinhole matches the optimal position where the center of the pinhole should be located, based on the optimum center position data and the position of the pinhole. Is achieved by adjusting and positioning the pinhole position of the confocal scanner.

According to the present invention, on the sample stage,
A diffuser is set, and a laser beam emitted from at least one laser excitation light source is irradiated on the diffuser, and the laser light diffused by the diffuser and collected by the confocal optical system is converted into a two-dimensional light. With a position sensor that can receive light
Receiving photoelectrically, generating light receiving position data corresponding to the laser light receiving position, digitizing the light receiving position data, and based on the digital light receiving position data obtained, the optimal position where the center of the pinhole should be located After calculating the optimum center position data corresponding to, the pinhole forming member is positioned in front of the position sensor, and a light source for pinhole position adjustment is positioned on the optical path of the fluorescent light downstream of the confocal optical system, Light emitted from a pinhole position adjusting light source is photoelectrically received by a position sensor via a pinhole formed in the pinhole forming member, and pinhole position data corresponding to the position of the pinhole. And the center of the pinhole is determined based on the digital pinhole position data and the optimum center position data obtained by digitizing. Move the pinhole forming member to adjust the position of the pinhole so that the center of the pinhole coincides with the optimal position to be located, and the position of the pinhole is configured to be positioned. The sample that emits fluorescence is set on the sample stage, and while changing the position of the pinhole, the excitation light is actually irradiated, and the fluorescence emitted from the sample is photoelectrically detected by the photodetector, and the signal is detected. Is generated, so that the position of the pinhole is positioned at the position where the most signal can be obtained.
The position of the pinhole can be adjusted and positioned as desired.

In a preferred embodiment of the present invention, the scanner includes a plurality of laser excitation light sources that emit laser lights having different wavelengths, and irradiates the diffusion plate with the laser light emitted from the plurality of laser excitation light sources. The laser light diffused by the diffusion plate and collected by the confocal optical system is photoelectrically received by the position sensor, and light-receiving position data corresponding to light-receiving positions of laser light having different wavelengths is obtained. Respectively, based on digital light receiving position data corresponding to the generated and digitized laser light receiving positions having different wavelengths, to generate optimum center position data corresponding to the optimum position where the center of the pinhole should be located. It is configured as follows.

According to a preferred embodiment of the present invention, the scanner includes a plurality of laser excitation light sources for emitting laser lights having different wavelengths, and irradiates the diffuser with laser light emitted from the plurality of laser excitation light sources; Diffused by the diffuser,
The laser light condensed by the confocal optical system is photoelectrically received by a position sensor, and light receiving position data corresponding to the light receiving positions of the laser light having different wavelengths is respectively generated, and the digitized wavelength is generated. Based on digital light receiving position data corresponding to the light receiving position of different laser light,
Since it is configured to generate optimal center position data corresponding to the optimal position where the center of the pinhole should be located,
A sample that is excited by the excitation light and emits fluorescence is set on the sample stage, and while changing the position of the pinhole, the excitation light is actually irradiated to detect the fluorescence emitted from the sample. Detector, photoelectrically detects and generates a signal, at the position where the most signal is obtained,
The operation of positioning the pinhole is repeated for each excitation light source, and the position of the pinhole can be determined much more easily and in a much shorter time than in the case of adjusting and positioning the pinhole. And the positioning can be performed.

In a further preferred aspect of the present invention, based on digital light receiving position data corresponding to digitized light receiving positions of laser light having different wavelengths, laser light having different wavelengths is received by the position sensor. Is determined as an optimum position where the center of the pinhole should be located.

In a further preferred aspect of the present invention, based on the optimum center position data and the digital pinhole position data, an optimum center position where the center of the pinhole should be located, and an image of the pinhole. Is displayed on display means, and the center of the image of the pinhole displayed on the display means is aligned with the optimum center position where the center of the pinhole displayed on the display means should be located. The position of the pinhole is adjusted and moved by moving the hole forming member.

According to a further preferred embodiment of the present invention, an optimum center position where the center of the pinhole should be located and an image of the pinhole are displayed based on the optimum center position data and the digital pinhole position data. To display,
The pinhole forming member is moved so that the center of the pinhole image displayed on the display means coincides with the optimal center position where the center of the pinhole displayed on the display means should be located, and the position of the pinhole is adjusted. The operator can adjust the position of the pinhole while observing the display means, so that when the excitation light is irradiated, the sample that emits fluorescence is placed on the sample stage. And changing the position of the pinhole, actually irradiating the excitation light, the fluorescence emitted from the sample is photoelectrically detected by the photodetector, and a signal is generated. The position of the pinhole can be easily and quickly adjusted, as compared to the case where the position of the pinhole is adjusted to position the pinhole at a position where a signal can be obtained, as desired. And integer, it is possible to position.

In still another preferred embodiment of the present invention, the center coordinates of the pinhole are calculated based on the digital pinhole position data, and the center coordinates of the pinhole are calculated based on the optimum center position data and the center coordinates of the pinhole. hand,
In order for the center of the pinhole to match the optimal position where the center of the pinhole should be located, calculate the distance to move the pinhole forming member, move the pinhole forming member, and The position of the hole is adjusted and positioned.

According to a further preferred embodiment of the present invention, the center coordinates of the pinhole are calculated based on the digital pinhole position data, and the center coordinates of the pinhole are calculated based on the optimum center position data and the center coordinates of the pinhole. The center is
In order to match the optimum position where the center of the pinhole should be located, calculate the distance to move the pinhole forming member, move the pinhole forming member, adjust the position of the pinhole, and position it Because it is composed
When the excitation light is irradiated, the sample that emits fluorescence is set on the sample stage, and while changing the position of the pinhole, the excitation light is actually irradiated, and the fluorescence emitted from the sample is detected by the photodetector. In comparison with the case where the position of the pinhole is adjusted so that the pinhole is positioned at the position where the most signal is obtained by detecting the photoelectrically and generating the signal, much easier and, In a very short time, the position of the pinhole can be adjusted and positioned as desired.

In a further preferred aspect of the present invention, the pinhole forming member is moved and fixed to the scanner so that the center of the pinhole coincides with an optimum position where the center of the pinhole should be located. By doing so, the pinhole is positioned.

In a further preferred aspect of the present invention, the pinhole forming member is configured to be movable by a pulse motor, and a center of the pinhole coincides with an optimum position where the center of the pinhole should be located. Optimum position data corresponding to a position is stored in a memory in the form of a drive pulse given to the pulse motor, and
Prior to exciting the fluorescent substance contained in the sample with the laser light emitted from the two laser excitation light sources, the drive pulse stored in the memory is read out and applied to the pulse motor. The position of the pinhole is adjusted and positioned.

According to a further preferred embodiment of the present invention, the pinhole forming member is configured to be movable by a pulse motor, and the center of the pinhole is located at a position corresponding to the optimum position where the center of the pinhole should be located. The corresponding optimal position data is stored in a memory in the form of a driving pulse to be supplied to a pulse motor, and the laser light emitted from at least one laser excitation light source is used to excite the fluorescent substance contained in the sample. Reading out the driving pulse stored in the memory and giving it to the pulse motor. Since the position of the pinhole is adjusted and positioned, the fluorescence from the fluorescent material carried on a gel support or the like that is not suitable for receiving light via the pinhole, or the accumulation property In a confocal scanner configured to be able to photoelectrically detect stimulable light emitted from the stimulable phosphor layer of the phosphor sheet, pinholes are positioned as desired, fluorescence is detected photoelectrically, and noise is reduced. It is possible to generate data for biochemical analysis with a small amount and a high S / N ratio.

In a further preferred aspect of the present invention, a plurality of pinholes are formed in the pinhole forming member.

[0030] In a further preferred aspect of the present invention, a position detecting diode is used as the position sensor.

[0031] The object of the present invention is also to provide at least one laser excitation light source, a sample stage for setting a sample containing a sample labeled with a fluorescent substance, and a laser light emitted from the at least one laser excitation light source. A scanning mechanism that scans the sample set on the sample stage and excites a fluorescent substance included in the sample by the laser light, and is excited by the laser light and emitted from the sample. A photodetector that photoelectrically detects light, and a confocal optical system that guides light emitted from the sample to the photodetector, wherein a pin is provided between the confocal optical system and the photodetector. The position of the pinhole of the confocal scanner where the pinhole forming member with the hole is arranged is adjusted, A hole adjustment and positioning device, at a position on the photoelectric detection surface of the photodetector, such that the photoelectric detection surface is located, and can be positioned, and receives light two-dimensionally, A position sensor capable of generating analog data, and configured to be located on the optical path of the fluorescent light downstream of the confocal optical system and to be retracted from the optical path of the fluorescent light downstream of the confocal optical system. A light source for adjusting a pinhole position capable of emitting light toward the position sensor, an A / D converter for digitizing analog data generated by the position sensor, and the at least one A laser beam emitted from a laser excitation light source is received and generated via the confocal optical system, and a light receiving position of the laser beam digitized by the A / D converter. Based on the light receiving position data corresponding to, the optimal center position data corresponding to the optimal position where the center of the pinhole should be located, the light emitted from the pinhole position adjusting light source by the position sensor Digital pinhole position data corresponding to the position of the pinhole which is photoelectrically received, generated and digitized by the A / D converter via the pinhole formed in the pinhole forming member. And the arithmetic processing means for comparing the optimum center position data with the optimum center position data.

According to the present invention, the pinhole position adjusting / positioning device of the confocal scanner can be positioned at the photoelectric detection surface of the photodetector so that the photoelectric detection surface is located, and And a position sensor capable of receiving light two-dimensionally and generating analog data, and a fluorescent sensor located on the downstream side of the confocal optical system and retracted from the optical path of fluorescent light on the downstream side of the confocal optical system. A light source for pinhole position adjustment capable of emitting light toward the position sensor, an A / D converter for digitizing analog data generated by the position sensor, and a position sensor Laser light emitted from at least one laser excitation light source is received and generated via a confocal optical system, and is converted by an A / D converter into a light receiving position of the digitized laser light. Optimum center position data corresponding to the optimum position where the center of the pinhole should be located is calculated based on the received light position data, and the light emitted from the pinhole position adjustment light source by the position sensor is transmitted to the pinhole forming member Digital pinhole position data corresponding to the position of the pinhole which is photoelectrically received, generated and digitized by the A / D converter through the pinhole formed in Is provided on the sample stage.
A diffusion plate is set, and a laser beam emitted from at least one laser excitation light source is irradiated on the diffusion plate. The laser beam diffused by the diffusion plate and collected by the confocal optical system is photoelectrically converted by the position sensor. Receiving light, and generating light receiving position data corresponding to the light receiving position of the laser beam, and processing the light receiving position data based on the digital light receiving position data obtained by digitizing the light receiving position data with an A / D converter. By calculating the optimal center position data corresponding to the optimal position where the center of the pinhole should be located,
The pinhole forming member is positioned in front of the position sensor, and a light source for pinhole position adjustment is positioned in the optical path of the fluorescent light downstream of the confocal optical system. Is photoelectrically received by a position sensor via a pinhole formed in the pinhole forming member, and pinhole position data corresponding to the position of the pinhole is generated. The calculated digital pinhole position data and the optimum center position data are compared by arithmetic processing means, and the pinhole formation is performed so that the center of the pinhole matches the optimum position where the center of the pinhole should be located. By moving the member, the position of the pinhole can be adjusted and positioned, so that the sample that emits fluorescence when irradiated with excitation light can be used. While setting it on the sample stage, changing the position of the pinhole, actually irradiating the excitation light, the fluorescence emitted from the sample is photoelectrically detected by the photodetector, and the signal is generated. To position the pinhole at a position where many signals can be obtained,
Compared with the case where the position of the pinhole is adjusted, the position of the pinhole can be adjusted and positioned as desired in a simple and short time.

In a preferred embodiment of the present invention, the apparatus further comprises display means capable of displaying visible data, wherein the arithmetic processing means determines an optimum position at which the center of the pinhole should be located based on the optimum center position data. A position is displayed on the display means, and an image of the pinhole is displayed on the display means based on the digital pinhole position data.

According to a preferred embodiment of the present invention, the pinhole position adjusting / positioning device of the confocal scanner further comprises a display means capable of displaying visible data, and the arithmetic processing means is configured to execute the processing based on the optimum center position data. Since the optimum position where the center of the pinhole should be located is displayed on the display means, and an image of the pinhole is displayed on the display means based on the digital pinhole position data. While observing the display means, the position of the pinhole can be adjusted, and thus, when irradiated with the excitation light, the sample that fluoresces,
While setting it on the sample stage, changing the position of the pinhole, actually irradiating the excitation light, the fluorescence emitted from the sample is photoelectrically detected by the photodetector, and a signal is generated. Adjust the pinhole position easily and in a short time, as compared to adjusting the pinhole position to position the pinhole at a position where many signals can be obtained. And positioning can be performed.

In still another preferred embodiment of the present invention, the arithmetic processing means calculates center coordinates of the pinhole based on the digital pinhole position data, and calculates the optimum center position data and the pinhole. The center of the pinhole is configured to calculate a distance to move the pinhole forming member in order to match an optimum position where the center of the pinhole should be located, based on the center coordinates of the pinhole. I have.

According to a further preferred aspect of the present invention, the arithmetic processing means calculates the center coordinates of the pinhole based on the digital pinhole position data, and calculates the center coordinates of the pinhole based on the optimum center position data and the center coordinates of the pinhole. Since the center of the pinhole is configured to calculate the distance by which the pinhole forming member is to be moved in order to match the optimum position where the center of the pinhole should be located, it is calculated by the arithmetic processing means. According to the distance to move the pinhole forming member, the position of the pinhole can be adjusted and positioned only by moving the pinhole forming member, and therefore, when the excitation light is irradiated,
The sample that emits fluorescence is set on the sample stage, and while changing the position of the pinhole, the excitation light is actually irradiated, and the fluorescence emitted from the sample is photoelectrically detected by the photodetector, and the signal is detected. Is generated and the pinhole position is adjusted much more easily and in a very short time than when the pinhole position is adjusted so that the pinhole is positioned at the position where the most signal can be obtained. The position can be adjusted and positioned as desired.

In a further preferred aspect of the present invention, the position sensor is constituted by a position detecting diode.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below in detail with reference to the accompanying drawings.

FIG. 1 is a schematic perspective view of a scanner in which the position of a pinhole is positioned by using the pinhole position adjusting / positioning method of a confocal scanner according to a preferred embodiment of the present invention.

As shown in FIG. 1, the scanner has 64
A first laser excitation light source 1 that emits a laser beam 4 having a wavelength of 0 nm, a second laser excitation light source 2 that emits a laser beam 4 of a wavelength of 532 nm, and a third laser excitation that emits a laser beam 4 having a wavelength of 473 nm. And a light source 3. In the present embodiment, the first laser excitation light source is constituted by a semiconductor laser light source, and the second laser excitation light source 2 and the third laser excitation light source 3 are configured to generate a second harmonic (Second harmonic).
Harmonic Generation) elements.

The laser light 4 generated by the first laser excitation light source 1 is collimated by a collimator lens 5 and then reflected by a mirror 6. In the optical path of the laser light 4 emitted from the first laser excitation light source 1 and reflected by the mirror 6, the first dichroic mirrors 7 and 532nm transmitting the 640nm laser light 4 and reflecting the 532nm wavelength light are provided. A second dichroic mirror 8 that transmits light having the above wavelength and reflects light having a wavelength of 473 nm is provided. The laser light 4 generated by the first laser excitation light source 1 is used as a first dichroic mirror. The light passes through 7 and the second dichroic mirror 8 and enters the optical head 15.

On the other hand, the laser light 4 generated from the second laser excitation light source 2 is collimated by the collimator lens 9 and then reflected by the first dichroic mirror 7 to change its direction by 90 degrees. Then, the light passes through the second dichroic mirror 8 and enters the optical head 15.

The laser light 4 generated from the third laser excitation light source 3 is collimated by the collimator lens 10 and then reflected by the second dichroic mirror 8 to change its direction by 90 degrees. After that, the light enters the optical head 15.

The optical head 15 includes a mirror 16, a perforated mirror 18 having a hole 17 formed in the center thereof, and a lens 19. The laser beam 4 incident on the optical head 15 is Reflected and perforated mirror 18
The sample carrier 21 set on the sample stage 20 through the hole 17 and the lens 19 formed in
Incident on top. The sample stage 20 is configured to be movable in the XY directions in FIG. 1 by a scanning mechanism (not shown).

The scanner shown in FIG. 1 uses a slide glass plate as a carrier, and a microarray in which a number of spots of a sample selectively labeled with a fluorescent dye are formed on the slide glass plate. Scans to excite the fluorescent dye, photoelectrically detects the fluorescence emitted from the fluorescent dye, and can generate data for biochemical analysis.

Further, the scanner shown in FIG. 1 uses a gel support as a carrier, and a large number of spots of a sample selectively labeled with a fluorescent dye are formed on one or a plurality of spots formed on the gel support. The sample carrier holding the microarray is scanned by the laser light 4 to excite the fluorescent dye, and the fluorescence emitted from the fluorescent dye is photoelectrically detected to generate data for biochemical analysis. Along with a carrier such as a membrane filter in which a number of spots of a sample selectively labeled with a radioactive labeling substance are formed, a stimulable phosphor sheet on which a stimulable phosphor layer containing a stimulable phosphor is formed. The stimulable phosphor layer of the stimulable phosphor sheet, on which positional information of the radioactive labeling substance obtained by exposing the stimulable phosphor layer is recorded, is scanned by the laser light 4. To excite the stimulable phosphor, by detecting the stimulated emission released from the stimulable phosphor photoelectrically, and is capable of generating structured data for biochemical analysis.

A microarray using a slide glass plate as a carrier is produced, for example, as follows.

First, the surface of the slide glass plate was
Pretreated with an L-lysine solution or the like, and then, at a predetermined position on the surface of the slide glass plate, cDNAs, which are a plurality of specific binding substances having different base sequences and different from each other.
Is dropped using a spotter device.

On the other hand, mRNA as a specimen is extracted from living cells, and RNA having poly A at the 3 ′ end is extracted from the mRNA. When synthesizing cDNA from the RNA having poly A at the end thus extracted, Cy-5 (registered trademark) which is a labeling substance is present to generate a probe DNA labeled with Cy-5.

The probe DNA labeled with Cy-5 thus obtained is prepared in a predetermined solution, gently placed on the surface of a slide glass onto which cDNA as a specific binding substance has been dropped, and hybridized.

When a gel support is used as a carrier, a microarray is similarly produced.

FIG. 2 is a schematic perspective view of the microarray 22 thus obtained. In FIG. 2, reference numeral 23 denotes the dropped cDNA.

On the other hand, in the stimulable phosphor layer of the stimulable phosphor sheet, for example, positional information of a radioactive labeling substance is recorded as follows.

The surface of a carrier such as a membrane filter is pre-treated, and cDNAs, which are a plurality of specific binding substances whose base sequences are known and are different from each other, are placed at predetermined positions on the surface of the carrier such as a membrane filter. Use the device to drip.

On the other hand, mRNA as a specimen is extracted from living cells, and RNA having poly A at the 3 ′ end is extracted from the mRNA. When synthesizing cDNA from the RNA having poly A at the terminal thus extracted, a radiolabeled substance is present to generate a probe DNA labeled with the radiolabeled substance.

The probe DNA labeled with the radiolabeled substance thus obtained is prepared in a predetermined solution, and gently placed on the surface of a carrier such as a membrane filter onto which cDNA as a specific binding substance has been dropped, and hybridized. .

Next, the stimulable phosphor layer formed on the stimulable phosphor sheet is superimposed on the surface of a carrier such as a membrane filter on which the hybridized sample is formed, and is kept in close contact for a predetermined time. By doing so, at least part of the radiation emitted from the radiolabeled substance on the carrier such as a membrane filter is absorbed by the stimulable phosphor layer formed on the stimulable phosphor sheet, and the position information of the radiolabeled substance is obtained. Is recorded in the stimulable phosphor layer.

When the laser beam 4 is incident on the sample 22 from the optical head 15, if the sample 22 is a microarray, the fluorescent material is excited by the laser beam 4 to emit fluorescent light 25. When 22 is a stimulable phosphor sheet, the stimulable phosphor contained in the stimulable phosphor layer is excited, and the stimulable phosphor 25 is emitted.

The fluorescence or stimulating light 25 emitted from the sample 22 is converted into parallel light by the lens 19 of the optical head 15 and is reflected by the perforated mirror 18.
Any one of the filters 28a, 28a, 28b, 28c, 28d of the filter unit 27 having four filters 28a, 28b, 28c, 28d.
The light is incident on 28b, 28c, 28d.

The filter unit 27 is configured to be movable in the left-right direction in FIG. 1 by a motor (not shown), and predetermined filters 28a, 28b, 28c, 28d are provided depending on the type of laser excitation light source used. , Or in the optical path of the fluorescent or stimulating light 25.

Here, the filter 28a is a filter used when the first laser excitation light source 1 is used to excite the fluorescent substance contained in the sample 22 and read the fluorescence 25, and has a wavelength of 640 nm. Cut the light of 64
It has the property of transmitting light having a wavelength longer than 0 nm.

The filter 28b is a filter used when the second laser excitation light source 2 is used to excite the fluorescent dye contained in the sample 22 and read the fluorescence 25, and has a wavelength of 532 nm. Cut light, 532
It has the property of transmitting light having a wavelength longer than nm.

Further, the filter 28c is a filter used to excite the fluorescent dye contained in the sample 22 by using the third laser excitation light source 3 and read the fluorescence 25, and has a wavelength of 473 nm. Cut the light, 47
It has the property of transmitting light having a wavelength longer than 3 nm.

When the sample 22 is a stimulable phosphor sheet, the filter 28d uses the first laser excitation light source 1 to excite the stimulable phosphor contained in the stimulable phosphor sheet. , Photostimulable light emitted from stimulable phosphor 25
This is a filter used when reading light, and has the property of transmitting only light in the wavelength region of stimulable light 25 emitted from the stimulable phosphor and cutting light having a wavelength of 640 nm.

Therefore, these filters 28a, 28a and 28a may be used in accordance with the type of the laser excitation light source to be used, that is, the type of the sample and the type of the fluorescent substance labeling the sample.
By selectively using 8b, 28c, 28d,
It becomes possible to cut off light in a wavelength region that becomes noise.

The filter 28a of the filter unit 27,
After passing through 28b and 28c and cutting light in a predetermined wavelength range, the fluorescence or stimulating light 25 enters a mirror 29, is reflected, and is condensed by a lens 30.

The lens 19 and the lens 30 constitute a confocal optical system. As described above, the confocal optical system is employed because, when the sample 22 is a microarray using a slide glass plate as a carrier, the fluorescence emitted from a minute spot-shaped sample formed on the slide glass plate is used. To
This is to enable reading at a high S / N ratio.

At the focal point of the lens 30, a confocal switching member 31 is provided.

FIG. 3 is a schematic front view of the confocal switching member 31.

As shown in FIG. 3, the confocal switching member 31 has a plate shape and has three pinholes 3 having different diameters.
2a, 32b and 32c are formed.

The pinhole 32a having the smallest diameter is arranged in the optical path of the fluorescent light emitted from the microarray when the sample 22 is a microarray using a slide glass plate as a carrier. Reference numeral 32c denotes a sample arranged in the optical path of the fluorescence emitted from the gel support when the sample 22 is a microarray using the gel support as a carrier.

The pinhole 32b having an intermediate diameter
When the sample 22 is a stimulable phosphor sheet, the sample 22 is arranged in the optical path of the stimulable phosphor 25 emitted from the stimulable phosphor layer.

As described above, at the focal position of the lens 30,
By providing the confocal switching member 31, when the sample 22 is a microarray using a slide glass plate as a carrier,
The pinhole 32a having the smallest diameter is located in the optical path of the fluorescent light 25 because the sample 22 is a microarray using a slide glass plate as a carrier. 25 is emitted from the surface of the slide glass plate, and the emission point is almost constant in the depth direction.
It is possible to form an image on the pinhole 32a having a small diameter by S / N
This is because it is desirable for improving the ratio.

On the other hand, when the sample 22 is a microarray using a gel support as a carrier, the pinhole 32c having the largest diameter is positioned in the optical path of the fluorescence 25 because the sample 22 is a gel support. In the case of a microarray using a carrier as a carrier, when the fluorescent dye is excited by the laser light 4, the fluorescent dye is distributed in the depth direction of the gel support, and the emission point fluctuates in the depth direction. Therefore, an image cannot be formed on a pinhole having a small diameter by the confocal optical system. If a pinhole having a small diameter is used, the fluorescence 25 emitted from the sample is cut, and the fluorescence 2
This is because a sufficient signal intensity cannot be obtained when 5 is photoelectrically detected, and it is necessary to use the pinhole 32c having a large diameter.

On the other hand, when the sample 22 is a stimulable phosphor sheet, the pinhole 32b having an intermediate diameter is located in the optical path of the stimulable phosphor by the laser light 4. When the stimulable phosphor contained in the layer is excited, the emission points of the stimulable phosphor 25 are distributed in the depth direction of the stimulable phosphor layer, and the emission points fluctuate in the depth direction. An image cannot be formed on a pinhole having a small diameter by the focusing optical system. If a pinhole having a small diameter is used, the photostimulated light 25 emitted from the sample is cut off, and the photostimulated light 25 is photoelectrically detected. Although sufficient signal intensity cannot be obtained when performing the method, the distribution in the depth direction of the light-emitting points and the fluctuation in the depth direction of the light-emitting points are not as large as those of a microarray using a gel support as a carrier. It is desirable to use the pinhole 32b having Is et al.

The positions of the pinholes 32a, 32b and 32c, and therefore the position of the confocal switching member 31, are determined as described later in detail.

The fluorescence or stimulating light 25 that has passed through the confocal switching member 31 is photoelectrically detected by the photomultiplier 33, and analog data is generated.

The analog data generated by the photomultiplier 33 is converted into digital data by the A / D converter 34 and sent to the data processing device 35.

The data processor 35 converts the visible data into C
It can be displayed on the screen of the RT 36.

FIG. 4 is a block diagram showing the detection system, drive system, input system and control system of the scanner.

As shown in FIG. 4, the detection system of the scanner includes a sample 22 set on a sample stage 20.
Carrier sensor 4 that detects the type of carrier that grips
0, and the driving system of the scanner includes a filter unit 27.
And a switching member motor 42 for moving the confocal switching member 31.

The input system of the scanner is a keyboard 4
The control system of the scanner includes a control unit 45 and a RAM 46.

FIG. 5 is a schematic side view of a scanner showing a first step of a pinhole position adjusting / positioning method of a confocal scanner according to a preferred embodiment of the present invention. The optical path of the laser light 4 from the second laser excitation light source 2 and the third laser excitation light source 3 to the optical head 15 is omitted.

Prior to adjusting and positioning the positions of the pinholes 32a, 32b, and 32c, and therefore the position of the confocal switching member 31, first, the filter unit 27 is set.
Is retracted from the optical path of the fluorescent or stimulating light 25, and the confocal switching member 31 is retracted from the optical path of the fluorescent or stimulating light 25 to the standby position.

Next, the position detecting diode 50 is arranged on the photoelectric detecting surface of the photomultiplier 33 to be provided later, such that the photoelectric detecting surface is located.
The diffusion plate 51 is placed on the sample stage 20.

A number of spots of a sample selectively labeled with a fluorescent dye using a slide glass plate as a carrier
The microarray formed on the slide glass plate is scanned by the laser beam 4 to excite the fluorescent dye,
Fluorescence 25 emitted from the fluorescent dye is detected photoelectrically,
When determining the position of the pinhole 32a used when generating data for biochemical analysis, first, the operator detects the position of the position detecting diode 5 on the keyboard 43.
When a light receiving position coordinate detection signal indicating that the position of the laser beam 4 received by the light source 0 should be detected is input and a light source selection signal indicating that the first laser excitation light source 1 is to be input, the light receiving position is detected. The coordinate detection signal and the light source selection signal
Output to the control unit 45.

Upon receiving a light source selection signal for selecting the first laser excitation light source 1, the control unit 45
The first laser excitation light source 1 is activated, and the first laser excitation light source 1 emits a laser beam 4 having a wavelength of 640 nm.

The light emitted from the first laser excitation light source 1
The laser light 4 having a wavelength of 40 nm is converted into parallel light by the collimator lens 5, reflected by the mirror 6, transmitted through the first dichroic mirror 7 and the second dichroic mirror 8, and transmitted to the optical head 15. Incident.

The laser beam 4 having a wavelength of 640 nm incident on the optical head 15 is reflected by the mirror 16, passes through the hole 17 formed in the perforated mirror 18, and passes through the lens 1.
The light is condensed by 9 and is incident on the diffusion plate 51 set on the sample stage 20.

The laser light 4 having a wavelength of 640 nm incident on the diffusion plate 51 set on the sample stage 20 is diffused by the diffusion plate 51, and the diffused laser light 4 is
The light is made into parallel light by the lens 19, and the perforated mirror 1 is formed.
8 reflected.

The laser light 4 having a wavelength of 640 nm, which is diffused by the diffuser plate 51 and reflected by the perforated mirror 18, is further reflected by the mirror 29, photoelectrically by the lens 30 and by the position detecting diode 50. 64 detected by the position detection diode 50
Analog light receiving position coordinate data corresponding to the light receiving position of the laser beam 4 having a wavelength of 0 nm is generated.

The analog light receiving position coordinate data generated by the position detecting diode 50 is digitized by the A / D converter 34 and output to the data processor 35, where the digital light receiving position of the laser light 4 having a wavelength of 640 nm is obtained. The coordinate data is stored in a memory (not shown) of the data processing device 35.

Next, a light receiving position coordinate detection signal indicating that the position of the laser beam 4 received by the position detecting diode 50 is to be detected is input to the keyboard 43 by the operator, and the second laser excitation light source 2 is turned on. When a light source selection signal for selection is input, a light receiving point position coordinate detection signal and a light source selection signal are output to the control unit 45.

When the control unit 45 receives a light source selection signal for selecting the second laser excitation light source 2,
The second laser excitation light source 2 is activated, and the second laser excitation light source 2 emits a laser beam 4 having a wavelength of 532 nm.

The light emitted from the second laser excitation light source 2
The laser light 4 having a wavelength of 32 nm is converted into parallel light by the collimator lens 9, reflected by the first dichroic mirror 7, transmitted through the second dichroic mirror 8, and enters the optical head 15.

The laser beam 4 having a wavelength of 532 nm incident on the optical head 15 is reflected by the mirror 16, passes through the hole 17 formed in the perforated mirror 18, and passes through the lens 1.
The light is condensed by 9 and is incident on the diffusion plate 51 set on the sample stage 20.

The laser beam 4 having a wavelength of 532 nm incident on the diffusion plate 51 set on the sample stage 20 is diffused by the diffusion plate 51, and the diffused laser beam 4
The light is made into parallel light by the lens 19, and the perforated mirror 1 is formed.
8 reflected.

The laser beam 4 having a wavelength of 532 nm, which is diffused by the diffusion plate 51 and reflected by the perforated mirror 18, is further reflected by the mirror 29, photoelectrically by the lens 30 and by the position detecting diode 50. 53 detected and received by the position detection diode 50
Analog light receiving position coordinate data corresponding to the light receiving position of the laser light 4 having a wavelength of 2 nm is generated.

The analog light receiving position coordinate data generated by the position detecting diode 50 is digitized by the A / D converter 34 and output to the data processing unit 35, where the digital light receiving position of the laser beam 4 having a wavelength of 532 nm is received. The coordinate data is stored in a memory (not shown) of the data processing device 35.

Further, a light receiving position coordinate detection signal indicating that the position of the laser beam 4 received by the position detecting diode 50 should be detected is input to the keyboard 43 by the operator, and the third laser excitation light source 3 is turned on. When a light source selection signal for selection is input, the light receiving position coordinate detection signal and the light source selection signal are transmitted to the control unit 4.
5 is output.

Upon receiving the light source selection signal for selecting the third laser excitation light source 3, the control unit 45
The third laser excitation light source 3 is activated, and a laser beam 4 having a wavelength of 473 nm is emitted from the third laser excitation light source 3.

The light emitted from the third laser excitation light source 3
The laser beam 4 having a wavelength of 73 nm is transmitted through the collimator lens 10.
After being converted into parallel light, the light is reflected by the second dichroic mirror 8 and enters the optical head 15.

The laser beam 4 having a wavelength of 473 nm incident on the optical head 15 is reflected by the mirror 16, passes through the hole 17 formed in the perforated mirror 18, and passes through the lens 1.
The light is condensed by 9 and is incident on the diffusion plate 51 set on the sample stage 20.

The laser beam 4 having a wavelength of 473 nm incident on the diffusion plate 51 set on the sample stage 20 is diffused by the diffusion plate 51, and the diffused laser beam 4
The light is made into parallel light by the lens 19, and the perforated mirror 1 is formed.
8 reflected.

The laser beam 4 having a wavelength of 473 nm, which is diffused by the diffusion plate 51 and reflected by the perforated mirror 18, is further reflected by the mirror 29, photoelectrically by the lens 30 and by the position detecting diode 50. 67 detected by the position detection diode 50
Analog light receiving position coordinate data corresponding to the light receiving position of the laser beam 4 having a wavelength of 30 nm is generated.

The analog light receiving coordinate position data generated by the position detecting diode 50 is digitized by the A / D converter 34 and output to the data processor 35, where the digital light receiving position of the laser beam 4 having a wavelength of 473 nm is output. The coordinate data is stored in a memory (not shown) of the data processing device 35.

Thus, the laser beam 4 having a wavelength of 640 nm
Digital light receiving position coordinate data of the laser beam 4 having a wavelength of 532 nm,
When the digital light receiving position coordinate data of the laser beam 4 having the wavelength of 3 nm is generated and stored in the memory of the data processing device 35, the control unit 45 sends the digital data of the laser beam 4 having the wavelength of 640 nm to the data processing device 35. Based on the light receiving position coordinate data, the digital light receiving position coordinate data of the laser light 4 having the wavelength of 532 nm, and the digital light receiving position coordinate data of the laser light 4 having the wavelength of 473 nm, it corresponds to the center of the light receiving position coordinates of the laser light 4 of each wavelength. A center position coordinate calculation instruction signal for instructing the calculation of coordinate values is output.

Upon receiving the center position coordinate calculation instruction signal, the data processor 35 sets the digital light receiving position coordinate data of the laser beam 4 having a wavelength of 640 nm stored in the memory as 5
The digital light receiving position coordinate data of the laser light 4 having a wavelength of 32 nm and the digital light receiving position coordinate data of the laser light 4 having a wavelength of 473 nm are read out, and the laser light 4 of each wavelength is read out.
Calculate the coordinate value corresponding to the center of the focal light receiving coordinates of
The light receiving position of the laser light 4 having a wavelength of 40 nm, the light receiving position of the laser light 4 having a wavelength of 532 nm, and the light receiving position of the laser light 4 having a wavelength of 473 nm, together with the central position of the light receiving position of the laser light 4 having each wavelength, are represented by visible data. As CRT36
On the screen.

FIG. 6 shows the CRT displayed at the center of the calculated light receiving position of the laser light 4 of each wavelength together with the light receiving position of the laser light 4 of each wavelength.
It is the schematic which shows the screen of 36.

As shown in FIG. 6, 640 nm light received by the position detecting diode 50 is displayed on the screen of the CRT 36.
The light receiving position 61 of the laser light 4 having the wavelength of 532 nm and the light receiving position 63 of the laser light 4 having the wavelength of 473 nm are displayed together with the light receiving position 62 of the laser light 4 having the wavelength of 473 nm. .

FIG. 7 is a schematic side view of the scanner showing the second step of the pinhole position adjusting / positioning method of the confocal scanner according to the preferred embodiment of the present invention.

In this way, the data processing device 35
When the center position at the center of the light receiving position of the laser light 4 of each wavelength is calculated and displayed on the screen of the CRT 36, the second step of the pinhole position adjustment / positioning method of the confocal scanner is executed. .

FIG. 7 is a schematic side view of the scanner showing the second step of the pinhole position adjustment / positioning method of the confocal scanner according to the preferred embodiment of the present invention.

As shown in FIG. 7, in the second step, first, a drive pulse signal is output to the switching member motor 42 by the control unit 45, and the switching member motor 42 The confocal switching member 31 is moved so that the hole 32a is located in front of the position detection diode 50, and the light source 5 is placed in the optical path of the fluorescence or stimulating light 25 near the lens 30 on the photomultiplier 33 side.
5 is set.

Next, a pinhole detection signal is input to the keyboard 43 by the operator and output to the control unit 45.

Upon receiving the pinhole detection signal, the control unit 45 turns on the light source 55 and the light source 55 emits light.

The control unit 45 outputs a pinhole detection signal to the data processor 35 at the same time.

The light emitted from the light source 55 passes through the pinhole 32a, is photoelectrically detected by the position detecting diode 50, and generates analog data. This analog data is generated by photoelectrically detecting the light passing through the pinhole 32a.
It shows the shape and position of “a”.

The analog data indicating the shape and position of the pinhole 32a generated by the position detecting diode 50 is digitized by the A / D converter 34 and sent to the data processing device 35.

When the pinhole detection signal is input, the data processor 35 displays a visible image of the pinhole 32a on the screen of the CRT 36 based on digital data indicating the shape and position of the pinhole 32a. .

FIG. 8 is a schematic diagram showing the screen of the CRT 36 on which the visible image of the pinhole 32a is displayed.

Therefore, the operator can know the relationship between the position of the pinhole 32a and the center position 65 from the image of the pinhole 32a displayed on the screen of the CRT 36, and the operator issues the switching member position adjustment signal. Is input to the control unit 45 via the keyboard 43, a drive pulse signal is supplied to the switching member motor 42 from the control unit 45, and the confocal switching member 31 is two-dimensionally moved. The relative positional relationship between the position of the hole 32a and the center position 65 changes, and the operator
Of the pinhole 32a and the center position 6 based on the image of the pinhole 32a displayed on the screen of FIG.
5 can be monitored.

Thus, the center position 65 is the pinhole 32
When it is confirmed that the image coincides with the center of the image 70 in a, a pinhole position determination signal is input to the keyboard 43 by the operator.

The pinhole position determination signal is input to the control unit 45 and the control unit 45
When the pinhole position determination signal is received, the position of the pinhole 32a whose center position 65 matches the center of the image of the pinhole 32a is determined based on the drive pulse signal given to the switching member motor 42. The set position data of the pinhole 32a at which the pinhole 32a is to be set is determined and stored in the RAM 46 in the form of the number of drive pulses to be given to the switching member motor 42.

Next, in the case of a microarray using a gel support as a carrier, the set position of the pinhole 32c arranged in the optical path of the fluorescence 25 emitted from the gel support is determined and switched in exactly the same manner. The set position data of the pinhole 32c is stored in the RAM 46 in the form of the number of drive pulses to be given to the member motor 42.

Further, the position of the pinhole 32b is determined. When the sample 22 is a stimulable phosphor sheet, the pinhole 32b has an optical path of the photostimulable light emitted from the photostimulable phosphor layer. The laser excitation light source used to excite the stimulable phosphor layer is 640.
Since only the first laser excitation light source 1 that emits the laser light 4 having a wavelength of nm is used, the calculation for determining the center position 65 is not required.

That is, when determining the position of the pinhole 32b, the 640 nm diffused by the diffusion plate 51 is used.
Is detected photoelectrically by the position detecting diode 50, and is converted by the A / D converter 34 into digital light receiving position coordinate data of the laser light 4 having a wavelength of 640 nm. The light receiving position 61 of the laser beam 4 having the wavelength is represented by C as visible data.
It is displayed on the screen of RT36.

Next, the confocal switching member 31 is moved so that the pinhole 32b is located in front of the position detecting diode 50, and the light source 55 is set near the lens 30 on the photomultiplier 33 side. Then, the light source 55 is turned on.

The pinhole 32b emitted from the light source 55
Is detected photoelectrically by the position detecting diode 50, analog data is generated, and digital data indicating the shape and position of the pinhole 32b obtained by being digitized by the A / D converter 34. , A visible image of the pinhole 32b is displayed on the screen of the CRT 36.

While observing the image displayed on the screen of the CRT 36, an operator inputs a switching member position adjustment signal to the control unit 45 via the keyboard 43, and drives the switching member motor 42 from the control unit 45. When a pulse signal is given, the confocal switching member 31 is moved two-dimensionally to 640 nm.
The light receiving position 61 of the laser light 4 having the wavelength of
When the image matches the center of the image b, the keyboard 43
A pinhole position determination signal is input.

The pinhole position determination signal is input to the control unit 45, and the control unit 45
Upon receiving the pinhole position determination signal, based on the drive pulse signal given to the switching member motor 42,
The light receiving position 61 of the laser beam 4 having a wavelength of m
The position of the pinhole 32b that matches the center of the image b
The set position data of the pinhole 32b to be set to the pinhole 32b is stored in the RAM 46 in the form of the number of drive pulses to be set to the pinhole 32b and to be given to the switching member motor 42.

As described above, the pinholes 32a, 3a
When the set positions of 2b and 32c are determined and the respective set position data are stored in the RAM 46, the pinhole position adjusting device of the confocal scanner including the position detecting diode 50 and the light source 55 is detached from the scanner. The photomultiplier 33 is set so that its photoelectric detection surface matches the photoelectric detection surface of the position detection diode 50, and the switching member motor 42 is driven,
The confocal switching member 31 is retracted to the standby position. Here, the set position data of the pinhole 32a, the set position data of the pinhole 32b and the set position data of the pinhole 32c are switched to move the confocal switching member 31 from the standby position to the respective set positions. The number of drive pulses to be given to the member motor 42 is generated by the control unit 45 and stored in the RAM 46.

In the scanner configured as described above, when the sample 22 is a microarray using a slide glass plate as a carrier, the laser beam 4
By scanning the entire surface of the sample 22, the fluorescent dye selectively labeling the sample is excited, the fluorescence 25 emitted from the fluorescent dye is photoelectrically detected, and digital data for biochemical analysis is obtained. Generate.

When the sample carrier 21 holding the sample 22 is placed on the sample stage 20, the type of the sample carrier 21 is detected by the carrier sensor 40, and the carrier detection signal is transmitted to the control unit 4.
5 is output.

When receiving the carrier detection signal from the carrier sensor 40, the control unit 45 determines the pinhole 3 stored in the RAM 46 based on the carrier detection signal.
2a is read out and the pinhole 32a is read out.
A driving pulse signal consisting of a predetermined driving pulse according to the set position data is output to the switching member motor 42, and the confocal switching member 31 is moved so that the pinhole 32a having the smallest diameter is located in the optical path. Move.

Next, when the type of the fluorescent substance as the labeling substance and the start signal are input to the keyboard 43 by the operator, an instruction signal is output from the keyboard 43 to the control unit 45.

For example, the type of fluorescent substance is Cy-
When 5 (registered trademark) is input, the control unit 45 outputs a drive signal to the filter unit motor 41 in accordance with the input instruction signal, moves the filter unit 27, and cuts light having a wavelength of 640 nm. Then, a filter 28a having a property of transmitting light having a wavelength longer than 640 nm is located in the optical path, and the first laser excitation light source 1 turns on a drive signal.

The laser light 4 emitted from the first laser excitation light source 1 is converted into parallel light by a collimator lens 5, then reflected by a mirror 6, and is reflected by a first dichroic mirror 7 and a second dichroic mirror. 8 and enter the optical head 15.

The laser beam 4 incident on the optical head 15 is
The sample 22 reflected by the mirror 16, passes through the hole 17 formed in the perforated mirror 18, is condensed by the lens 19, and is set on the sample stage 20.
Is incident on the microarray.

Since the sample stage 20 is moved in the X and Y directions in FIG. 1 by a scanning mechanism (not shown), the entire surface of the microarray set on the sample carrier 21 is scanned by the laser beam 4. You.

When the laser beam 4 is irradiated, the probe D
A fluorescent dye that labels NA, for example, Cy-5 is excited, and fluorescence 25 is emitted. When a slide glass plate is used as a carrier for the microarray,
Since the fluorescent dye is distributed only on the surface of the slide glass plate, the fluorescence 25 is also emitted only from the surface of the slide glass plate.

The fluorescent light 25 emitted from the surface of the slide glass plate is converted into parallel light by the lens 19, reflected by the perforated mirror 18, and filtered by the filter unit 27.
Incident on.

The filter unit 27 includes a filter 28a
Has been moved so as to be located in the optical path,
5 enters the filter 28a, cuts light having a wavelength of 640 nm, and transmits only light having a wavelength longer than 640 nm.

The fluorescent light 25 transmitted through the filter 28a is reflected by the mirror 29 and is imaged by the lens 30.

Prior to the irradiation of the laser beam 4, the confocal switching member 31 is moved so that the pinhole 32a having the smallest diameter is located in the optical path.
The set position of a is 6 detected by the position detection diode 50.
Light receiving position 61, 532n of laser beam 4 having a wavelength of 40 nm
Receiving positions 62 and 473 nm of laser light 4 having a wavelength of m
The center position 65 corresponding to the center of the light receiving position 63 of the laser light 4 having the wavelength of is set so as to coincide with the center of the pinhole 32a, so that the fluorescent light 25 is imaged on the pinhole 32a and the photomultiplier is formed. The prior data is photoelectrically detected by the prior 33 and analog data is generated.

As described above, using the confocal optical system, the fluorescence 25 emitted from the fluorescent dye on the surface of the slide glass plate was used.
Is guided to the photomultiplier 33 and photoelectrically detected, so that noise in data can be minimized.

The analog data generated by the photo multiplier 33 is converted into digital data by the A / D converter 34 and sent to the data processing device 35.

In this embodiment, first, the position detecting diode 50 is arranged at a position corresponding to the photoelectric detection surface of the photomultiplier 33 so that the photoelectric detection surface is located. The plate 51 is placed, the filter unit 27 is retracted from the optical path of the fluorescent or stimulating light 25, and the confocal switching member 31 is retracted from the optical path of the fluorescent or stimulating light 25 to the standby position. In this state, the first laser excitation light source 1 is turned on, the laser light 4 having a wavelength of 640 nm is emitted, and the diffused laser light 4 is photoelectrically detected by the position detection diode 50 by the diffusion plate 51. , 6
Digital light receiving position coordinate data of the laser beam 4 having a wavelength of 40 nm is generated. Similarly, the second laser excitation light source 2 is turned on, and 532n
The laser beam 4 having the wavelength of m is photoelectrically detected by the position detecting diode 50 to generate digital light receiving position coordinate data of the laser beam 4 having the wavelength of 532 nm, and the third laser excitation light source 3 is turned on. And the diffusion plate 51
The laser light 4 having a wavelength of 473 nm diffused by the photoelectric conversion device is photoelectrically detected by the position detection diode 50, and
Digital light receiving position coordinate data of the laser beam 4 having a wavelength of 73 nm is generated.

The digital light receiving position coordinate data of the laser beam 4 having a wavelength of 640 nm thus generated, 532 nm
The light receiving position 61 of the 640 nm wavelength laser light 4 received by the position detecting diode 50 based on the digital light receiving position coordinate data of the laser light 4 having the wavelength of 473 nm and the digital light receiving position coordinate data of the laser light 4 having the wavelength of 473 nm. Receiving position 62 of laser beam 4 having a wavelength of 532 nm
And a center position 65 corresponding to the center of the light receiving position 63 of the laser beam 4 having a wavelength of 473 nm is calculated, and the CRT 36
Will be displayed on the screen.

Next, the switching member motor 42
The confocal switching member 31 is moved so that the pinhole 32a is positioned in front of the position detection diode 50, and the lens 30 on the side of the photomultiplier 33 is moved.
, A light source 55 is set, and the light emitted from the light source 55 and passing through the pinhole 32b is photoelectrically detected by the position detection diode 50 to generate analog data, and the A / D converter 34 Thus, a visible image of the pinhole 32a is displayed on the screen of the CRT 36 based on digital data indicating the shape and position of the pinhole 32a obtained by digitization.

As described above, while observing the image displayed on the screen of the CRT 36, the switching member position adjustment signal is input to the control unit 45 via the keyboard 43 by the operator.
The drive pulse signal is given to the switching member motor 42 from the above, the confocal switching member 31 is moved two-dimensionally, and when the center position 65 matches the center of the image of the pinhole 32a, A hole position determination signal is input.

The pinhole position determination signal is input to the control unit 45. When the pinhole position determination signal is received by the control unit 45, the center position 65 is determined based on the drive pulse signal given to the switching member motor 42. The position of the pinhole 32a corresponding to the center of the image of 32a is determined as the position where the pinhole 32a is to be set, and the pinhole 32a is to be set in the form of the number of drive pulses to be given to the switching member motor 42 32a is stored in the RAM 46.
Is stored.

Therefore, according to the present embodiment, when the sample 22 is a microarray using a slide glass plate as a carrier, the wavelength that can be excited most efficiently according to the type of the fluorescent substance labeling the sample. Even if the laser light 4 is selected to excite the fluorescent substance, it is possible to guarantee that the fluorescent light 25 emitted from the fluorescent substance forms an image in the pinhole 32a. And data with an improved S / N ratio can be generated.

Further, according to the present embodiment, the setting position of the pinhole 32c used in the case of the microarray using the gel support as the carrier is determined in the same manner, so that the fluorescent light Even if the fluorescent light is excited by selecting the laser beam 4 having a wavelength that can be excited most efficiently according to the type of the substance, the fluorescent light 25 emitted from the fluorescent substance is cut by the pinhole 32c. Therefore, it is possible to detect the fluorescence 25 photoelectrically and generate data having a high signal intensity.

Further, according to this embodiment, sample 2
The position detecting diode 50 can efficiently excite the stimulable phosphor diffused by the diffusion plate 51 to the set position of the pinhole 32b having an intermediate diameter used when 2 is a stimulable phosphor sheet. The position 61 where the laser beam 4 having a wavelength of 640 nm is received is the image 7 of the pinhole 32b.
And the pinhole 32b is positioned, so that the photostimulable light emitted from the stimulable phosphor layer is not cut off by the pinhole 32b. Can be photoelectrically detected to generate data having a high signal strength.

The present invention is not limited to the above embodiments, and various changes can be made within the scope of the invention described in the claims, and these are also included in the scope of the present invention. It goes without saying that it is a thing.

For example, in the above embodiment, CR
Observe the image of the center position 65 and the pinholes 32a and 32b displayed on the screen of T36, or the image of the light receiving position 61 of the 640 nm wavelength laser beam 4 and the image of the pinhole 32b displayed on the screen of the CRT 36. Meanwhile, the operator inputs the switching member position adjustment signal to the keyboard 43, causes the control unit 45 to output a drive pulse signal corresponding to the switching member position adjustment signal to the switching member motor 42, and displays it on the screen of the CRT 36. The confocal switching member 31 is moved two-dimensionally until the center position 65 thus set and the center of the image of the pinholes 32a and 32b match, or the laser light 4 of 640 nm wavelength displayed on the screen of the CRT 36 is displayed. The confocal switching member 31 is moved until the light receiving position 61 of By moving dimensionally,
Although the setting positions of the pinholes 32a, 32b, and 32c are determined, the data processing device 35
The center coordinates are calculated based on the digital image data of 32b and 32c, and the number of drive pulses to be given to the switching member motor 42 is calculated according to the distance between the center coordinates of the pinholes 32a and 32c and the center position coordinates. By moving the confocal switching member 31 two-dimensionally, the setting positions of the pinholes 32a, 32b, and 32c can be determined. In such a case, the CRT 36 must be used. There is no.

Further, in the above embodiment, the scanner uses a microarray having a slide glass plate as a carrier and a large number of spots of a sample selectively labeled with a fluorescent dye formed on the slide glass plate. Scanning with light 4 excites the fluorescent dye, photoelectrically detects the fluorescent light 25 emitted from the fluorescent dye, and is capable of generating data for biochemical analysis. Further, the gel support is used as a carrier. A lot of spots of a sample selectively labeled with a fluorescent dye are scanned with a laser beam 4 on a sample carrier holding one or more microarrays formed on a gel support to excite the fluorescent dye. The apparatus is configured to photoelectrically detect the fluorescence 25 emitted from the fluorescent dye to generate data for biochemical analysis. A carrier such as a membrane filter on which a number of spots of a sample selectively labeled by quality are formed is brought into close contact with a stimulable phosphor sheet on which a stimulable phosphor layer containing a stimulable phosphor is formed. The stimulable phosphor layer of the stimulable phosphor sheet on which the positional information of the radioactive labeling substance obtained by exposing the stimulable phosphor layer was recorded was
Scans to excite the stimulable phosphor, photoelectrically detects the stimulable emission emitted from the stimulable phosphor, and is configured to generate data for biochemical analysis. ,
Pinholes 32a, 32b, and 32c having different diameters are formed, and a confocal switching member 31 movable by a switching member motor 42 is provided.
a, 32b, and 32c are stored in the RAM 46 in the form of the number of drive pulses to be given to the switching member motor 42. The scanner uses a slide glass plate as a carrier and a fluorescent dye. The microarray formed on the slide glass plate is scanned with the laser beam 4 to excite the fluorescent dye, and the fluorescent light 25 emitted from the fluorescent dye is photoelectrically scanned on the microarray formed on the slide glass plate. When configured to detect and generate data for biochemical analysis,
The center position 65 displayed on the screen of the CRT 36 is manually set.
The member on which the pinhole 32a is formed may be moved until the center of the image of the pinhole 32a matches the center of the image of the pinhole 32a, and the member on which the pinhole 32a is formed may be fixed to the scanner.

Further, in the above embodiment, the light receiving position 61 of the laser light 4 having the wavelength of 640 nm detected by the position detecting diode 50 and the light receiving position 62 of the laser light 4 having the wavelength of 532 nm and the laser light 4 having the wavelength of 473 nm are detected. A central position 65 corresponding to the center of the light receiving position 63;
6, the position where the center of the image of the pinhole 32a displayed on the screen coincides with the position where the pinhole 32a should be set is determined.
It is not always necessary to determine the position where the center of the image of the pinhole 32a displayed on the screen 6 coincides with the position where the pinhole 32a should be set. Based on the light receiving position 61 of the laser light 4 having the wavelength of 532 nm and the light receiving position 62 of the laser light 4 having the wavelength of 532 nm, and based on the light receiving position 63 of the laser light 4 having the wavelength of 473 nm, the optimum reference position where the center of the pinhole 32a should be located is determined. This reference position is obtained by calculation and the pinhole 32 displayed on the screen of the CRT 36.
The position where the center of the image a coincides with the pinhole 32a
May be determined as the position to be set.

In the above embodiment, the pinhole 32a of the scanner provided with the first laser excitation light source 1, the second laser excitation light source 2, and the third laser excitation light source 3 is used.
In the case where the position is adjusted and the position is adjusted, the present invention is applied to the case where the pinholes 32 a and 32 of the confocal scanner provided with the laser excitation light sources having three different emission wavelengths are used.
It is not limited to the position adjustment and positioning of b, 32c, but also when adjusting and positioning the pinhole position of a confocal scanner provided with four or more laser excitation light sources, a single laser excitation It can also be applied when adjusting and positioning the position of the pinhole of a confocal scanner with a light source, when determining the position of the pinhole of a confocal scanner with a single laser excitation light source. The position of the pinhole may be determined in exactly the same way as when determining the pinhole 32b used when the sample 22 is a stimulable phosphor sheet.

Further, in the above embodiment, the position detecting diode 50 is used. However, if it is possible to photoelectrically receive light to detect a two-dimensional light receiving position, the position detecting diode 50 is used. Other photodetectors can be used, for example, a CCD area sensor can be used.

In the above-described embodiment, the CRT 36 is used as the display means of the visible data.
Instead of T36, a flat display panel such as a liquid crystal display panel or an organic EL display panel can be used, and the display means is not particularly limited.

In the above embodiment, the confocal switching member 31 has three pinholes 32 having different diameters.
a, 32b, 32c are formed and a number of spots of the sample selectively labeled with a fluorescent dye are scanned by a laser beam 4 on a microarray formed on a slide glass plate to excite the fluorescent dye. When the fluorescence emitted from the fluorescent dye is photoelectrically detected to generate data for biochemical analysis, the pinhole 32a is provided with the position information of the radiolabeled substance obtained by exposing the stimulable phosphor layer. The stimulable phosphor layer of the stimulable phosphor sheet on which is recorded is scanned by the laser light 4 to excite the stimulable phosphor, and the stimulable phosphor emitted from the stimulable phosphor is photoelectrically irradiated. To generate data for biochemical analysis,
2b is electrophoresed on the gel support and scans with a laser light 4 a fluorescent sample using a gel support containing a sample selectively labeled with a fluorescent dye,
When the fluorescent dye is excited and the fluorescence emitted from the fluorescent dye is photoelectrically detected to generate data for biochemical analysis, the pinholes 32c are used, but the confocal switching member 31 is used. And pinholes 32a, 3
A large number of spots of the sample which form only 2b and are selectively labeled with a fluorescent dye are scanned by a laser beam 4 on a microarray formed on a slide glass plate to excite the fluorescent dye, When photoelectrically detecting the fluorescence emitted from the phosphor and generating data for biochemical analysis, the fluorescence 25 is received via the pinhole 32a, and the stimulable luminescence emitted from the stimulable phosphor layer is emitted. When photoelectrically detecting 25 and generating data for biochemical analysis, the photostimulable light is received via the pinhole 32b, and the fluorescence 25 emitted from the gel support is photoelectrically detected. When generating data for biochemical analysis, the confocal switching member 31 may be retracted from the optical path of the fluorescent light 25 to increase the amount of light received by the photomultiplier 33, And, a confocal switching member 31, only to form pinholes 32a, a number of spots of selectively labeled sample by fluorescent dye, a microarray formed on a slide glass plate, the laser beam 4
By scanning, the fluorescent dye is excited, the fluorescence emitted from the fluorescent dye is photoelectrically detected, and only when data for biochemical analysis is generated, via the pinhole 32a,
The fluorescent light 25 is received, and the photostimulable light 25 emitted from the stimulable phosphor layer is photoelectrically detected to generate the data for biochemical analysis, and the fluorescent light 25 emitted from the gel support is photoelectrically detected. When the data for biochemical analysis is to be detected and detected, the confocal switching member 31 can be retracted from the optical path of the fluorescent light 25 so that the amount of light received by the photomultiplier 33 can be increased.

[0163]

According to the present invention, in a scanner using a confocal optical system, the position of a pinhole can be adjusted and positioned as desired in a simple and short time. It is possible to provide a method and an apparatus for adjusting and positioning a pinhole position of a scanner.

Further, according to the present invention, in a scanner using a confocal optical system, even if the scanner has a plurality of light sources, the position of the pinhole can be easily and quickly determined.
It is possible to provide a pinhole position adjustment / positioning method and apparatus of a confocal scanner that can be adjusted and positioned as desired.

[Brief description of the drawings]

FIG. 1 is a schematic perspective view of a scanner in which the position of a pinhole is positioned using a pinhole position adjustment / positioning method of a confocal scanner according to a preferred embodiment of the present invention.

FIG. 2 is a schematic perspective view of a microarray.

FIG. 3 is a schematic front view of a confocal switching member.

FIG. 4 is a block diagram showing a detection system, a drive system, an input system, and a control system of the scanner.

FIG. 5 is a schematic side view of the scanner showing a first step of a pinhole position adjustment / positioning method of the confocal scanner according to the preferred embodiment of the present invention.

FIG. 6 is a schematic diagram showing a screen of the CRT in which the center position of the calculated light receiving position of the laser light of each wavelength is displayed together with the light receiving position of the laser light of each wavelength. It is.

FIG. 7 is a schematic side view of the scanner showing a second step of the pinhole position adjustment / positioning method of the confocal scanner according to the preferred embodiment of the present invention.

FIG. 8 is a view showing C in which a visible image of a pinhole is displayed.
It is a schematic diagram showing a screen of RT.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 1st laser excitation light source 2 2nd laser excitation light source 3 3rd laser excitation light source 4 laser beam 5 collimator lens 6 mirror 7 1st dichroic mirror 8 2nd dichroic mirror 9 collimator lens 10 collimator lens 15 optical head Reference Signs List 16 mirror 17 hole 18 perforated mirror 19 lens 20 sample stage 21 sample carrier 22 sample 23 dropped cDNA 25 fluorescence or stimulating light 27 filter unit 28a, 28b, 28c, 28d filter 29 mirror 30 lens 31 confocal switching member 32a, 32b, 32c Pinhole 33 Photomultiplier 34 A / D converter 35 Data processor 36 CRT 40 Carrier sensor 41 Filter unit motor 42 Switching member motor 43 Keybo Mode 45 control unit 46 RAM 50 position detecting diode 51 diffuser 55 light source 61 light receiving position of laser light 4 having wavelength of 640 nm 62 light receiving position of laser light 4 having wavelength of 532 nm 63 light receiving position of laser light 4 having wavelength of 473 nm 65 Image of 70 pinhole at center position

Continued on the front page F term (reference) 2G043 AA06 BA16 CA03 DA02 DA06 EA01 EA19 FA02 GA04 GB01 HA02 HA09 HA15 JA02 KA02 KA05 KA09 LA02 LA03 NA06 2H043 AA03 AA09 AA21 AA24 AA27 2H052 AA08 AA09 AC25 AC24 AC14

Claims (13)

[Claims] At least one laser excitation light source, a sample stage for setting a sample containing a sample labeled with a fluorescent substance, and a laser light emitted from the at least one laser excitation light source set on the sample stage. A scanning mechanism that scans the sample and excites a fluorescent substance contained in the sample by the laser light, and photoelectrically detects light emitted from the sample when excited by the laser light. A photodetector, and a confocal optical system that guides light emitted from the sample to the photodetector, and a pin having a pinhole formed between the confocal optical system and the photodetector. Pinhole position adjustment and position of confocal scanner to adjust and position pinhole of confocal scanner where hole forming member is placed A determination method, comprising setting a diffusion plate on the sample stage, irradiating the diffusion plate with laser light emitted from the at least one laser excitation light source, diffusing the diffusion plate, The laser light condensed by the optical system is photoelectrically received by a position sensor capable of two-dimensionally receiving the light, and light receiving position data corresponding to the light receiving position of the laser light is generated. After calculating the optimal center position data corresponding to the optimal position where the center of the pinhole should be located, based on the digital light receiving position data obtained by digitizing the above, the pinhole forming member is placed on the front surface of the position sensor. And a light source for pinhole position adjustment on the optical path of the fluorescence downstream of the confocal optical system, and a light source for pinhole position adjustment. Light emitted from a source is photoelectrically received by the position sensor via the pinhole formed in the pinhole forming member, and pinhole position data corresponding to the position of the pinhole is generated. Then, based on the digital pinhole position data obtained by digitization and the optimum center position data, the pin is adjusted so that the center of the pinhole matches the optimum position where the center of the pinhole should be located. A pinhole position adjustment / positioning method for a confocal scanner, wherein a position of the pinhole is adjusted and moved by moving a hole forming member. 2. The scanner according to claim 1, wherein the scanner includes a plurality of laser excitation light sources that emit laser lights having different wavelengths, and irradiates the laser light emitted from the plurality of laser excitation light sources to the diffusion plate, and diffuses the laser light by the diffusion plate. The laser light condensed by the confocal optical system is photoelectrically received by the position sensor, and light-receiving position data corresponding to the light-receiving positions of the laser lights having different wavelengths is generated, respectively. And generating, based on the digital light receiving position data corresponding to the light receiving positions of the laser beams having different wavelengths, optimum center position data corresponding to an optimum position where the center of the pinhole should be located. 2. The method for adjusting and positioning a pinhole position of a confocal scanner according to item 1. 3. The method according to claim 1, wherein the position sensor receives the laser beams having different wavelengths based on digital light receiving position data corresponding to the digitized laser light receiving positions having different wavelengths. 3. The method according to claim 2, wherein the center is determined as an optimum position to be located. 4. Displaying an image of the pinhole and an optimum center position where the center of the pinhole should be located on a display means based on the optimum center position data and the digital pinhole position data, By moving the pinhole forming member so that the center of the image of the pinhole displayed on the display means matches the optimum center position where the center of the pinhole displayed on the display means should be located, 4. The method for adjusting and positioning a pinhole position of a confocal scanner according to claim 1, wherein the position of the pinhole is adjusted and positioned. 5. The center coordinates of the pinhole are calculated based on the digital pinhole position data, and the center of the pinhole is determined based on the optimum center position data and the center coordinates of the pinhole. In order to match the optimal position where the center of the pinhole should be located, calculate the distance to move the pinhole forming member, move the pinhole forming member, adjust the position of the pinhole, and position The method for adjusting and positioning a pinhole position of a confocal scanner according to any one of claims 1 to 3, wherein: 6. The pinhole forming member is moved and fixed to the scanner such that the center of the pinhole coincides with an optimum position where the center of the pinhole should be located. 6. The pinhole position adjustment / positioning method for a confocal scanner according to any one of 1 to 5. 7. The pinhole forming member is configured to be movable by a pulse motor, and an optimum position data corresponding to a position where the center of the pinhole coincides with the optimum position where the center of the pinhole should be located. Storing in a memory in the form of a drive pulse applied to the pulse motor, and prior to exciting the fluorescent substance contained in the sample by the laser light emitted from the at least one laser excitation light source, Read out the driving pulse stored in the pulse motor and give it to the pulse motor. The pinhole position adjustment / positioning method for a confocal scanner according to any one of claims 1 to 5, wherein the position of the pinhole is adjusted and positioned. 8. The pinhole forming member according to claim 1, wherein a plurality of pinholes are formed.
8. The pinhole position adjustment / positioning method for a confocal scanner according to any one of items 7 and 7. 9. The method for adjusting and positioning a pinhole position of a confocal scanner according to claim 1, wherein a position detection diode is used as the position sensor. 10. At least one laser excitation light source;
A sample stage for setting a sample including a sample labeled with a fluorescent substance, and scanning on the sample set on the sample stage by laser light emitted from the at least one laser excitation light source,
A scanning mechanism that excites a fluorescent substance contained in the sample by the laser light, a photodetector that is excited by the laser light and photoelectrically detects light emitted from the sample, and emitted from the sample. A confocal optical system that guides the received light to the photodetector, wherein a pinhole forming member having a pinhole is disposed between the confocal optical system and the photodetector. A pinhole position adjusting / positioning device of a confocal scanner for adjusting and positioning the position of a pinhole of a confocal scanner, wherein the position is located on the photoelectric detection surface of the photodetector, such that the photoelectric detection surface is located. And can receive light two-dimensionally, and a position sensor capable of generating analog data, and positioned in the optical path of the fluorescence downstream of the confocal optical system, the position of the confocal optical system A light source for adjusting the position of a pinhole capable of emitting light toward the position sensor and digitized analog data generated by the position sensor are configured to be able to be retracted from the optical path of the fluorescent light on the upstream side. A laser beam emitted from the at least one laser excitation light source is received and generated by the A / D converter and the position sensor via the confocal optical system, and the A / D converter Based on the light receiving position data corresponding to the digitized laser light receiving position,
Optimal center position data corresponding to an optimal position where the center of the pinhole should be located, and light emitted from the pinhole position adjusting light source is formed on the pinhole forming member by the position sensor. Digital pinhole position data corresponding to the position of the pinhole, which is photoelectrically received, generated and digitized by the A / D converter via the pinhole, and the optimum center position data. An apparatus for adjusting and positioning a pinhole position of a confocal scanner, comprising arithmetic processing means for comparison. 11. A display unit capable of displaying visible data, wherein the arithmetic processing unit displays, on the display unit, an optimum position where the center of the pinhole should be located based on the optimum center position data. 11. The pinhole position of a confocal scanner according to claim 10, wherein the pinhole position is displayed on the display means while displaying the image of the pinhole based on the digital pinhole position data. Adjustment / positioning device. 12. The arithmetic processing unit calculates center coordinates of the pinhole based on the digital pinhole position data, and calculates the center coordinates of the pinhole based on the optimum center position data and the center coordinates of the pinhole. The center of the hole is configured to calculate a distance by which the pinhole forming member is to be moved in order to match an optimum position where the center of the pinhole should be located. Pinhole position adjustment / positioning device for confocal scanners. 13. The pinhole position adjustment / positioning device for a confocal scanner according to claim 10, wherein said position sensor is constituted by a position detection diode.