JP2000338035A - Bio-chip reading device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To permit miniaturization, cost reduction and the enhancement of accuracy by arranging a plurality of the spectroscopic data of a sample to the empty region between sample images in the image corresponding to the sample. SOLUTION: The light (exciting light) emitted from a light source 1 becomes parallel light by a lens 2 to be reflected by a dichroic mirror 3, and the reflected light is condensed through an object lens 4 to irradiate the surface of a sample 5. The sample 5 emits fluorescence (different from exciting light in wavelength) by this irradiation light and this fluorescence again returns to the object lens 4 to be incident on the dichroic mirror 3. The fluorescence from the sample 5 transmitted through the dichroic mirror 3 is diffracted by a diffraction lattice 6 and the angle of diffraction thereof corresponds to a wavelength. The light diffracted by the diffraction lattice 6 is condensed on a photodetector 8 through a lens 7. As the photodetector 8, for example, a camera is used. For example, when spots of a plurality of samples are arranged to a bio-chip, spectroscopic images (spectra) are obtained on the photodetector 8 at positions spatially shifted at every samples.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reading device for labeling a DNA or protein sample with a fluorescent substance, exciting the fluorescent substance with a laser beam or the like to generate fluorescence, and reading the wavelength of the fluorescence. The present invention relates to improvements for cost reduction and high accuracy.

[0002]

2. Description of the Related Art Conventionally, there is a technique of labeling DNA or protein with a fluorescent substance, irradiating a laser beam to excite the fluorescent substance, reading the fluorescence generated thereby, and detecting and analyzing DNA or protein. In this case, a biochip in which DNAs and proteins labeled with a fluorescent substance are spotted in an array on the biochip is used.

[0003] Reading of a biochip is performed as follows. For example, the laser light is oscillated in the horizontal axis direction to irradiate and irradiate the fluorescent substance of each spot in the array, and the emitted fluorescent light is condensed by, for example, an optical fiber, and the light is received by a light receiver via an optical filter. Extract the wavelength. When the reading operation of one line (spot row) is completed in this way, the biochip is driven in the vertical direction, and the same operation as described above is performed. This operation is repeated to read the entire biochip.

[0004]

However, such a conventional reading apparatus has the following problems. 1) The biochip has many spots and large external dimensions. There are many arrays. 2) Since the wavelength of the fluorescent light is separated by the optical filter, in the case of multi-color, the spectra are hardly separated by mixing depending on the concentration of each dye. 3) Quantitative property deteriorates due to mixing of auto-fluorescence, background light, etc., resulting in poor precision. 4) When switching the optical filter and the light receiver according to the fluorescent color, the switching operation takes time. 5) Instead of switching the optical filter and the light receiver, if a plurality of these are prepared and light is received at the same time, the speed is increased, but there is a disadvantage that it is expensive. 6) When a scanning confocal microscope is used, the number of parts is large, so that it is expensive and large, and the measurement takes time.

[0005] An object of the present invention is to solve the above-mentioned problems, and to provide a biochip reader which can be reduced in size, price, and accuracy at a glance.

[0006]

In order to achieve the above object, according to the first aspect of the present invention, a biochip in which a plurality of samples are arranged in a spot or line array is irradiated with light, and a light receiving device is provided. A biochip reader that reads image information corresponding to the plurality of samples, comprising means for arranging a plurality of pieces of spectral information of the target sample in a space between sample images in an image corresponding to the sample. It is characterized by having.

According to such a configuration, the spectral information of the sample can be output between the samples of the image of the light receiving device, and simultaneous multi-wavelength measurement can be easily realized. Also, multi-wavelength information can be obtained compactly.

[0008] In this case, as the second aspect, the means has a configuration in which a diffraction grating, a combination of an optical filter and an optical shift means, or a Fourier spectroscopy means is disposed between the sample and the light receiver. And various mechanisms can be used as appropriate.

Further, when the sample is a spot, the spectral information can be developed two-dimensionally on the light receiving device. According to such an invention, a large amount of information can be measured with high accuracy while being compact.

Further, in the present invention, a scanning or non-scanning confocal microscope or a two-photon excitation microscope can be appropriately used for the measurement.

Further, the accuracy of the signal of the spectral information can be improved by using means for separating the signal of the spectral information from noise by a regression method using a known spectrum.

Further, when the opening for limiting the spectral target area is matched with the position of each sample or with a part of each sample, information with less noise can be obtained. .

According to a seventh aspect of the present invention, the reading means is a non-scanning confocal microscope having an opening which coincides with the image position of the specimen or which coincides with a part of the specimen. Even with such a configuration, information with less noise can be easily obtained.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings. FIG. 1 is a configuration diagram showing an embodiment of the reading apparatus of the present invention. In the figure, 1 is a light source that generates laser light, 2 is a lens that converts light from the light source into parallel light, 3 is a dichroic mirror, 4 is an objective lens, 5 is a sample, 6 is a diffraction grating, 7 is a lens, and 8 is It is a light receiver.

The light (excitation light) generated from the light source 1 is converted into parallel light by the lens 2, reflected by the dichroic mirror 3, condensed through the objective lens 4, and illuminates the surface of the sample 5.
The sample emits fluorescence (having a wavelength different from that of the excitation light) by the irradiation light, and the fluorescence returns to the objective lens 4 again and enters the dichroic mirror 3.

The fluorescence from the sample transmitted through the dichroic mirror 3 is diffracted by the diffraction grating 6. The diffraction angle corresponds to the wavelength. The light diffracted by the diffraction grating 6 is condensed on a light receiver 8 via a lens 7. As the light receiver 8, for example, a camera or the like is used.

When, for example, spots of four samples S1, S2, S3, and S4 are arranged on the biochip as shown in FIG. 2, the photodetector 8 has a spatial arrangement for each sample as shown in FIG. The spectral images (spectrum) of the wavelengths λ1 to λn are obtained at the positions deviated from the above. Although the spectral image is spectral information, the spectral information can be sufficiently measured with a monochrome camera. In this case, as is clear from the figure, the gaps between the spots are skillfully used.

Although the above embodiment is directed to a biochip in which spots are scattered in an array, the present invention is not limited to this, and it is also applicable to a fluorescent pattern of an electrophoresis pattern arranged in a line. In that case, an image as shown in FIG. 4 is obtained. That is, spectral images of λ1 to λn are generated at positions spatially shifted in the horizontal axis direction with respect to the migration pattern (vertical axis direction) of each lane.

FIG. 5 is a block diagram showing another embodiment of the present invention. FIG. 5 shows an example in which two diffracted photons are arranged at right angles. According to such a configuration, a two-dimensional spectrum is obtained as shown in FIG. For example, the horizontal axis direction (X-axis direction) is incremented by 100 nm, and the vertical axis direction (Y-axis direction) is 10 n.
With m increments, a high dynamic range and high-precision measurement is possible.

FIG. 7 shows an embodiment in which a dichroic mirror is used instead of the diffraction grating. This is a combination of an optical filter and an optical shifting means. As shown, dichroic mirrors 31 and 3 having different transmission wavelengths are provided.
2, 33 (optical filters) are stacked on the optical axis. In this case, the angles of the dichroic mirrors are set so that the light reflected by the dichroic mirrors is reflected at the same angle as the one just diffracted by the diffraction grating (corresponding to an optical shift means).

FIG. 8 shows an example in which a non-movable Fourier spectroscopic means 81 of the Savart type or Michelson type is used in place of the diffraction grating or the dichroic mirror. In this case, the image obtained on the receiver is not the spectrum itself,
It is an interference fringe image. Therefore, a spectrum can be obtained by subjecting the interference fringe image to Fourier transform processing by a calculating means (not shown).

When using a confocal or two-photon microscope in addition to a normal fluorescence microscope or camera for the measurement,
Higher resolution is obtained. Further, even when the thickness of each sample varies due to the confocal slicing effect, a constant volume of the sample can always be measured, so that the quantitative property is improved. In this case, the confocal microscope may be a non-scanning type.

Further, as shown in FIG. 9, autofluorescence having a wavelength slightly different from the original fluorescence can be easily removed because the characteristics of the reagent used are known. If necessary, the signal spectrum may be separated by a regression method. In this case, high precision and high sensitivity can be easily realized.

In the case of spectroscopy, it is necessary to limit the measurement area by light shielding means such as a slit. Therefore, for example, FIG.
As shown in FIG. 0, by aligning the opening A with a certain region of the sample S1 or with a part of the sample,
The area can be used most effectively.

This is also effective for removing an error due to disturbance of the edge of the sample. Note that the shape of the opening may be not only a round shape but also a rectangular shape.

When the aperture shown in FIG. 10 or the rectangular aperture as described above is used as a pinhole or a slit of a non-scanning confocal microscope, it is inexpensive and small, but has high confocal high resolution. And quantification by slice. In this case, the detection means is not limited to the spectral method as shown in FIG. 1, but may be a general filter method.

[0027]

As described above, according to the present invention, the following effects can be obtained. 1) Fluorescence of multiple wavelengths can be measured simultaneously without switching between filters and light receivers, and a compact reader can be realized. 2) The spectrum displayed on the light receiver can be photographed with a black-and-white camera, which is inexpensive. 3) The spectrum displayed on the light receiver can easily be a two-dimensional spectrum, and high accuracy can be easily achieved.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing one embodiment of a biochip reader according to the present invention.

FIG. 2 is a diagram for explaining the arrangement of samples on a biochip.

FIG. 3 is an explanatory diagram for explaining spectral information displayed on a light receiver.

FIG. 4 is an explanatory diagram for explaining spectral information when measuring samples arranged in a line array.

FIG. 5 is a configuration diagram showing another embodiment of the present invention.

FIG. 6 is an explanatory diagram of a spectral image when spectral information is developed two-dimensionally.

FIG. 7 is a configuration diagram showing still another embodiment of the present invention.

FIG. 8 is a configuration diagram showing still another embodiment of the present invention.

FIG. 9 is an explanatory diagram showing a distribution state of autofluorescence and the like.

FIG. 10 is an explanatory diagram relating to a relationship between a sample and an opening.

[Explanation of symbols]

 Reference Signs List 1 light source 2 lens 3, 31, 32, 33 dichroic mirror 4 objective lens 5, S1, S2, S3, S4 sample 6, 61 diffraction grating 7 lens 8 light receiver 81 Fourier spectroscopic means A opening

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Claims (7)

[Claims] 1. A biochip reader for irradiating a biochip on which a plurality of samples are arranged in a spot or a line array with light and reading image information corresponding to the plurality of samples by a light receiver, In the empty area between the sample images in the image corresponding to the sample,
A biochip reader comprising means for arranging a plurality of pieces of spectral information of a target sample. 2. The apparatus according to claim 1, wherein said means has a structure in which a diffraction grating, a combination of an optical filter and an optical shift means, or a Fourier spectroscopic means is arranged between the sample and the light receiving device. The biochip reader according to the above. 3. The method according to claim 2, wherein the sample is a spot.
The biochip reader according to claim 1, wherein the biochip reader is configured to two-dimensionally expand spectral information on the light receiver. 4. The biochip reader according to claim 1, wherein said means uses a scanning or non-scanning confocal microscope or a two-photon excitation microscope. 5. The biochip reader according to claim 1, further comprising means for separating said spectral information signal from noise by a regression method using a known spectrum. 6. An opening for limiting a spectral target area,
2. The biochip reader according to claim 1, wherein the biochip reader matches the position of each sample or a part of each sample. 7. A biochip reader for irradiating a biochip on which a plurality of samples are arranged in a spot or a line array with light and reading image information corresponding to the plurality of samples by a light receiver, A biochip reader comprising: a non-scanning confocal microscope having an opening corresponding to an image position of a sample or a part of the sample as reading means.