JP3689901B2 - Biochip reader - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a reading device that labels a fluorescent substance on a DNA or protein sample, excites it with a laser beam or the like to generate fluorescence, and reads the wavelength of the fluorescence. It is about the improvement for conversion.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, there is a technique for labeling a fluorescent substance on DNA or protein, irradiating laser light to excite the fluorescent substance, reading the generated fluorescence, and detecting and analyzing DNA or protein. In this case, a biochip in which DNA or protein labeled with a fluorescent substance is spotted in an array on the biochip is used.
[0003]
The biochip is read as follows. For example, a laser beam is shaken and irradiated in the horizontal axis direction to excite the fluorescent material in each spot of the array, and the emitted fluorescence is collected by, for example, an optical fiber, and received by a light receiver through an optical filter. Extract the wavelength. When the reading operation for one line (spot row) is completed in this way, the biochip is driven in the vertical axis direction and the same operation as described above is performed. This operation is repeated to read the entire biochip.
[0004]
[Problems to be solved by the invention]
However, such a conventional reading apparatus has the following problems.
1) Biochip has many spots and large external dimensions. There are many arrays.
2) Since the wavelength of fluorescence is separated by an optical filter, it is difficult to separate the spectrum by mixing with the concentration of each dye in the case of multiple colors.
3) Since autofluorescence and background light are mixed, the quantitativeness is deteriorated and the accuracy is poor.
4) When the optical filter and the light receiver are switched according to the fluorescent color, the switching operation takes time.
5) Instead of switching between the optical filter and the light receiver, if a plurality of these are prepared and light is received simultaneously, 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 the scanning confocal microscope is expensive and large in size and takes a long time for measurement.
[0005]
SUMMARY OF THE INVENTION An object of the present invention is to solve the above-described problems, and to provide a biochip reader that can be reduced in size, reduced in price, and improved in accuracy.
[0006]
[Means for Solving the Problems]
In order to achieve such an object, in the present invention,
A plurality of samples arranged two-dimensionally at positions spaced apart from each other by a predetermined distance on the substrate are simultaneously irradiated with measurement light, and fluorescence from the sample is simultaneously measured with one two-dimensional light receiver. A biochip reader,
With a means for arranging a plurality of spectral information of the target sample in the space between the sample images in the image corresponding to the sample ,
The opening for limiting the spectroscopic object region is configured to coincide with the position of each sample or a part of each sample .
[0007]
According to such a configuration, the spectral information of the sample can be output between the samples of the image of the light receiver, and simultaneous multi-wavelength measurement can be easily realized. In addition, multi-wavelength information can be obtained in a compact manner.
In addition, since the opening matches the position of each sample or a part of each sample, information with less noise can be easily obtained.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing an embodiment of a reading apparatus according to the present invention.
In the figure, 1 is a light source that generates laser light, 2 is a lens that collimates light from the light source, 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 a lens. It is a light receiver.
[0015]
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 irradiated onto 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.
[0016]
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 collected on the light receiver 8 through the lens 7. For example, a camera or the like is used as the light receiver 8.
[0017]
For example, when the spots of four samples S1, S2, S3, and S4 are arranged on the biochip as shown in FIG. 2, the light receiver 8 is spatially shifted for each sample as shown in FIG. A spectral image (spectrum) of wavelengths λ1 to λn is obtained at the position. Although this spectral image is spectral information, the spectral information can be measured sufficiently with a black and white camera. In this case, as clearly shown in the figure, the gaps between the spots are skillfully used.
[0018]
In the above embodiment, biochips with spots scattered in an array are targeted. However, the present invention is not limited to this, and fluorescent patterns of electrophoresis patterns arranged in a line can also be targeted. 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.
[0019]
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, if the horizontal axis direction (X-axis direction) is incremented by 100 nm and the vertical axis direction (Y-axis direction) is incremented by 10 nm, measurement with a wide dynamic range and high accuracy becomes possible.
[0020]
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 shift means. As illustrated, dichroic mirrors 31, 32, and 33 (optical filters) having different transmission wavelengths are stacked on the optical axis. In this case, the angle of each dichroic mirror is set so that the light reflected from the dichroic mirror is reflected at the same angle as that diffracted by the diffraction grating (corresponding to the optical shift means).
[0021]
FIG. 8 shows an example in which a Savart type or Michelson type non-movable Fourier spectroscopic means 81 is used in place of the diffraction grating and the dichroic mirror. In this case, the image obtained by the light receiver is not a spectrum itself but an interference fringe image. Therefore, a spectrum can be obtained by subjecting this interference fringe image to Fourier transform processing by a calculation means (not shown).
[0022]
In addition, when using not only a normal fluorescence microscope and a camera but also a confocal or two-photon microscope, the resolution becomes higher. Further, even when the thickness of each sample varies due to the confocal slicing effect, it is possible to always measure a sample having a constant volume, so that quantitativeness is improved.
In this case, the confocal microscope may be a non-scanning type.
[0023]
In addition, 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 way, high accuracy and high sensitivity can be easily realized.
[0024]
In spectroscopy, it is necessary to limit the measurement region with a light shielding means such as a slit. Therefore, for example, as shown in FIG. 10, the area can be used most effectively by matching the opening A with a certain region of the sample S1 or with a part of the sample.
[0025]
This is also effective for removing errors due to the disturbance of the edge of the sample. The shape of the opening may be not only a round shape but also a rectangle.
[0026]
Further, when an opening as shown in FIG. 10 or a rectangular opening as described above is used as a pinhole or slit of a non-scanning confocal microscope, it is inexpensive and small, but with high confocal resolution and slices. Quantitative properties can be obtained.
In this case, the detection means is not limited to the spectroscopic method as shown in FIG.
[0027]
【The invention's effect】
As described above, the present invention has the following effects.
1) It is possible to simultaneously measure multi-wavelength fluorescence without switching filters and light receivers, and a compact reader can be realized. In addition, since the opening matches the position of each sample or matches a part of each sample, information with less noise can be easily obtained.
2) The spectrum displayed on the light receiver can be captured with a black and white camera, and can be inexpensive.
3) The spectrum displayed on the light receiver can easily be a two-dimensional spectrum, and high accuracy is easy.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing an 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 samples arranged in a line array are measured.
FIG. 5 is a block 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 block diagram showing still another embodiment of the present invention.
FIG. 8 is a block 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]
DESCRIPTION OF SYMBOLS 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 spectroscopy means A Aperture

Claims (4)

A plurality of samples arranged two-dimensionally at positions spaced apart from each other by a predetermined distance on the substrate are simultaneously irradiated with measurement light, and fluorescence from the sample is simultaneously measured with one two-dimensional light receiver. A biochip reader,
With a means for arranging a plurality of spectral information of the target sample in the space between the sample images in the image corresponding to the sample ,
A biochip reader characterized in that an opening for limiting a spectroscopic object region is configured to coincide with a position of each sample or a part of each sample . Said means, between said sample and the light receiver, grating or optical filter and the combination of the optical shift means, or Bio placing serial to claim 1, characterized in that the configuration of arranging the Fourier spectral means, Chip reader. It said means when said sample spot, the light receiver on the spectral information two-dimensionally configured and becomes possible biochip reader of the mounting serial to claim 1, wherein to deploy. It said means scanning or non-scanning confocal microscope, or two-photon excitation microscope the serial mounting of the biochip reader to claim 1, characterized by using.

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