JP4345739B2 - Biochip reader - Google Patents

Description

  The present invention relates to a biochip reader, and more particularly to a biochip reader using a coherent light source such as a laser as excitation light.

  Prior art documents related to this type of biochip reader include the following.

New Optical Microscope, Volume 2, “Application of Confocal Laser Microscope to Medicine and Biology”, Interdisciplinary Planning Co., Ltd., published on March 28, 1995, P132-133 Experimental medicine separate volume, post-genome era experimental lecture 3, "GFP and bioimaging", Yodosha Co., Ltd., published on October 25, 2000, P158

  FIG. 17 is a principle configuration diagram based on the confocal optical system described in Non-Patent Document 1. In the biochip reader based on such a principle configuration diagram, the laser light emitted from the light source 1 emitted through the pinhole PH provided in the light shielding plate 2 becomes parallel light by the lens 3 and passes through the dichroic mirror 4. Then, the light enters the objective lens 5. The objective lens 5 collects the excitation light and irradiates the sample (biochip) 6.

  The fluorescent substance attached to the sample of the biochip 6 is excited by excitation light to emit light, and the fluorescence is reflected by the dichroic mirror 4 through the objective lens 5 and then is narrowed down by the condenser lens 7 to be blocked by the light shielding plate 8. The image is formed on the pinhole PH provided in the. This image is a fluorescent image of the sample 6 and is detected by the light receiver 9.

  In order to observe the fluorescent image (two-dimensional image) of the sample 6 surface, it is necessary to scan the surface of the sample 6 with excitation light. In this case, normally, the side of the stage (not shown) on which the sample 6 is placed is scanned in a direction perpendicular to the optical axis (referred to as stage scanning) instead of scanning the excitation light side.

  In such a biochip reader, laser light is used because the amount of light is insufficient when white light is used as the light source. In addition, since the confocal type is formed using a pinhole, the detection image is not affected by dust adhering to the sample 6 and speckle noise is not generated because the excitation light is focused on the sample. It is like that.

FIG. 18 is a principle configuration diagram showing another example of a conventional biochip reader. Such a principle is described in Non-Patent Document 2, for example.
In FIG. 18, excitation light from a parallel light source (not shown) such as a laser is focused by the condenser lens 11, then reflected by the dichroic mirror 12 and incident on the objective lens 13. At this time, the excitation light forms an image at a position of the focal length f of the objective lens 13 and enters the objective lens 13 as a second light source.

  The sample 14 is irradiated with excitation light that has passed through the objective lens 13. An example of the sample is a DNA chip in which DNA is arranged on a slide glass having a flat surface. At each site 15 of the DNA chip, the fluorescent substance of the labeled DNA is excited by excitation light and emits fluorescence.

  The fluorescence forms an image on the light receiver 18 through an image forming optical system. That is, the fluorescent light becomes parallel light by the objective lens 13 as shown by a solid line, passes through the dichroic mirror 12 and the barrier filter 16 and enters the lens 17. The image of the sample 14 formed by the lens 17 is detected by the light receiver 18.

  In this case, the behavior of the excitation light irradiated over the entire surface of the sample is considered as follows. As shown by the broken line, the excitation light reflected by the flat sample surface (this excitation light is referred to as reflected excitation light or return excitation light) is focused by the objective lens 13 and converges to the position of the focal length f. Then, the light enters the light receiver 18 through the lens 17 having the focal position as an intermediate image plane.

  The reflected excitation light passes through the dichroic mirror 12 and the barrier filter 16 before passing through the lens 17. The barrier filter 16 is formed so as to pass fluorescence but remove (attenuate) reflected excitation light. By passing through the barrier filter 16, reflected excitation light mixed into the light receiver 18 as background light is reduced.

However, such a conventional biochip reader has the following problems.
(1) The biochip reader shown in FIG. 17 has a problem that adjustment of pinholes and the like is difficult and expensive. Further, the stage is required to be durable for scanning, and has a drawback of becoming expensive.

(2) Although the apparatus shown in FIG. 18 attenuates reflected excitation light with a barrier filter, sufficient attenuation was not obtained. In fluorescent molecule measurement and the like, it is necessary to reduce the background light to about 10 ⁻⁹ , but with this apparatus, only an attenuation rate of about 10 ⁻⁷ can be obtained, and there is a problem that the attenuation is clearly insufficient.

  Moreover, when measuring the expression of mRNA in a biochip with cDNA, there is a significant difference in the expression level, and there are the following problems. For example, if there is a difference of 10 to 100 times in the expression levels of gene A and gene B shown in FIG. 19 (a), if the expression level is measured as it is with high accuracy, However, the analog / digital converters and amplifiers used are required to have a large dynamic range and high accuracy, and are expensive.

  In addition, a method of measuring a plurality of times by changing the gain of an analog / digital converter or an amplifier is conceivable, but this method has a problem that it takes a long time to measure, and a variation in measured values and a fading of a biochip increase.

  An object of the present invention is to solve such a problem and to provide a biochip reader capable of sufficiently attenuating reflected excitation light as background light.

In order to achieve such a problem, in the invention according to claim 1 of the present invention,
In the biochip reader configured to irradiate the sample with a flat surface with excitation light and read the fluorescence generated from the fluorescent substance in the sample with a light receiver,
An imaging lens that is formed in a plano-convex shape and has a fluorescent interference film that transmits the fluorescence and attenuates the excitation light on the plane side thereof, and the fluorescent interference film receives the light in front of the light receiver. is disposed to face the vessels, with imaging the fluorescence arising from the illuminated before Symbol samples the excitation light as parallel light to the light receiving surface of the photodetector, incident on the photodetector-side from the sample side and wherein the to Turkey into parallel light excitation light.

  If such a lens is used, the same effect as the barrier filter can be realized simply, small and inexpensively.

The present invention has the following effects.
(1) By using a plano-convex lens as the imaging lens and attaching a fluorescent interference film to the imaging lens, it is possible to easily realize the same effects as using the barrier filter without using the barrier filter.

(2) Since the reflected excitation light beam focused by the objective lens is shielded or reflected, it is possible to easily prevent the reflected excitation light from entering the light receiver without using a barrier filter.
(3) Also in the transmission type fluorescence reading biochip reader, the excitation light entering the light receiver can be easily attenuated by arranging a barrier filter.

  Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a main part configuration diagram showing an example of a biochip reader related to the present invention.

  In FIG. 1, 31 is a light source that generates coherent light such as a laser (hereinafter referred to as laser light) as excitation light, 32 is a lens that converts the laser light into parallel light, and 33 is a microlens array. The microlens array 33 is an array of a plurality of microlenses ML, and is attached to the rotating plate 34.

  35 is a motor for rotating the rotating plate 34, 36 is a sample, 37 is a lens, 38 is a barrier filter, 39 is a condenser lens, and 40 is a camera using a CCD or the like as a light receiving element.

  The laser light emitted from the light source 31 is collimated by the lens 32 and enters the microlens array 33. Each microlens ML collects the laser beam and irradiates the sample 36. In the sample 36, a plurality of cells are two-dimensionally arranged, and a sample is injected into each cell.

  When the rotating plate 34 is rotated by the motor 35, the excitation light beam focused by each microlens ML scans the sample 36. The microlens ML is disposed on the rotating plate 34 in such a spatial positional relationship that each beam can individually scan each cell of the sample.

Fluorescence from each sample enters the lens 37 and then enters the lens 39 via the barrier filter 38. The barrier filter 38 has an effect of passing the fluorescence from the sample 36 but attenuating the excitation light entering through the sample 36, and is used to remove background light of the sample image.
The sample image condensed and imaged by the lens 39 is received by a light receiving element (not shown) of the camera 40.

With such a configuration, it is possible to easily obtain a two-dimensional image of a sample by obtaining a multi-beam with a plurality of microlenses and scanning the sample with the beam.
Note that since dust attached to the sample can be drastically reduced by using a cartridge or the like, it is not necessary to use a confocal reader as in the prior art. However, the amount of excitation light is necessary. In the present invention, since laser light is used as excitation light, a sufficient amount of light can be obtained with high brightness.

  In the present invention, since the excitation light is focused by the microlens, speckle noise does not occur in the sample image. In addition, since a pinhole is not used as in the prior art, adjustment is easy, and a moving mechanism for moving the stage for optical scanning is not required. Therefore, a highly durable and small-sized apparatus can be easily realized.

  FIG. 2 is a block diagram showing the main part of another example. The biochip reader of FIG. 1 is a transmissive type, while FIG. 2 is a reflective type. A dichroic mirror 41 is disposed between the microlens array 33 and the sample 36, and the fluorescence emitted from the sample 36 is reflected by the dichroic mirror 41 and enters the lens 39. The lens 39 collects this fluorescence and forms an image on the light receiving element surface of the camera 40. Other configurations and operations are the same as those in FIG.

  FIG. 3 is another embodiment of the present invention in which excitation light is incident on the barrier filter at an incident angle of ± 5 degrees or less. The difference from the conventional example of FIG. 18 is the arrangement position of the barrier filter. In FIG. 3, the barrier filter 16 is disposed between the lens 17 and the light receiver 18.

As shown in FIG. 4, the attenuation rate of the barrier filter 16 (the ratio of the outgoing light intensity to the incident light intensity) depends on the incident angle of light. To obtain an attenuation factor of 10 ⁻⁷ or more, the incident angle to the barrier filter is Must be ± 5 degrees (deg) or less.

  The data shown in FIG. 4 is an example in which the barrier filter is measured using parallel laser light. Since a general spectrophotometer uses divergent or convergent light, such a value cannot be obtained.

With this arrangement of the barrier filter 16, the excitation light is incident on the barrier filter 16 at an incident angle of ± 5 degrees or less, and is attenuated by approximately 10 ⁻⁹ . Therefore, the background light of the observation image on the surface of the light receiver 18 is sufficiently reduced.

In addition, this invention is not limited to the said Example. For example, the following configuration may be employed.
(1) Case Related to Size of Light Source Image FIG. 5 shows a configuration diagram of a main part of a Koehler illumination system when non-laser light such as white light is used. Light from the light source 51 (the diameter of the light source is a) enters the condenser lens 11 to create a second light source 52 having a diameter a ′. The sample 14 is irradiated by the second light source 52. The angle β of the excitation light that irradiates the sample 14 at this time (here, this angle is referred to as the irradiation angle) β corresponds to the diameter of the second light source 52.

  A transmission-type or reflection-type microscope cannot obtain a necessary and sufficient resolution without a large angle β by Koehler illumination. Similarly, in the case of the fluorescence microscope, the diameter a ′ of the light source image 52 is enlarged to about 10 times the diameter a of the original light source 51, and the angle β is increased.

However, when the angle β is increased, the incident angle to the barrier filter 16 is also increased, and the attenuation rate of the excitation light is lowered. Therefore, the angle β has a limit. There is the following relationship between the light source image and the focal length f of the objective lens 13.
a ′ / 2 = fβ
That is,
β = a ′ / (2f)
Where f is the focal length of the objective lens 3

  In order to obtain a sufficient attenuation rate, β needs to be ± 5 degrees or less. In order to suppress it to ± 5 degrees or less, for example, the size of the light source or the focal length f ′ of the excitation optical system may be adjusted.

(2) Case of Light Source Array FIG. 6 is a configuration diagram in the case where a light source array composed of two light sources having different wavelengths such as green G and red R is used. The G light source is placed on the optical axis at a distance f ′ from the lens 11, and the R light source is placed at a position away from the optical axis by b. In such an arrangement, the light from the G light source enters perpendicular to the horizontally placed sample surface, and the light from the other R light source enters at an irradiation angle γ (= f′b).

  After the excitation light reflected on the sample surface passes through the objective lens 3 and the dichroic mirror 12, the light on the short wavelength side (G light source side) is reflected on the second dichroic mirror 12a, and the other long wavelength side (R light source side) ) Passes through the second dichroic mirror 12a.

  The former forms an image on the light receiver 18 through the imaging lens 17 and the barrier filter 16, and the latter forms an image on the light receiver 18a through the imaging lens 17a and the barrier filter 16a.

In this case, when the barrier filter 16a is mounted horizontally, the reflected excitation light of the R light source enters at an angle inclined by γ degrees. Therefore, the barrier filter 16a is disposed so as to be inclined by γ degrees from the horizontal plane so as to be incident at a right angle.
As a result, the reflected excitation light on the sample surface is incident on the barrier filters 16 and 16a at a right angle, and is sufficiently attenuated.

(3) Modified Example of Imaging Lens FIG. 7 is a configuration diagram of a main part of the imaging lens. This is an example in which a fluorescent filter lens 60 having both a barrier filter function and an imaging lens function is used.
The fluorescent filter-equipped lens 60 is obtained by attaching a fluorescent interference film 62 to the plane side of the plano-convex lens 61. The interference film 62 attenuates reflected excitation light in the same manner as a barrier filter.

(4) Modification to Point Mask Type As shown in FIG. 8, a light shielding member 71 is arranged at a location where reflected excitation light from the sample is converged by the objective lens 13 to cut the reflected excitation light. The light shielding member 71 is supported by, for example, a transparent plate or three support pillars extending radially in the horizontal direction.

  If the diameter φ of the light shielding member 71 is about 1.22λ / NA (where λ is the wavelength of the excitation light and NA is the numerical aperture of the objective lens 13) (in other words, the area is approximately equal to the beam diameter of the excitation light). 80% or more of the reflected excitation light can be cut. In this case, it is not necessary to use a barrier filter as in the above embodiment. Alternatively, an inexpensive filter with a low attenuation factor may be used.

(5) Modification to Point Mirror Type Instead of the dichroic mirror shown in the above embodiment, a small mirror 72 is arranged in the second light source part of Koehler illumination as shown in FIG. The mirror 72 has an area substantially equal to the beam diameter of the excitation light, reflects the excitation light from the light source 81 as in the dichroic mirror, irradiates the sample 14, and reflects the excitation excited by the objective lens 13. Light also reflects to the light source side. Therefore, the reflected excitation light does not enter the light receiver (not shown).
The mirror 72 is held by a support 73 made of a transparent substrate or a column, and the mounting position and angle thereof are maintained.

(6) In the case of a transmission fluorescence type FIG. 10 is a diagram showing an embodiment of a biochip reader of a transmission fluorescence reading system. In this case, similarly, the transmitted excitation light can be attenuated by using the barrier filter. Barrier filters 94 and 97 are disposed between the sample 93 and the objective lens 95, and between the imaging lens 96 and the light receiver 18, respectively. Note that only one of the barrier filters may be used.
In this case, the objective lens 95 and the imaging lens 96 are telecentric.

FIG. 11 shows a configuration in which a barrier filter 38 is arranged between the lens 39 and the camera 40 in the configuration shown in FIG. As shown by the dotted line in FIG. 11, the excitation light transmitted through the sample 36 enters the imaging optical system composed of lenses 37 and 39 as substantially parallel light, and then is substantially parallel to the camera 40. Incident.
At this time, the excitation light enters the barrier filter 38 at an incident angle of ± 5 degrees or less, and is attenuated by approximately 10 ⁻⁷ or more.

  Further, as a solution to the problem pointed out in FIG. 19 of the conventional example, the following configuration is desirable. Excitation light consisting of a portion having a high light intensity and a portion having a low light intensity is generated from the light source, and the shading of the excitation light is intentionally increased.

  For example, as shown in FIG. 12, the excitation light has a light intensity pattern in which the center is strong and the periphery is weak, and the ratio α of the minimum value to the maximum value of the light intensity I within the effective range R of the parallel light is set to about 90%. To do. Excitation light having such a light intensity distribution irradiates the sample 23 with a similar light intensity distribution pattern via the microlens array 21 and the dichroic mirror 22.

  On the other hand, in the sample 23, as shown in FIG. 13, a sample with a higher expression level is arranged in the outer cell, and a sample with a lower expression level is placed in the inner cell, so Arrange it to be a pattern.

  If the light intensity distribution of the excitation light and the expression level distribution of the sample are in this relationship, a sample with low expression level, in other words, a sample with low fluorescence, is irradiated with excitation light with strong light intensity, and the expression level is high. In this case, excitation light with weak light intensity is irradiated to a sample having a lot of fluorescence.

  As a result, even if there is a large difference in the expression level, there is not much difference in fluorescence from each sample incident on the detector, so the analog / digital converter and amplifier in the detector have a large dynamic range and high accuracy. It is not required and a low-precision and inexpensive one can be used. The excitation light distribution is measured separately, and the fluorescence from the sample is corrected by the excitation light distribution and used.

  Such a biochip reader is not limited to the above embodiment. For example, as shown in FIG. 14, when only a part of the excitation light is used using a light shielding or dimming mask 100, the excitation light applied to the biochip has a light intensity distribution as shown. In this way, if a mask having a light-shielding or dimming pattern (herein also referred to as a density pattern) is used, the degree of freedom of sample arrangement on the biochip is greatly improved.

  Further, as shown in FIG. 15, the shading amount of the excitation light may be made variable. In the figure, the difference from the configuration of FIG. 12 is the zoom mechanism. The zoom mechanism 110 is a mechanism using a normal zoom lens, and applies a parallel light beam from a light source (not shown) to the microlens array 21 by appropriately enlarging or reducing it.

  When the zoom mechanism unit 110 expands the beam, the light intensity distribution pattern of the excitation light emitted from the light source spreads horizontally, and the intensity distribution pattern of the excitation light incident on the microlens array 21 becomes a flatter pattern. On the other hand, if the image is reduced, the pattern becomes steeper.

  As described above, the shading amount changes due to enlargement / reduction, so that the shading amount can be arbitrarily set according to the sample 23 by operating the zoom mechanism unit 110.

In addition, the fluorescence image of the sample is detected using a detector composed of light-receiving elements whose input / output characteristics are in a logarithmic relationship as shown in FIG. 16 without changing the light intensity distribution of the excitation light or the arrangement of the sample. It is good also as composition to do.
According to such a configuration, saturation in the analog / digital converter or amplifier on the detector side can be prevented even if there is a large difference in the input (fluorescence amount), and the analog / digital converter or amplifier has a large dynamic range. High accuracy is no longer required.

According to the invention of the embodiments described above, even if there is a large difference in the expression level of each sample of the biochip, the detector does not require a large dynamic range or high accuracy, and is inexpensive and low-accuracy analog-digital conversion. Satisfactory sample images can be detected with a detector using a detector or amplifier.
In addition, since the shading amount of the light intensity distribution of the light source can be increased, there is an effect that the efficiency of the light amount can be significantly increased.

It is a principal part block diagram which shows one Example of the biochip reader concerning this invention. It is a principal part block diagram which shows the other Example of this invention. It is a principal part block diagram which shows the further another Example of this invention. It is an attenuation factor characteristic view of a barrier filter. It is a principal part block diagram which shows another example of the biochip reader of this invention. It is explanatory drawing at the time of using a light source array. It is a block diagram which shows the other Example of the lens for imaging. It is a block diagram at the time of using a light shielding member. It is a block diagram at the time of using a mirror. 1 is a configuration diagram of an embodiment according to a transmission type fluorescence reading type biochip reading apparatus; FIG. It is a principal part block diagram which shows the other Example of this invention. It is a principal part block diagram which shows the further another Example of this invention. It is a figure for demonstrating the mode of arrangement | positioning of a sample. It is a principal part block diagram which shows the other Example of this invention. It is a principal part block diagram which shows the other Example of this invention. It is a figure which shows the input-output characteristic of a detection element. It is a principle block diagram of the conventional biochip reader. It is a principle block diagram which shows another example of the conventional biochip reader. It is explanatory drawing about the difference in the expression level of a biochip and a sample.

Explanation of symbols

11, 92, 95, 96 Lens 12, 12a, 41 Dichroic mirror 13 Objective lens 14, 36, 93 Sample 15 Site 16, 16a, 38, 94, 97 Barrier filter 17, 17a, 25, 32, 37, 39 Lens 18 , 18a Light receiver 21, 33 Micro lens array 23, 36 Sample 31, 51, 81, 91 Light source 34 Rotating plate 35 Motor 40 Camera 52 Second light source 60 Lens with fluorescent filter 61 Plano-convex lens 62 Fluorescent interference film 71 Light shielding member 72 Mirror 100 Mask 110 Zoom mechanism

Claims (1)

In the biochip reader configured to irradiate the sample with a flat surface with excitation light and read the fluorescence generated from the fluorescent substance in the sample with a light receiver,
An imaging lens that is formed in a plano-convex shape and has a fluorescent interference film that transmits the fluorescence and attenuates the excitation light on the plane side thereof, and the fluorescent interference film receives the light in front of the light receiver. is disposed to face the vessels, with imaging the fluorescence arising from the illuminated before Symbol samples the excitation light as parallel light to the light receiving surface of the photodetector, incident on the photodetector-side from the sample side biochip reader, wherein the to Turkey into parallel light excitation light.

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