JP2010032400A - Biochip reader - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biochip reader capable of suppressing the change in the quantity of exciting light based on the positional shift in the optical axis direction of the biochip to suppress the lowering of reading precision. <P>SOLUTION: An illumination optical system 20 is constituted so that an exciting light beam vertically illuminates the whole surface 11 of the biochip 1. Accordingly, the intensity of the exciting light beam is not changed even if the position of the surface 11 of the biochip 1 is shifted from a regular position in the P-direction (optical axis direction) shown by Fig.1. As a result, a change in the intensity of the exciting light or the lowering of detection precision caused by intensity irregularity can be prevented. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biochip reader that includes an illumination optical system that irradiates the surface of a biochip with excitation light and reads a fluorescent image based on the excitation light.

There is known a biochip reader that irradiates the surface of a biochip with excitation light and reads a fluorescence image formed by fluorescence excited by the excitation light. In this apparatus, a fluorescence image of the biochip surface is formed on a light receiver, and the fluorescence intensity of the sample is detected based on the amount of light received detected by the light receiver.
JP 2003-28799 A

  However, in the conventional apparatus, unless the surface of the biochip is accurately arranged at the focal position of the excitation light, sufficient fluorescence intensity cannot be obtained, and high reading accuracy cannot be obtained.

  That is, when the biochip surface deviates from the focal position of the excitation light, the light intensity per unit area of the excitation light irradiated on the biochip surface decreases, so the intensity of the excited fluorescence also decreases. The fluorescent image formed on the screen becomes dark.

  The cause of the position of the biochip is the processing accuracy of the sample holder holding the biochip and the assembly accuracy of the sample holder and the optical system, but the dimensional accuracy of the biochip itself is also greatly related.

  In general, as shown in FIG. 5A, the biochip reader is arranged so that the biochip 100 is held by a sample holder 400 and the surface of the biochip 100 is at the focal position of the optical system (objective lens) 300. ing.

  Here, as shown in FIG. 5B, when a foreign substance 450 such as dust adheres to the sample holder 400, the surface of the biochip 100 is tilted and deviates from the focal position depending on the location.

  5C to 5E show the dimensional accuracy of the biochip 100. FIG. 5 (c) shows the flatness of the biochip 100, FIG. 5 (d) shows the tolerance of the thickness of the biochip 100, and FIG. 5 (e) shows the deformation of the biochip 100 due to distortion and weight. is there.

  As described above, when the surface position of the biochip deviates from the focus due to various causes, the fluorescence intensity to be detected is greatly reduced, so that high reading accuracy cannot be obtained.

  According to JIS R3703, in the case of a glass slide for a microscope that is also used as a material for a biochip, the thickness tolerance is +0.1 mm in the thick direction and -0.2 mm in the thin direction. Is defined as ± 0.1 mm, and the flatness is also defined as 0.05 mm or less. For this reason, in order to constantly irradiate the biochip surface with excitation light having a certain intensity or more, the reading device has a depth of focus of ± 50 μm even when considering only flatness, and ± 100 μm or more considering thickness tolerance. There is a need.

  FIG. 6 shows a conventional excitation method. FIG. 6A shows a single beam case, and FIG. 6B shows a multi-beam case. In general, in the conventional reader, since the lens that irradiates the biochip with the excitation light and the lens that receives the fluorescence are shared, the focal depth of the lens cannot be increased. Could not have In other words, as shown in the figure, the spread angle φ or the incident angle δ of the excitation light is large.

  An object of the present invention is to realize a biochip reader with a simple configuration that eliminates the drawbacks of the conventional device as described above and can suppress a decrease in reading accuracy based on a positional deviation of the biochip in the optical axis direction. It is what.

The biochip reader of the present invention includes an illumination optical system that irradiates the surface of the biochip with excitation light, and in the biochip reader that reads a fluorescent image based on the excitation light, the illumination optical system includes the biochip. The excitation light is irradiated perpendicularly and parallel to the surface.
According to this biochip reader, since the excitation light is irradiated perpendicularly to the surface of the biochip, it is possible to suppress the change in the amount of excitation light based on the displacement of the biochip in the optical axis direction and to suppress the decrease in reading accuracy. .

  The illumination optical system is configured to adjust the divergence angle of the excitation light to a narrow angle and irradiate at an incident angle close to perpendicular to the biochip surface, and the focal depth of the excitation optical system is affected in the range of variation of the slide glass. It may be configured to have a depth of focus of ± 50 μm or more so as not to receive light.

  According to the biochip reader of the present invention, since the excitation light is irradiated perpendicularly to the surface of the biochip, it is possible to suppress the change in the amount of excitation light based on the positional deviation in the optical axis direction of the biochip and to reduce the reading accuracy. Can be suppressed.

  Hereinafter, an embodiment of a biochip reader according to the present invention will be described with reference to FIGS.

  FIG. 1 is a diagram illustrating a configuration of an optical system in the biochip reader according to the present embodiment.

  As shown in FIG. 1, the biochip reader of this embodiment includes an illumination optical system 20 that irradiates the surface 11 of the biochip 1 with excitation light, and a fluorescent image based on the excitation light irradiated by the illumination optical system 20. An imaging optical system 30 that forms an image and a mechanism (sample holder) 40 that holds the biochip 1 are provided.

  A plurality of sites 12 made of a sample labeled with a fluorescent reagent are two-dimensionally arranged on the surface 11 of the biochip 1. The biochip 1 is held on a sample holder 40 for holding a biochip.

The illumination optical system 20 includes a laser light source 21 that generates laser light as excitation light, a lens 22 that converts the laser light from the laser light source 21 into parallel light, a transparent rotating plate 23 on which a microlens 23a is formed, Lenses 24 and 25 for light, and a dichroic mirror 26 that selectively reflects only excitation light and selectively transmits only fluorescence. The rotating plate 23 is attached to the rotating shaft 28 of the motor 27 and is rotated by the driving force of the motor 27. When the parallel light from the lens 22 enters the rotating plate 23, the laser light is condensed by each microlens 23a and irradiates the biochip 1.

  When the rotating plate 23 is rotated by the motor 27, the surface 11 of the biochip 1 is scanned with the excitation light beam narrowed by each microlens 23a. The microlens 23a is arranged on the rotating plate 23 in a spatial positional relationship such that each beam can individually scan each site 12 of the biochip 1. With such a configuration, generation of speckle noise due to laser light can be prevented.

  The imaging optical system 30 includes a lens 31, a barrier filter 32 that removes excitation light and selectively transmits only fluorescence, and an objective lens 33. The fluorescence from the site 12 of the biochip 1 excited by the excitation light is incident on the light receiver 4 through the lens 31, the barrier filter 32, and the objective lens 33 and forms an image on the light receiver 4.

  In the biochip reader of the present embodiment, the illumination optical system 20 is configured so that the excitation light beam is irradiated perpendicularly to the entire surface 11 of the biochip 1. For this reason, even if the position of the surface 11 of the biochip 1 is shifted from the normal position in the P direction (optical axis direction) shown in FIG. 1, the intensity of the excitation light beam does not change. Therefore, it is possible to prevent a decrease in detection accuracy due to fluctuations in the intensity of excitation light and intensity unevenness.

  FIG. 2 shows the relationship of the depth of focus when the excitation light having the divergence angle φ is irradiated onto the biochip surface at the incident angle δ. FIG. 3 illustrates the relationship between the depth of focus and the maximum incident angle under the condition of a wavelength of 640 nm, which is the excitation wavelength of the Cy5 dye often used in biochips. As shown in FIG. 3, even if the excitation light is not parallel, the maximum incident angle is ± 4.6 by adjusting the divergence angle of the excitation light to a narrow angle and the incident angle close to perpendicular to the biochip surface. It is possible to obtain a focal depth of ± 50 μm in the case of degrees and ± 100 μm in the case of ± 3.3 degrees.

  The fluorescence excited by the excitation light is emitted radially from the sample at the site 12 in all directions. In the biochip reader according to the present embodiment, the excitation system and the light receiving system have different paths, so that the numerical apertures of the excitation system and the light receiving system can be set separately. For this reason, the imaging optical system 30 is configured such that the numerical aperture of the light receiving system of the biochip reader increases as much as possible in order to increase the amount of received light and increase the detection sensitivity.

  FIG. 4 is an actual measurement example illustrating the relationship between the amount of deviation from the normal focal position and the amount of received light. In FIG. 4, a solid line 51 represents the relationship in the biochip reader according to the present invention, and a dotted line 52 represents the relationship in the comparative example using the conventional reader. Looking at the range of points where the amount of received light at the regular focal position decreases by 10%, it can be seen that the present invention achieves a deep depth of focus with 250 μm for the present invention and 18 μm for the conventional type.

  As described above, according to the biochip reader of the present invention, the excitation light is irradiated perpendicularly to the surface of the biochip, or the excitation light is irradiated at a divergence angle and an incident angle that give an allowable depth of focus. Therefore, a change in the amount of excitation light based on the positional deviation of the biochip in the optical axis direction can be suppressed, and a decrease in reading accuracy can be suppressed.

  The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied to a biochip reader that includes an illumination optical system that irradiates the surface of a biochip with excitation light and reads a fluorescent image based on the excitation light.

The block diagram which shows one Example of the optical system in the biochip reader of this invention. The figure which shows the theoretical formula of a focal depth. The figure which shows the relationship between the focal depth and incident angle in an example wavelength. The figure which illustrates the relationship between the deviation | shift amount from a regular position, and light reception amount. The figure which shows the structure of the conventional biochip reader, and the dimensional accuracy of a biochip. The figure which shows the excitation system in the conventional biochip reader.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,100 Biochip 11 Surface 20 Illumination optical system 30 Imaging optical system 40 Biochip holding mechanism

Claims (2)

In a biochip reader that includes an illumination optical system that irradiates the surface of the biochip with excitation light and reads a fluorescent image based on the excitation light,
The biochip reader is characterized in that the illumination optical system irradiates the excitation light perpendicularly to the surface of the biochip. In a biochip reader that includes an illumination optical system that irradiates the surface of the biochip with excitation light and reads a fluorescent image based on the excitation light,
The irradiation optical system is configured to adjust the divergence angle of excitation light to a narrow angle and irradiate at an incident angle near to the biochip surface perpendicularly, and is configured so that the focal depth of the excitation optical system is 50 μm or more. A biochip reader characterized by that.

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