JPH09229862A - Fluorescence photometer - Google Patents

Abstract

(57) [Abstract] [Problem] To accurately measure a plurality of wells in a short time. SOLUTION: A plurality of wells (O1,
O2 ... O6 ...) is a fluorescence measurement device that measures the fluorescence emitted from inside the imaging optical system (G2-G5) that collects the fluorescence from multiple wells to form a reduced image. And a photoelectric detector (5) for photoelectrically detecting an image formed by the imaging optical system. And
The imaging optical system comprises, in order from the well side, a telecentric first objective lens system (G2-G4) on the well side, and a second objective lens that forms an image by condensing the light flux from this first objective lens system. It is composed of the system (G5).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence photometric device for photometric analysis of fluorescence emitted from a sample placed in a plurality of wells, and more particularly to a fluorescence photometric device suitable for HLA typing.

[0002]

2. Description of the Related Art In a fluorescence photometric device, particularly an HLA typing device for analyzing human white blood cells, a reagent is put into a sample placed in a well plate provided with a plurality of wells, and at that time, from each sample. The emitted fluorescence is measured. In this apparatus, the difference in HLA type is determined based on the intensity of fluorescence from the sample and the spectral characteristics. Such a conventional fluorescence photometric device is composed of a bundle fiber and a photoelectric detector for measuring the fluorescence from the well via the bundle fiber, and one of the plurality of wells on the well plate, After the end face of the bundle fiber is positioned to measure the fluorescence from the well, the bundle fiber and the well plate are relatively moved to position the end face of the bundle fiber in another well.

[0003]

In the conventional fluorescence photometric device as described above, it is necessary to position the end face of the bundle fiber in the well and perform photometry of the well for the number of wells provided in the well plate. However, there is a problem that it takes a very long time to measure one well plate.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a fluorescence photometric device capable of accurately measuring a plurality of wells in a short time.

[0005]

In order to achieve the above-mentioned object, a fluorescence photometric apparatus according to one aspect of the present invention is a fluorescence measuring apparatus for measuring fluorescence generated in a plurality of wells into which an object to be measured is injected. The measuring apparatus includes an imaging optical system that collects fluorescence from a plurality of wells to form a reduced image, and a photoelectric detector that photoelectrically detects an image by the imaging optical system. The imaging optical system includes, in order from the well side, a telecentric first objective lens system on the well side, and a second objective lens system that collects the light flux from the first objective lens system to form the image. Composed of.

The fluorescence photometric device according to a preferred embodiment of the present invention is further configured to further include a flat glass disposed between the first objective lens system and the well, and the first objective lens system is a positive lens. When the effective diameter of this lens group is D, the distance from the object plane of the imaging optical system to the image plane is L, and the distance from the object plane to the lens group is W, , 2D <f <4D (1) 0.4L <f <0.8L (2) 0.05L <W <0.2L (3).

[0007] In a preferred embodiment of the present invention,
An image intensifier for photoelectrically amplifying the intensity of the image is arranged on the image plane formed by the second objective lens system, and the photoelectric detector detects the fluorescence through the image intensifier. Is. Further, it is preferable that the second objective lens system is image-side telecentric.

In a preferred aspect of the present invention, the light beam between the first objective lens system and the second objective lens system is afocal.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is an apparatus schematically showing the configuration of a fluorescence photometric device according to an embodiment of the present invention. In FIG. 1, a plurality of wells O1, O2, ... 06, ...
The well plate 1 provided with ... Is provided with a plurality of recesses in a translucent member such as glass. The well plate 1 is supported by a support member 2 having an opening. Below the opening, an image forming optical system including a plurality of lens groups G2 to G5 and having a reduction magnification is arranged, and these lens groups are held by the metal object 3. Further, in the optical path between the image forming optical systems G2 to G5 and the well plate 1, a drip-proof flat glass G1 made of a plane-parallel plate made of quartz glass is provided.

Here, the plurality of wells O1, O2, ...
Samples are loaded in each of the wells 06, ..., When a reagent is added to the sample in this well from a dispenser (not shown), each sample emits fluorescence corresponding to the characteristics of each sample. Is emitted. Here, the object planes of the imaging optical systems G2 to G5 are set so as to be located near the bottom surfaces of the wells of the well plate 1,
The fluorescence from this sample is imaged as a fluorescence image I on the image planes of the imaging optical systems G2 to G5. An image intensifier 4 is arranged on this image plane,
The image intensifier 4 has the fluorescence image I described above.
Is amplified photoelectrically. The light emitted from the image intensifier 4 is captured by the image sensor 5. The image pickup information from the image pickup device 5 is transmitted to the processing device 6 for obtaining the characteristic of the sample from the image pickup information.

The image forming optical systems G2 to G5 are composed of a positive first lens group G2, a negative second lens group G3, and a positive third lens group G4 in this order from the well plate 1 side. As the first objective lens system G2 to G4 having a positive refractive power
And a second objective lens system G5 including a positive fourth lens group G5. Here, the first objective lens system G2
In the optical path between G4 and the second objective lens system G5, a light beam from one point on the object plane is a parallel light beam (afocal). In addition, the first objective lens systems G2 to G4
A diaphragm S is arranged at the focal position on the rear side (second objective lens system side). Therefore, the first objective lens systems G2 to G
5 is a telecentric optical system on the well side. Further, the diaphragm S is arranged so as to be substantially located at the focal position in front of the second objective lens system G5 (on the side of the first objective lens systems G2 to G5). Therefore, the second objective lens system G5 is image-side telecentric.

As described above, since the fluorescence from the plurality of wells is guided to the photoelectric detector by the image forming optical systems G2 to G5 having the reduction magnification, the fluorescence emitted from the sample accumulated at the bottom of each well is collected at once. It can be led to a photoelectric detector and a reduction in measurement time can be achieved. further,
Since the first objective lens systems G2 to G5 are telecentric to the well side, the fluorescence can be measured from the same direction regardless of the position of the well on the well plate 1, so that there is no variation in measurement accuracy. There are advantages.

Further, in the above-described embodiment, since the flat glass G1 for drip-proof is arranged just below the well side, liquids such as samples and reagents are not mixed in the optical system part. Is playing. In the embodiment as described above, the first objective lens system G2
The positive first lens group G2 closest to the well in G4 has a focal length f of the first lens group G2, an effective diameter D of the first lens group G2, and an object-to-image plane of the imaging optical system. Is 2D <f <4D (1) 0.4L <f <0.8L (2) 0, where L is the distance from the object surface to W and the distance from the object surface to the first lens group is W. It is preferable to satisfy the condition of 0.05 L <W <0.2 L (3).

The above-mentioned conditional expressions (1) to (3) are conditions for suppressing an increase in cost while realizing compactness of the image forming optical system and for realizing good aberration correction. By configuring the imaging optical system so as to satisfy these conditional expressions (1) to (3), it is possible to make the lens group arranged subsequent to the first lens group G2 compact, and the first lens group G2 can be made compact. Good aberration correction can be achieved without complicating the configuration of the group G2 itself, and further, it becomes possible to measure a large well plate without increasing the size of the first lens group.

In the conditional expression (1), f <2D
In this case, since the aberration generated in the first lens group G2 becomes extremely large, the lens configuration of the first lens group G2 cannot be simplified, and a lens element having a large effective diameter is used as the first lens group G2. Since it is necessary, cost reduction cannot be achieved, which is not preferable. On the other hand, when f> 4D,
Since the first lens group G2 cannot sufficiently bend the light flux from the peripheral portion (well in the peripheral portion of the well plate 1) on the object plane, it is not preferable because the second lens group G3 and thereafter are upsized.

In conditional expression (2), f <0.4
In the case of L, the aberration generated in the first lens group G2 becomes extremely large, so that the lens configuration of the first lens group G2 cannot be simplified and the cost cannot be reduced, which is not preferable. On the other hand, when f> 0.8L, the focal length of the first lens group G2 becomes longer than the entire length of the imaging optical systems G2 to G5, and the light flux from the peripheral portion on the object plane is sufficiently bent. This is not preferable because it is impossible to increase the size of the second lens group G3 and thereafter.

Next, in conditional expression (3), W <0.0
When it becomes 5L, the first lens group and the well plate 1
Is too short, which makes it difficult to dispose the flat glass G1 for protecting the imaging optical system, which is not preferable. On the other hand, when W> 0.2L, the light beam emitted from the sample in the well is excessively diverged when entering the first lens group G2, so that the first lens group G2 is extremely diverged. Since it is necessary to make the effective diameter large, it is not possible to reduce the cost, and further, it is necessary to take a large space for guiding the first lens group G2 to the second lens group G3 and thereafter, so that it is possible to achieve compactness. It is not possible and not preferable.

In the present embodiment, the image intensifier 4 for photoelectrically amplifying the image intensity is arranged on the image plane of the image forming optical system. It has an effect that even if the fluorescence of is weak, it can be detected. Also, the second
Since the objective lens system G5 is telecentric on the image side, the luminous flux guided to the image intensifier 4 is in the same state at any position on the image intensifier 4, that is, there is no difference depending on the position of the visual field. Therefore, the light amount can be captured extremely uniformly regardless of the directionality of the image intensifier 4. Therefore, it is possible to detect the well at any position in the well plate 1 with high accuracy.

In the present embodiment, the first objective lens system G2 to G4 composed of the lens groups G2 to G4.
And a second objective lens system G5 composed of the lens group G5 are in a parallel system (afocal), it is possible to easily adjust the distance between the first and second objective lens systems. , Which is advantageous in manufacturing. With this configuration, for example, the telecentricity can be easily adjusted.

[0020]

EXAMPLES Next, numerical examples will be described. In the following numerical examples, the well plate 1 has a size of 80 × 120 mm.
The size of the flat glass G1 for drip-proof is 150mm
Has an effective diameter of. The first lens group G2 arranged subsequent to the flat glass G1 has an effective diameter of 150 mm and an outer diameter of 160 mm.

Here, as shown in FIG. 1, the first lens group G2 is composed of a plano-convex lens having a convex surface facing the well side, and the second lens group G3 is composed of a cemented lens composed of a biconcave lens and a biconvex lens. The fourth lens group G4 includes a cemented lens including a plano-concave lens and a plano-convex lens, and a biconvex lens arranged on the image side of the cemented lens. The fourth lens group G5 includes, in order from the well side, a cemented lens including a biconvex lens and a negative meniscus lens having a concave surface facing the well side, a plano-convex lens, a positive meniscus lens having a convex surface facing the well side, and the well side. It is composed of a cemented lens including a negative meniscus lens having a convex surface, a cemented lens including a biconcave lens and a biconvex lens, a biconvex lens, and a negative meniscus lens having a convex surface facing the well side.

Table 1 below shows lens data of numerical examples. In each table, L is the total length of the imaging optical system (distance between object images), ri is the radius of curvature of the i-th surface, and d0 is the first from the object plane.
The distance to the surface (working distance), di is the surface distance between the i-th surface and the (i + 1) th surface, and ni is the F-line (λ = 486.1 nm) of the medium between the i-th surface and the (i + 1) th surface. Refractive index (blank indicates medium is air). In addition, in each table, the values corresponding to the conditions of the numerical examples are also shown, where f is the focal length of the first lens group G2 (the positive lens group in the first objective lens system), and W is the object. Face and first
The distance from the lens group G2 (the positive lens group in the first objective lens system) is shown.

[0023]

L = 750 mm d0 = 70.0 r1 = 220.0 d1 = 20.0 n1 = 1.5224 r2 = ∞ d2 = 300.0 r3 = −82.77 d3 = 6.0 n3 = 1. 5224 r4 = 220.0 d4 = 9.0 n4 = 1.7587 r5 = 692.5 d5 = 125.0 r6 = -230.8 d6 = 6.0 n6 = 1.7645 r7 = ∞ d7 = 13.0 n7 = 1.5020 r8 = −130.0 d8 = 1.0 r9 = 666.1 d9 = 10.0 n9 = 1.6100 r10 = −309.68 d10 = 60.0 r11 = 156.1 d11 = 14 .0 n11 = 1.52020 r12 = −76.16 d12 = 4.5 n12 = 1.7556 r13 = −226.3 d13 = 0.5 r14 = 59.99 d14 = 10.7 n14 = 1.6 75 r15 = ∞ d15 = 0.5 r16 = 36.507 d16 = 20.0 n16 = 1.520 r17 = 1901 d17 = 3.4 n17 = 1.75656 r18 = 20.73 d18 = 14.0 r19 = − 29.16 d19 = 5.8 n19 = 1.765 45 r20 = 33.5 d20 = 15.3 n20 = 1.52020 r21 = -33.5 d21 = 0.5 r22 = 50.12 d22 = 9.4 n22 = 1.7581 r23 = -68.1 d23 = 0.6 r24 = 31.357 d24 = 19.8 n24 = 1.6304 r25 = 22.77 d25 = 6.5 (Numerical Example) Conditional corresponding value ( 1) f = 2.5D (2) f = 0.56L (3) W = 0.093L In order to show the imaging performance of the above-mentioned numerical example, FIG. 2 shows various aberration diagrams of the numerical example. In each aberration diagram, H indicates the incident height, Y indicates the image height, and A indicates the incident angle. In the astigmatism diagram, the meridional image plane is shown by a broken line and the sagittal image plane is shown by a solid line. In the lateral aberration diagram, the aberration due to the skew ray is also shown by a broken line.

As shown in FIG. 2, in the image forming optical system according to the numerical example, each aberration is well corrected, and a reduced image of fluorescence generated from the sample in the well is formed under good image forming performance. Can be caught.

[0025]

According to the present invention as described above, since the luminous flux from the large well plate is taken into the well side by the telecentric imaging optical system, the well may be located at any position in the well plate. It is possible to accurately perform photometry in a short time. This makes it possible to efficiently analyze a large amount of samples in a short time.

If the image forming optical system is a telecentric optical system on the image side and the image intensifier is arranged on the image plane, the image intensifier will be uniform regardless of the directional characteristics of the image intensifier. Since the image can be photoelectrically amplified, the photometric uniformity of the entire well plate can be further enhanced.

[Brief description of drawings]

FIG. 1 is a diagram showing an overall configuration of a fluorescence photometric device according to an embodiment of the present invention.

FIG. 2 is a diagram of various types of aberration in the imaging optical system of the numerical example.

[Explanation of symbols]

 1 Well plate G1 Flat glass G2, G3, G4 First objective lens system G5 Second objective lens system 4 Image intensifier 5 Image sensor

Claims (5)

[Claims] 1. A fluorescence measuring apparatus for measuring fluorescence emitted from a plurality of wells into which a measurement target is injected, and an imaging optical system for condensing fluorescence from the plurality of wells to form a reduced image. And a photoelectric detector that photoelectrically detects an image formed by the image forming optical system, wherein the image forming optical system has a first objective lens system telecentric to the well side in order from the well side. (1) a second light-condensing light beam from the objective lens system to form the image
A fluorescence photometric device comprising an objective lens system. 2. A flat glass arranged between the first objective lens system and the well, wherein the first objective lens system includes a lens group having a positive focal length f, 2D <f <4D 0.4L <f, where D is the effective diameter of the group, L is the distance from the object plane of the imaging system to the image plane, and W is the distance from the object plane to the lens group. The fluorescence photometric device according to claim 1, wherein the condition <0.8L 0.05L <W <0.2L is satisfied. 3. An image forming plane formed by the second objective lens system,
An image intensifier for photoelectrically amplifying the intensity of the image is arranged, and the photoelectric detector detects the fluorescence through the image intensifier. Fluorescence photometer. 4. The fluorescence photometric apparatus according to claim 1, wherein the second objective lens system is image-side telecentric. 5. The fluorescence photometric device according to claim 1, wherein the light beam between the first objective lens system and the second objective lens system is afocal.