JPH03115959A - Microplate reader - Google Patents

Abstract

PURPOSE:To measure transmitted fluorescence in the state of high S/N by inserting an optical system for changing the incident direction of the exciting light into the optical path of the exciting light and changing the incident direction of the exciting light to a direction diagonal with a microplate so that the exciting light is not made incident to a detector and the transmitted fluorescence is made incident thereto at the time of making fluorescent measurement. CONSTITUTION:The optical system 5 for changing the incident direction of the exciting light 6 makes the exciting light 6 incident perpendicularly to the microplate by deviating the optical system 5 from the optical path of the exciting light 6 at the time of making transmission and absorption measurement. On the other hand, the optical system 5 to change the incident direction of the exciting light 6 is inserted into the optical path of the exciting light 6 at the time of making fluorescent measurement. The incident direction of the exciting light 6 is made into the direction diagonal with the microplate and the exciting light 6 is no longer made incident to the detector 10 by inserting this optical system 5. The fluorescence 24 is released in the direction of the detector 10 as well as against this and, therefore, the fluorescence 24 is made incident to the detector 10 and is received therein. The transmitted fluorescence is measured in the state of the high S/N in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a microplate reader that optically measures a sample in a well of a microplate by making excitation light enter the microplate in a vertical direction. be. Microplate readers are used in fields such as medicine and biochemistry.

(Prior Art) In a microplate reader, excitation light is generally made incident on a microplate to perform transmission and absorption measurements. Therefore, an excitation optical system is provided on one side of the microplate to allow excitation light to enter the microplate in a vertical direction, and a detector that receives transmitted light is provided on the opposite side of the microplate.

Recently, there has been a demand for not only transmission and absorption measurements using microplates but also fluorescence measurements. For example, this is the case when FIA (fluorescence labeled immunoreaction) 8'I11 determination is performed using a microplate.

A microplate reader shown in FIG. 2 is used to enable not only transmission absorption measurement but also reflection fluorescence measurement with the microplate 1 reader. 2 is a well of a microplate, in which a sample is placed. An ellipsoidal mirror 4 is provided above the microplate, and excitation light 6 passes through a hole in the ellipsoidal mirror 4 and enters the well 2 in a vertical direction.

A detector 10 for receiving transmitted light 8 is provided below the microplate. In order to receive fluorescence, a detector 16 is provided which receives reflected light 12 from the ellipsoidal mirror 4 through a fluorescence selection filter 14.

In addition, in order to be able to perform not only transmission absorption measurement but also transmission fluorescence measurement with a microplate reader, a third
As shown in Figure (A), an apparatus is also being considered in which the fluorescence contained in the transmitted light 8 of the well 2 is selected by a fluorescence selection filter 14 and detected by a detector 10.

(Problem to be solved by the invention) When a method of receiving reflected fluorescence as shown in Fig. 2 is adopted,
In addition to the conventional transmission side detector 10.

It is necessary to provide a detector 16 for detecting reflected fluorescence, which makes the device expensive.

As shown in FIG. 3, the excitation light E is
In the method of separating x and fluorescence FQ and extracting only fluorescence, even though the wavelengths of excitation light Ex and fluorescence FQ are different as shown in (B), fluorescence selection that completely blocks intense excitation light is necessary. It is difficult to make a filter, and a lot of stray light enters the detector 10, resulting in measurements with a poor S/N ratio.

Therefore, an object of the present invention is to provide a microplate reader that can perform both transmission absorption measurement and fluorescence measurement, without increasing the cost too much, and that can perform measurements with a high S/N ratio. It is something to do.

(Means for Solving the Problems) The present invention includes an excitation optical system that allows excitation light to enter the microplate 1- in a vertical direction, and a detector that is provided on the opposite side of the microplate to the excitation optical system. An optical system for changing the direction of incidence of excitation light to an oblique direction with respect to the microplate was removably provided between the excitation optical system and the microplate.

(Function) The optical system that changes the direction of incidence of the excitation light is removably installed, so when performing transmission absorption measurements, the optical system is removed from the optical path of the excitation light and the excitation light is incident vertically onto the microplate. let

On the other hand, when performing fluorescence measurement, an optical system that changes the direction of the incident power of the excitation light is inserted into the optical path of the excitation light. By inserting this optical system, the direction of incidence of the excitation light becomes oblique to the microplate, and the excitation light no longer enters the detector. On the other hand, since the fluorescence is also emitted toward the detector, the fluorescence enters the detector and is received.

(Example) FIG. 1 shows an example. The same symbols are used for the same parts as in FIGS. 2 and 3.

2 is a well of a microplate, in which a sample is placed. An excitation optical system 5 including a light source and a spectrometer is provided above the microplate, and excitation light 6 enters the microplate in a direction perpendicular to the microplate.

Above the microplate, a mirror unit 20 is removably inserted into the optical path of the excitation light 6 as an optical system for changing the direction of incidence. The mirror unit 20 includes two mirrors 20a and 20b, and the excitation light 6 is directed to the mirror 20a.
, is reflected, enters the mirror 20b, and is reflected by the mirror 20b.
Mirrors 20a and 20b are arranged so that the light reflected by 0b enters the well 2 from an oblique direction with respect to the microplate.
The position and angle of are set. Mirror unit 20
can be manually attached to or removed from the excitation light path by an operator, or may be driven by a motor or the like so that it can be moved between the excitation light path and a position removed from the excitation light path.

A detector 10 is provided on the side opposite to the excitation optical system 5 with respect to the microplate. The position of the detector 10 is set at such a position that it receives the transmitted light of the well 2 when the mirror unit 20 is removed from the excitation light optical path. Further, a mask 22 is provided on the incident side of the detector 10 to prevent the transmitted light 8 of the excitation light incident in an oblique direction from entering the detector 10 when the mirror unit 20 is placed on the excitation light optical path. Space is limited. 14 is a fluorescence selection filter, and when the mirror unit 2o is inserted into the excitation light optical path, the fluorescence selection filter 14 is also inserted into the incident side of the detector 10, and when the mirror unit 20 is removed from the excitation light optical path, the fluorescence selection filter 14 is unit] 4 is also removed from the incident side of the detector 10.

Next, the operation of this embodiment will be explained.

When performing fluorescence measurement, the mirror unit 20 is inserted into the incident optical path of the excitation light 6, and the fluorescence selection filter 14 is inserted on the incident side of the detector 100. Excitation light 6 is transmitted through two mirrors 20
The optical path is changed at points a and 20b so that the light enters the microplate in an oblique direction, and the light enters the well 2. The excitation light 6 excites the fluorescent substance in the well 2 to generate fluorescence. The excitation light 8 that has passed through the well 2 does not enter the detector 1o. Of the fluorescence generated from the fluorescent substance in well 2,
The solid angle portion 24 of the detector 10 passes through the fluorescence selection filter 14 and enters the detector 10 for detection.

Since stray light of the excitation light 8 that enters the detector 10 is reduced during fluorescence measurement, the fluorescence selection filter 14 can be used even if it does not have strict wavelength selection characteristics.

When performing transmission absorption measurement, the mirror unit 7 20 is removed from the excitation light optical path, and the fluorescence selection filter 14 is also removed from the incident side of the detector 10. As a result, the excitation light 6 enters the well 2 from a direction perpendicular to the microplate, and the excitation light that has passed through the well 2 enters the detector 10 and is detected.

The optical system for changing the direction of incidence of the excitation light is not limited to the one using two mirrors as shown in the embodiment.

(Effects of the Invention) In the present invention, in a microplate reader capable of performing transmission absorption measurement, when performing fluorescence measurement, an optical system that changes the direction of incidence of the excitation light is inserted in the excitation light optical path to change the direction of incidence of the excitation light. Since the excitation light is not incident on the detector and the transmitted fluorescence is made to be incident on the detector, the transmitted absorption measurement and the transmitted fluorescence measurement can be performed using the same detector. It is possible to measure with a high ratio. Compared to the conventional method of measuring reflected fluorescence, only one detector is required, resulting in lower costs.

In addition, the S/N is higher than conventional equipment that uses only a filter to separate excitation light and fluorescence from light transmitted through a microplate.
ratio becomes higher.

[Brief explanation of drawings]

Figure 1 is a schematic configuration diagram showing one embodiment, Figure 2 is a schematic configuration diagram showing an example of a conventional microplate reader, and Figure 3 is a schematic configuration diagram showing an example of a conventional microplate reader.
Figure (A) is a schematic configuration diagram showing another conventional microplate reader, and Figure (B) is a waveform diagram showing excitation light and fluorescence. 2...Wells of microplate, 5...
... Excitation optical system, 6 ... Excitation light, 8 ... Transmitted light, 10 ... Detector, 14 ... Fluorescence selection filter, 20 ... Mirror Unit, 20a
, 20 b --Mirror, 22...Mask, 2
4...Transmitted fluorescence.

Claims (1)

[Claims] (1) comprising an excitation optical system that makes excitation light enter the microplate in a vertical direction; and a detector provided on the opposite side of the microplate to the excitation optical system, and
The microplate reader is provided with an optical system that is removably installed between the excitation optical system and the microplate to change the direction of incidence of excitation light to a diagonal direction with respect to the microplate.