JP2000241350A - Method for evaluating fluorescence observation apparatus and transmission filter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize the subject method capable of stably and accurately evaluating excitation or detection efficiency. SOLUTION: An exciting light optical system evaluating filter 21 and a power meter 22 are arranged at a position where a sample must be placed. The exciting light optical system evaluating filter 21 has a transmission spectrum equal to the absorption spectrum of a fluorescent substance contained in the sample and may be inserted at any position on the light path of an exciting light optical system. The power meter 22 is arranged at the position where the sample 1 must be placed. The exciting light emitted from an exciting light source 11 to pass through the exciting light optical system and the exciting light optical system evaluating filter 21 is detected by the power meter 22 to evaluate the exciting light source 11 or the exciting light optical system on the basis of the detection result.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating a fluorescence observation apparatus for irradiating a sample with excitation light and observing fluorescence generated from a fluorescent substance contained in the sample, and an evaluation method for the fluorescence observation apparatus. It relates to a transmission filter suitable for use.

[0002]

2. Description of the Related Art A fluorescence observation apparatus typified by a fluorescence microscope irradiates a sample with excitation light output from an excitation light source via an excitation light optical system, and emits fluorescence generated from a fluorescent substance in the sample with the irradiation. It is observed by a camera or the like via a fluorescent optical system. In such a fluorescence observation apparatus, it is important to evaluate each of the excitation light source, the excitation light optical system, and the fluorescence optical system and to select suitable components in order to observe the sample with high accuracy.

Conventionally, the intensity of fluorescence observed under the same excitation conditions has been detected, and a fluorescence observation apparatus has been evaluated based on the detection results. In addition, an important evaluation item is a ratio of light absorbed by the fluorescent substance to light output from the excitation light source and reaching the sample (hereinafter simply referred to as “excitation efficiency”).
And). This is the efficiency with which the fluorescence generated from the fluorescent substance contained in the sample is detected by a camera or the like via the fluorescent optical system (hereinafter simply referred to as “detection efficiency”).

In evaluating the pumping efficiency, generally, the pumping light intensity is measured by a power meter, the transmission spectrum of a limited removable part in the pumping light optical system is measured, or the pumping light source is measured. They refer to the catalog values of the emission spectrum and the optical characteristics of other components. The excitation light source and the optical component are selected based on the result of the measurement or the evaluation.

On the other hand, when the detection efficiency is evaluated, a fluorescent bead or a fluorescent glass piece simulating a fluorescent substance is used as a standard fluorescent sample, and this standard fluorescent sample is placed at a sample mounting position of a fluorescence observation apparatus. The intensity of the fluorescence generated from the standard fluorescent sample is detected by a high-sensitivity photodetector via the fluorescent optical system, and the fluorescent optical system is evaluated based on the detection result.

[0006]

However, the above-mentioned conventional evaluation method has the following problems.

In the evaluation of the excitation efficiency, it is difficult to measure the emission spectrum of the excitation light source. Therefore, the excitation light in the excitation light optical system is not based on the actually measured values but on the basis of the descriptions in catalogs or chronological tables. The filter was being selected. In addition, since it is difficult to evaluate the change in the emission spectrum due to the deterioration of the excitation light source, it is necessary to determine the replacement time of the excitation light source only based on the usage time of the excitation light source without grasping the actual deterioration state of the excitation light source. I was

Further, only limited components in the excitation light optical system are removed for evaluation, and the whole excitation light optical system has not been comprehensively evaluated. Furthermore, the intensity of the excitation light applied to the sample from the excitation light optical system is measured with a power meter placed at the sample mounting position, and the measured value is normalized as the intensity of the excitation light. The evaluation of the excitation efficiency in consideration of the efficiency of exciting the fluorescent substance has not been made.

On the other hand, when the detection efficiency is evaluated, fluorescent beads or fluorescent glass pieces simulating a fluorescent substance are used as a standard fluorescent sample, but any fluorescent substance can avoid the occurrence of fading. Therefore, when comparing various states with each other using the same standard fluorescent sample, or when re-evaluating using the same standard fluorescent sample after a long time has elapsed since the first evaluation, The evaluation results lacked stability.

Further, the fluorescence spectrum of a fluorescent substance shifts to the long wavelength side or the short wavelength side depending on the protein molecules bound thereto, the conditions of the solution, and the like. Can not do. Therefore, when accurate evaluation of the fluorescent optical system is required, an actual valuable sample has to be used as a sample for evaluation.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and has been made in consideration of the above-mentioned problems, and has provided a fluorescent observation apparatus evaluation method capable of stably and accurately evaluating excitation efficiency and detection efficiency.
It is an object to provide a transmission filter suitable for use in this evaluation.

[0012]

According to a method for evaluating a fluorescence observation apparatus according to the present invention, a sample is irradiated with excitation light output from an excitation light source via an excitation light optical system, and is generated from a fluorescent substance contained in the sample. A method for evaluating a fluorescence observation apparatus for observing fluorescence, comprising inserting a transmission filter having a transmission spectrum equal to the absorption spectrum of a fluorescent substance on the optical path of an excitation light optical system, outputting an excitation light optical system from an excitation light source, and An excitation light having passed through a transmission filter is detected by a photodetector, and an excitation light source or an excitation light optical system is evaluated based on the detection result.

According to the method for evaluating a fluorescence observation apparatus, the excitation light output from the excitation light source reaches the photodetector via the excitation light optical system and the transmission filter, and is detected by the photodetector. Since the transmission filter inserted on the optical path of the excitation light optical system has a transmission spectrum equal to the absorption spectrum of the fluorescent substance contained in the sample, the excitation light detected by the photodetector is effective for the fluorescent substance. It is a component that can be excited. Therefore, the excitation light source or the excitation light optical system can be evaluated based on the detection result by the photodetector.

Another method for evaluating a fluorescence observation device according to the present invention is a method for evaluating a fluorescence observation device for observing fluorescence generated from a fluorescent substance contained in a sample irradiated with excitation light at a predetermined position via a fluorescence optical system. A white light source that outputs a light that is white in a predetermined wavelength band including a peak wavelength of the fluorescence spectrum by inserting a transmission filter having a transmission spectrum equal to the fluorescence spectrum of the fluorescent substance on the optical path of the fluorescence optical system. The output light that has passed through the transmission filter and the fluorescent optical system is detected by a photodetector, and the fluorescent optical system is evaluated based on the detection result.

According to this fluorescence observation apparatus evaluation method, the light output from the white light source is detected by the photodetector via the transmission filter and the fluorescence optical system. The transmission filter inserted on the optical path of the fluorescence optical system has a transmission spectrum equal to the fluorescence spectrum of the fluorescent substance contained in the sample, and the light output from the white light source has a fluorescence spectrum of the fluorescent substance. Since the light is white in a predetermined wavelength band including the peak wavelength, the light output from the white light source and transmitted through the transmission filter simulates the fluorescence generated from the fluorescent substance contained in the sample. Therefore, the fluorescent optical system can be evaluated based on the detection result by the photodetector.

According to still another aspect of the present invention, there is provided a method for evaluating a fluorescence observation apparatus for observing fluorescence generated from a fluorescent substance contained in a sample irradiated with excitation light at a predetermined position via a fluorescence optical system. A method comprising disposing an isotropic scattering object at a position where a sample is to be placed, and transmitting white light in a predetermined wavelength band including a peak wavelength of a fluorescent spectrum of the fluorescent substance, having a transmission spectrum equal to the fluorescent spectrum. Irradiates an isotropic scattering object through a filter, detects light scattered by the isotropic scattering object and passes through a fluorescent optical system with a photodetector, and evaluates the fluorescent optical system based on the detection result. And

According to this method for evaluating a fluorescence observation apparatus, the light output from the white light source passes through the transmission filter and irradiates the isotropic scattering object placed at the position where the sample is to be placed. Scattered by scattering objects. Light scattered by the isotropic scattering object is detected by a photodetector via a fluorescent optical system. The transmission filter has a transmission spectrum equal to the fluorescence spectrum of the fluorescent substance contained in the sample, and the light output from the white light source emits white light in a predetermined wavelength band including the peak wavelength of the fluorescent spectrum of the fluorescent substance. Therefore, the light output from the white light source and transmitted through the transmission filter simulates the fluorescence generated from the fluorescent substance contained in the sample. The isotropic scattering object is arranged at a position where the sample is to be placed, and scatters the incident light isotropically. Therefore, it is possible to simulate a situation in which fluorescence isotropically generated from the fluorescent substance contained in the sample at the time of evaluation, and thus it is possible to accurately evaluate the fluorescence optical system based on the detection result by the photodetector.

The transmission filter according to the present invention is characterized in that it has a transmission spectrum equal to the absorption spectrum of the fluorescent substance, and the above-mentioned method for evaluating a fluorescence observation apparatus for evaluating an excitation light source or an excitation light optical system of the fluorescence observation apparatus. It is preferably used. Further, another transmission filter according to the present invention is characterized in that it has a transmission spectrum equal to the fluorescence spectrum of the fluorescent substance, and is suitably used in the above fluorescence observation apparatus evaluation method for evaluating the fluorescence optical system of the fluorescence observation apparatus. Can be

The fact that the transmission spectrum of the transmission filter used for evaluating the excitation light optical system and the absorption spectrum of the fluorescent substance are equal to each other means that the respective peak wavelengths coincide with an accuracy of ± 5 nm, and the respective peak values are reduced to 1. In the wavelength range where the relative transmittance of the transmission filter is 1 to 0.3 when normalized, the relative error between the two is ± 20% or less, and the relative transmittance of the transmission filter is 0.3 or less. Means that the absolute error between the two is ± 0.1 or less. The absorption spectrum of the fluorescent substance is obtained by measuring the absorbance A (λ) of the fluorescent substance at a wavelength λ under the same environment (solution composition and the like) under a microscope by another measuring instrument, and

[0020]

(Equation 1)

The absorption rate T of the fluorescent substance represented by the following formula:
(Λ).

Further, the fact that the transmission spectrum of the transmission filter used for the evaluation of the fluorescence optical system and the fluorescence spectrum of the fluorescent substance are equal to each other means that each peak wavelength coincides with an accuracy of ± 5 nm and each peak value is 1 In a wavelength range where the relative transmittance of the transmission filter is 1 to 0.3 when standardized to the following, the relative error between the two is ± 20% or less, and the relative transmittance of the transmission filter is 0.3 or less. It means that the absolute error between the two is within ± 0.1 within the range. Note that the fluorescence spectrum of the fluorescent substance is measured by a fluorometer.

[0023]

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

First, a description will be given of a fluorescence observation apparatus to which the fluorescence observation apparatus evaluation method and the transmission filter according to the present embodiment are suitably applied. FIG. 1 is a schematic configuration diagram of the fluorescence observation device. In this figure, a lens, an excitation filter, a shutter, and the like inserted on the optical path of each optical system are omitted. In this fluorescence observation apparatus, the excitation light output from the excitation light source 11 is reflected by the dichroic mirror 12, condensed by the objective lens 13, and irradiates the sample 1 on the slide glass 14. The fluorescent light generated from the fluorescent substance contained in the sample 1 upon irradiation of the sample 1 with the excitation light passes through the slide glass 14 and the objective lens 13, passes through the dichroic mirror 12, is reflected by the mirror 15, and is reflected by the camera 16. Is imaged.

In this figure, the excitation light optical system includes an excitation light source 11
This is an optical system of excitation light from the light source to the sample 1 via the dichroic mirror 12, the objective lens 13, and the slide glass 14. The fluorescent optical system is a fluorescent optical system from the sample 1 to the camera 16 via the slide glass 14, the objective lens 13, the dichroic mirror 12, and the mirror 15.

FIG. 2 is an explanatory diagram of the method for evaluating a fluorescence observation apparatus according to this embodiment. Hereinafter, the evaluation method of the excitation light source 11 or the excitation light optical system and the evaluation method of the fluorescence optical system will be described separately.

The evaluation of the excitation light source 11 or the excitation light optical system is performed as follows. In place of the sample 1, an excitation light optical system evaluation filter 21 and a power meter 22 are arranged. The excitation light optical system evaluation filter 21 may be inserted at any position on the optical path of the excitation light optical system, but if it is an interference filter, the position of the parallel optical system is desirable. The power meter 22 is arranged at a position where the sample 1 is to be placed. It is assumed that the wavelength characteristics of the power meter 22 can be ignored within a target wavelength range. The excitation light output from the excitation light source 11 and having passed through the excitation light optical system and the excitation light optical system
And the excitation light source 11 based on the detection result.
Or evaluate the excitation light optics.

The excitation light optical system evaluation filter 21 is a transmission filter having a transmission spectrum equal to the absorption spectrum of the fluorescent substance contained in the sample 1. That is, FIG.
When a fluorescent substance (rhodamine in this figure) having an absorption spectrum as shown in FIG. 4 is contained in the sample 1, an excitation light optical system evaluation filter 21 having a transmission spectrum as shown in FIG. . The fluorescent substance includes Hoechst 333 in addition to rhodamine.
42, bodipi, DAPI, dansyl, fluoro3, acridine orange, fluorescein, Dil, Cy3,
Cy5, Cy7, etc. are conceivable. (Examples of various fluorescent dyes are "Fluorescence microscopy Vol. II", FWDRost,
Cambridge University Press 1995, Chapter 7, Fluoro
chromes, pp.198-392). The fact that the absorption spectrum of the fluorescent substance and the transmission spectrum of the excitation light optical system evaluation filter 21 are equal to each other means that each peak wavelength is ± 5 nm.
And the relative error between the two is within ± 20% in a wavelength range where the relative transmittance of the transmission filter is 1 to 0.3 when each peak value is normalized to 1. Mean that the absolute error between the two is ± 0.1 or less in a wavelength range where the relative transmittance of the two is 0.3 or less.

The excitation light optical system evaluation filter 21 for rhodamine is as follows. One surface of a glass plate (CM-500, manufactured by HOYA) having a thickness of 1 mm is coated with an anti-reflection film, and the other surface is a dichroic film (for a wavelength λ = 652 nm, (SiO ₂ λ / 8 +
(Ti0 ₂ λ / 4 + SiO ₂ λ / 8) was coated for 12 periods. Further, as another excitation light optical system evaluation filter 21 for rhodamine, one surface of a 1 mm thick glass plate (Y-46 manufactured by HOYA) is coated with an antireflection film, and the other surface is a dichroic film. (For wavelength λ = 420 nm, (TiO ₂ λ / 8 + Si
(0 ₂ λ / 4 + TiO ₂ λ / 8) for 5 cycles. Then, the two were combined to form the excitation light optical system evaluation filter 21.

The emission spectrum of the excitation light source 11 is shown in FIG.
It is assumed that the transmission spectrum of the excitation light optical system evaluation filter 21 is as shown in FIG. 5B. At this time, power meter 2
The spectrum of the excitation light reaching FIG. 2 (FIG. 5C) includes the emission spectrum of the excitation light source 11 (FIG. 5A) and the transmission spectrum of the excitation light optical system evaluation filter 21 (FIG. 5A).
(B) and the optical characteristics of the excitation light optical system are superimposed on each other. Further, as described above, the transmission spectrum of the excitation light optical system evaluation filter 21 is equal to the absorption spectrum of the fluorescent substance contained in the sample 1.

Therefore, it is possible to evaluate the effective excitation efficiency based on the value measured by the power meter 22, and to evaluate and select the excitation light source 11 and the components (excitation filter and the like) in the excitation light optical system. it can. That is, it is possible to select an optimal combination of the components in the excitation light source 11 and the excitation light optical system so that the value measured by the power meter 22 is maximized. Further, even in the case where components in the excitation light optical system cannot be removed, such as a fluorescence microscope, the excitation efficiency can be quantitatively evaluated for the entire excitation light source 11 and the excitation light optical system.

When the excitation light optical system evaluation filter 21 is removed, the spectrum of the excitation light reaching the power meter 22 is the emission spectrum of the excitation light source 11 (FIG. 5).
(A) and the optical characteristics of the excitation light optical system are superimposed on each other. Since the transmission spectrum of the excitation light optical system evaluation filter 21 is equal to the absorption spectrum of the fluorescent substance, the intensity is measured by the power meter 22 when the excitation light optical system evaluation filter 21 is inserted and when it is removed. When the ratio between the two is obtained, the ratio is
It shows the ratio of the component effective for exciting the fluorescent substance in the excitation light output from 1.

Therefore, based on the above ratio, it is possible to evaluate the ratio of the component effective for exciting the fluorescent substance in the excitation light output from the excitation light source 11, and to evaluate the excitation efficiency and select an excitation filter. be able to. Further, by comparing the above ratio when the pump light source 11 is newly started to be used and the above ratio after use, it is possible to determine the deterioration state and the replacement time of the pump light source 11. When the output of the excitation light source 11 is adjusted to match the excitation light output with the excitation light optical system evaluation filter 21 inserted, the camera 1
By evaluating the amount of light (the amount of noise) detected by 6, the amount of noise of the excitation light optical system with respect to the intensity of the excitation light can be evaluated.

On the other hand, the evaluation of the fluorescent optical system is performed as follows. A fluorescent optical system evaluation filter 31 and a white light source 32 are arranged in place of the sample 1. The fluorescent optical system evaluation filter 31 may be inserted at any position on the optical path of the fluorescent optical system, but in the case of an interference filter, the parallel optical system position is desirable. The white light source 32 is disposed at a position where the sample 1 is to be placed. The light output from the white light source 32 and passed through the fluorescent optical system evaluation filter 31 and the fluorescent optical system is detected by the camera 16, and the fluorescent optical system is evaluated based on the detection result. It should be noted that a photomultiplier tube or a photodiode may be used instead of the camera 16.

The filter 31 for evaluating the fluorescent optical system is the sample 1
Is a transmission filter having a transmission spectrum equal to the fluorescence spectrum of the fluorescent substance contained in the filter. That is, when a fluorescent substance (rhodamine in this figure) having a fluorescence spectrum as shown in FIG. 3 is included in the sample 1, the fluorescent optical system evaluation filter 31 having a transmission spectrum as shown in FIG. Is used. The fact that the fluorescence spectrum of the fluorescent substance and the transmission spectrum of the fluorescence optical system evaluation filter 31 are equal to each other means the same as described above. As a filter 31 for evaluating the fluorescent optical system for rhodamine, a glass plate (H
Antireflection films were coated on both sides of a glass plate (manufactured by OYA, CM-500) and a 0.5 mm thick glass plate (manufactured by HOYA, O-58), and both were used.

The white light source 32 is preferably white in a predetermined wavelength band including the peak wavelength of the fluorescence spectrum of the fluorescent substance contained in the sample 1, and has an adjusting means for keeping the luminance always constant. Used. When the fluorescent substance is rhodamine, its fluorescence spectrum has a wavelength of 550 nm.
Since the intensity is large in the range of 〜650 nm, the white light source 32 has a wavelength band of 500 nm as shown in FIG.
A xenon lamp that has a flat emission spectrum and a white color at a wavelength of about 700 nm is preferably used.

The emission spectrum of the white light source 32 is shown in FIG.
It is assumed that the transmission spectrum is as shown in FIG. 7A, and the transmission spectrum of the fluorescent optical system evaluation filter 31 is as shown in FIG. 7B. At this time, the spectrum of the fluorescence reaching the camera 16 (FIG. 7C)
The emission spectrum (FIG. 7A), the transmission spectrum of the fluorescent optical system evaluation filter 31 (FIG. 7B), and the optical characteristics of the fluorescent optical system are superimposed on each other.

The spectrum of the light output from the white light source 32 and passing through the fluorescent optical system evaluation filter 31 is the emission spectrum of the white light source 32 (FIG. 7A).
And the transmission spectra of the fluorescent optical system evaluation filter 31 are superimposed on each other. As described above, the emission spectrum of the white light source 31 is white in a predetermined wavelength band including the peak wavelength of the fluorescence spectrum of the fluorescent substance contained in the sample 1, and the transmission spectrum of the fluorescence optical system evaluation filter 31 is Equivalent to the fluorescence spectrum of the fluorescent substance contained in 1. Therefore, the light output from the white light source 32 and transmitted through the fluorescent optical system evaluation filter 31 simulates the fluorescence generated from the fluorescent substance contained in the sample 1.

Accordingly, since the problem of fading does not occur, the detection efficiency can be stably evaluated.
Further, even when the characteristics of the fluorescent optical system change, the amount of light output from the white light source 32 and transmitted through the fluorescent optical system evaluation filter 31 and the amount of light output from the white light source 32 and transmitted through the fluorescent optical system evaluation filter 31 are determined. Further, by comparing the amount of light passing through the fluorescent optical system with the light amount, even if a long time has elapsed since the initial evaluation, the fluorescent optical system, that is, the detection efficiency can be stably evaluated.

As shown in FIG. 8, it is preferable to provide an aperture 33 having an arbitrary opening shape between the white light source 32 and the position where the sample is to be placed. in this case,
The light output from the white light source 32 and passing through the aperture of the aperture 33 reaches the camera 16 via the fluorescent optical system evaluation filter 31 and the fluorescent optical system. Therefore, it is possible to evaluate the fluorescence optical system, that is, the detection efficiency, only for the fluorescence generated from the region defined by the opening of the aperture 33. In addition, it is also possible to evaluate a pinhole by directly placing the pinhole on the sample surface.

As shown in FIG. 9, when the excitation light optical system evaluation filter 21 is formed as an interference filter, the angle between the normal direction of the excitation light optical system evaluation filter 21 and the optical axis is determined. As the angle is increased to 0 °, 10 °, 20 °,..., The transmission spectrum shifts to shorter wavelengths. Similarly, when the fluorescence optical system evaluation filter 31 is created as an interference filter as shown in FIG.
As the angle between the normal direction of the fluorescent optical system evaluation filter 31 and the optical axis increases to 0 °, 10 °, 20 °,..., The transmission spectrum shifts to the shorter wavelength side. Therefore, even when the absorption spectrum or the fluorescence spectrum of the fluorescent substance shifts due to the protein molecules binding to the fluorescent substance or the conditions of the solution, etc., the excitation light optical system evaluation filter 21 or the fluorescent optical system By inclining the evaluation filter 31, it is possible to accurately evaluate the excitation light optical system and the fluorescence optical system.

Further, as shown in FIG.
The isotropic scattering object 34 is irradiated with the light output from the filter and transmitted through the fluorescent optical system evaluation filter 31.
It is also preferable to detect light scattered isotropically by the camera 16 via the fluorescent optical system. This isotropic scattering object 34 is placed on the slide glass 14 at a position where the sample 1 is to be placed. In this case, by generating light isotropically from the isotropic scattering object 34, it is possible to simulate a situation in which fluorescent light is generated from the fluorescent substance contained in the sample 1 at the time of evaluation. That is, the detection efficiency can be more accurately evaluated.

[0043]

As described above in detail, according to the present invention, a transmission filter having a transmission spectrum equal to the absorption spectrum of a fluorescent substance contained in a sample is used as a filter for evaluating an excitation light optical system, and the excitation of the fluorescence observation apparatus is performed. By inserting it into the optical optics and detecting the excitation light output from the excitation light source through the excitation light optics and the transmission filter with the photodetector, the excitation efficiency, that is, the excitation light source and the excitation light optics Can be evaluated.

A transmission filter having a transmission spectrum equal to the fluorescence spectrum of the fluorescent substance contained in the sample is inserted into the fluorescence optical system of the fluorescence observation device as a filter for evaluating the fluorescence optical system, and the light output from the white light source is reflected. Detection by the photodetector via the fluorescent optical system and the transmission filter allows stable and accurate evaluation of the detection efficiency, that is, the fluorescent optical system.

Alternatively, an isotropic scattering object is arranged at a position where a sample is to be placed, and the light output from the white light source is irradiated on the isotropic scattering object through the transmission filter, and is scattered by the isotropic scattering object. The detected light is detected by the photodetector through the fluorescent optical system and the transmission filter, whereby the detection efficiency, that is, the fluorescent optical system can be evaluated stably and accurately.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram of a fluorescence observation device.

FIG. 2 is an explanatory diagram of a fluorescence observation device evaluation method according to the embodiment.

FIG. 3 shows an absorption spectrum and a fluorescence spectrum of a fluorescent substance, respectively.

FIG. 4 is a diagram showing transmission spectra of an excitation light optical system evaluation filter and a fluorescence optical system evaluation filter.

FIG. 5 is an explanatory diagram of a method for evaluating an excitation light optical system.

FIG. 6 is a diagram showing an emission spectrum of a xenon lamp.

FIG. 7 is an explanatory diagram of a method for evaluating a fluorescent optical system.

FIG. 8 is an explanatory diagram of a fluorescence observation device evaluation method according to another embodiment.

FIG. 9 is a diagram showing a transmission spectrum when an excitation light optical system evaluation filter is inclined.

FIG. 10 is a diagram showing a transmission spectrum when a fluorescence optical system evaluation filter is inclined.

FIG. 11 is an explanatory diagram of a fluorescence observation device evaluation method according to still another embodiment.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Sample, 11 ... Excitation light source, 12 ... Dichroic mirror, 13 ... Objective lens, 14 ... Slide glass, 15 ...
Mirror, 16: Camera, 21: Excitation light optical system evaluation filter, 22: Power meter, 31: Fluorescence optical system evaluation filter, 32: White light source, 33: Aperture, 34: Isotropic scattering object.

Continued on front page F-term (reference) 2G020 AA04 CA01 CB24 CB43 CB51 CC27 CC47 CD03 CD11 CD23 DA12 DA24 DA66 2G043 AA06 EA01 FA02 GA02 GA04 GB01 GB18 HA01 HA09 JA03 KA02 LA02 LA03 NA01 2H052 AA09 AC04 AC05 AC25 AC29 AF30 AD30

Claims (5)

[Claims] 1. A method for evaluating a fluorescence observation device that irradiates a sample with excitation light output from an excitation light source via an excitation light optical system and observes fluorescence generated from a fluorescent substance contained in the sample. Inserting a transmission filter having a transmission spectrum equal to the absorption spectrum of the fluorescent substance into the optical path of the excitation light optical system, and optically detecting excitation light output from the excitation light source and passing through the excitation light optical system and the transmission filter. A method for evaluating the excitation light source or the excitation light optical system based on the detection result. 2. A method for evaluating a fluorescence observation device for observing fluorescence generated from a fluorescent substance contained in a sample irradiated with excitation light at a predetermined position via a fluorescent optical system, comprising: A transmission filter having an equal transmission spectrum is inserted on the optical path of the fluorescence optical system, and the transmission filter and the fluorescence output from a white light source that outputs white light in a predetermined wavelength band including a peak wavelength of the fluorescence spectrum. A method for evaluating a fluorescence observation apparatus, comprising: detecting light having passed through an optical system with a photodetector; and evaluating the fluorescent optical system based on a result of the detection. 3. A method for evaluating a fluorescence observation device for observing fluorescence generated from a fluorescent substance contained in a sample irradiated with excitation light at a predetermined position via a fluorescent optical system, wherein a position where the sample is to be placed Arrange an isotropic scattering object to, the white light in a predetermined wavelength band including the peak wavelength of the fluorescence spectrum of the fluorescent substance, the isotropic scattering object through a transmission filter having a transmission spectrum equal to the fluorescence spectrum. Irradiating, detecting light scattered by the isotropic scattering object and passing through the fluorescent optical system by a photodetector, and evaluating the fluorescent optical system based on the detection result, evaluating a fluorescence observation apparatus. Method. 4. A transmission filter having a transmission spectrum equal to the absorption spectrum of a fluorescent substance. 5. A transmission filter having a transmission spectrum equal to the fluorescence spectrum of a fluorescent substance.