JPS606485B2 - Epi-fluorescence photometric microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an epi-illuminated light microscope, and more particularly to an epi-illuminated light microscope in which an apparatus function for determining the excitation wavelength spectrum of a sample is removed.

Nowadays, crab light microscopes are widely used, and along with the development of macro spectroscopic technology, the emission intensity of crab light, the crab light wavelength spectrum, or the excitation wavelength spectrum can be determined at the microscopic level, and it is possible to determine the identification of substances and crab photopigments, and to determine the identity of crab light pigments and scales. Microscopic twilling technology that performs folding has come to be widely used. An epi-illuminated side light microscope used in such a revealing side light technique will be explained with reference to FIG. In Figure 1, 1 is a light source, 2 is an excitation side monochromator, 3 is a dichroic mirror, 4 is an objective lens, 5 is a stage, 6 is a sample, and 7
is an observation prism that can be inserted into and removed from the optical path of the microscope, 8 is a rear eye lens, 9 is a monochromator on the honeycomb side, 10 is a detector consisting of a photo mark, etc. 11 is a recorder. In such a microscope, To obtain the excitation wavelength spectrum of the sample 6, insert the observation prism 7 into the optical path to determine the field of view, then remove the observation prism 7 from the optical path, and place the monochrome light side 9 on the largest crab of the sample 6. The excitation side monochromator 2 is scanned by setting a certain wavelength input F near the optical wavelength.At this time, the spectrum S, input obtained from the detector 10 is an instrument function that is affected by the wavelength characteristics of each element of the microscope. and the crab light wavelength spectrum of sample 6.
Since the crab light side monochromator 9 transmits only the light of the wavelength F of the crab light from the sample 6, the objective lens 4 and the turquoise mirror 3 for the crab light from the sample 6 are used.
There is no need to consider the wavelength characteristics of the crab light side monochromator 9 and detector 10. Therefore, as device functions, it is sufficient to consider the wavelength characteristics of the light source 1, excitation side monochromator 2, dichroic mirror 3, and objective lens 4 related to the excitation light. Now, if the wavelength characteristics of the light source 1, the excitation side monochromator 2, the dichroic mirror 3, and the objective lens 4 are L input, M, and BX 0 input for the wavelength input of the excitation light east, then the detector The following formula (1) holds between the spectrum S, obtained in step 10, and the excitation wavelength spectrum of sample 6, ST input, F input. SI input = L person ■ MI input, ST person F
1) In other words, in the above-mentioned conventional technology, the spectrum S obtained from the detector 10 is "in addition to the excitation wavelength spectrum ST of the sample 6, there are The value will include the wavelength characteristics of the lens 4.

The present invention aims to provide an epi-illumination type crab-light side light microscope that is capable of removing the device function included in the signal from the detector and extracting only the excitation wavelength spectrum of the sample when determining the excitation wavelength spectrum of the sample. purpose.

An embodiment of the present invention will be described below based on FIG.

Light from a light source 2 such as a xenosol lamp enters the excitation side monochrome end 22.

The excitation side monochromator 22 selects and emits only the light having a preset wavelength out of the incident light from the light source 21. reflected, objective lens 2
4 to illuminate the object 26 on the stage 25. Light from the object to be measured 26 is transmitted through the dichroic mirror 23 by the objective lens 24 and focused onto the slit 27 . The light transmitted through the pinhole provided in the slit 27 enters the crab light side monochromator 29. The monochromator 29 on the crab light side selects and emits only light having a preset wavelength. The light emitted from the monochromator 29 on the crab light side is converted into an electrical signal proportional to the light intensity by three detectors. The switch SW selectively applies the signal from the detector 30 to one input terminal a of the divider circuit 31 or the input terminal b of the memory circuit 32. A signal read from the storage circuit 32 is applied to the other input terminal c of the division circuit 31. Reading of signals from the storage circuit 32 is performed by signals from the synchronizing means 33. A divider circuit 31 takes the ratio of both input signals and drives a recorder 34. The measurement is carried out as follows.

First, as an object to be measured on the stage 25, Rhodamine B3g
'Use an ethylene glycol solution. Rhodamine B3
G' The ethylene glycol solution is a light-emitting substance that has a constant luminous efficiency regardless of the wavelength of the excitation light (approximately 30 m to 60 m). This rhodamine B/so ethylene glycol solution is placed in a well-known microcell and placed on a stage. In this state, crab light side monochrome makeup 2
9 is set to a certain value F near the maximum light wavelength of the sample, and the excitation side monochromator 22 is scanned. At this time, switch SW is connected to terminal b. Enter the set wavelength of the excitation side monochromator 22. A light source 21, an excitation side monochromator 22, a dichroic mirror 23, an objective lens 24
The wavelength characteristics of L^, , M people, B input, 0 input, respectively.
Then, the spectrum intensity S2 obtained by the detector 30 is calculated by the following formula '2, where k is the crab light spectrum of Rhodamine B.
} is given. S2^・ = 1 in L, 1 in MI, 1 in B.

. Input 1 0k . Next, the sample is placed on the stage 25. At this time, the light-side monochromator 29 remains set to wavelength input F, and the switch SW is switched to be connected to terminal a.
At this time, when the excitation side monochromator 22 is scanned, when the set wavelength of the excitation side monochromator 22 is on 2, the detector 30
From, the spectral intensity S3 input 2 given by the following equation (3)
occurs. S3 input 2 = L^2 ・MI^2 ・B^2
・. Input 2・ST person F input 2...(3, However, ST input F input 2 is the excitation wavelength spectrum of the sample when the set wavelength of the excitation side monochromator is ^2.

When the sample is placed on the stage 25, the synchronization means 33
The contents stored in the storage circuit 32 at a wavelength synchronized with the set wavelength input 2 of the excitation side monochromator 22 are read out and applied to the division circuit 31. In other words, the dividing circuit 31 operates when the set wavelength of the excitation side monochromator 22 is the same when the sample is placed on the stage 25 and when the Rhodamine B3g/〆ethylene glycol solution is spread, that is, ON, = ON2.
Take the ratio between the spectral intensities at =^. Therefore, the output of the division circuit 31 is given by the following equation {4}. Trunk-S Gakuenhada [4) Formula {4}' Koitek! Since it is a known constant, the excitation wavelength spectrum ST and F of the sample can be easily determined.

Note that some modifications are possible in the above embodiments.

For example, first, a sample is placed on the stage 25 for measurement, the results are stored in the memory circuit 32, and then a rhodamine B3g' and ethylene glycol solution is placed on the stage 25.
Similar results can be obtained even if the measurement is carried out by remounting the sample on top.
Furthermore, the excitation wavelength spectrum of the sample can be determined by replacing the sample with the rhodamine B3g' quetylene glycol solution for each set wavelength of the excitation side monochromator 22.
As described above, according to the present invention, when determining the excitation wavelength spectrum of a sample using a radiation type crab light side light microscope,
Instrument functions contained in the signal from the detector can be removed.

[BRIEF DESCRIPTION OF THE DRAWINGS] Fig. 1 is an explanatory diagram of a side-light microscope of a brush type used in the conventional 9-light technology, and Fig. 2 is an explanatory diagram of an embodiment of the present invention. . Explanation of symbols of main parts, SW...Switch, 3
0...detector, 31...divider circuit, 32...
・Memory circuit, 33... Synchronization means, 34...
Recorder, 25. ．． ...Stage, 26...
Object to be measured. Figure 2

Claims (1)

[Scope of Claims] 1. Light source means for sequentially setting and emitting excitation light of different wavelengths, detection means for detecting light of a predetermined wavelength and photoelectrically converting the detected light, and excitation from the light source means. In an epi-illumination type fluorescence photometric microscope having an optical means for epi-illuminating a sample with light and making the fluorescent light from the sample enter the detection means, a photoelectric conversion signal from an input terminal is converted to the wavelength of the excitation light. a storage means for storing data in a corresponding manner; a dividing means having two input terminals, one of which is connected to the output terminal of the storage means; a selection means for selectively inputting an input to the other input terminal of the dividing means; and a synchronization means for calling a stored value corresponding to the set wavelength from the stored value of the storage means in synchronization with the wavelength setting of the light source means. A fluorescence photometric microscope characterized in that the excitation wavelength spectrum of the sample is determined by the output of the dividing means.