JPS606486B2 - Transmission type fluorescence photometric microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a transmission type crab light side light microscope, and more particularly to a transmission type crab light side light microscope that eliminates the device function when determining the crab light wavelength spectrum of a sample.

Nowadays, crab light microscopes are widely used, and along with the development of macro spectroscopic technology, it is possible to determine the emission intensity of crab light, crab light wavelength spectrum, or excitation wavelength spectrum at the microscopic level, and to identify and analyze substances and halo pigments. Microscopic crab light storage technology that performs folding has come to be widely used. A transmission-type gauze-light microscope used in such a revealing light-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 quartz total reflection mirror, 4 is a quartz type field-of-field condenser, 5 is a sample, 6 is an objective lens, 7 is an observation prism that can be inserted into and removed from the optical path of the microscope, 8 numeral 9 is a saddle-eye lens, numeral 9 is a monochromator on the light side, a detector consisting of a photo-maru, etc., and numeral 11 is a recorder. In order to obtain the afterlight wavelength spectrum of the sample 5 in such a microscope, the observation prism 7 is inserted into the optical path to determine the field of view, and then the observation prism 7 is removed from the optical path, and the excitation side monochromator 2 is moved to the sample 5. A certain wavelength ^E near the maximum excitation wavelength is set, and the crab light side monochromator 9 is scanned. At this time, the spectrum S obtained from the detector 10 is given by the product of the instrument function, which is the influence of the wavelength characteristics of each element of the microscope, and the pot light wavelength spectrum of the sample 5. Since the light coming from is a single wavelength input E, light source 1 and excitation side monochromator 2 are used.
It is not necessary to consider the wavelength characteristics of , and it is sufficient to consider the wavelength characteristics of the objective lens 6 , the crab light side monochromator 9 , and the detector quantity 0 as device functions. Therefore, “Monochrome Ending on the Sunrise Side – Evening 9th
When the set wavelength is on, the wavelength characteristics of objective lens 6, monochromator 9, and detector 10 are set to ○on and M2, respectively.
If a person enters D, then "spectrum S obtained by detector 9,
The following equation {1} holds true between the light wavelength spectrum ST in and E in of sample 5. SI entry=.

input, M2 input, D input, ST person E input... (1) That is, in the above-mentioned conventional technology, the spectrum S obtained from the detector 10,
Well, the crab light wavelength spectrum of the sample 5 becomes a value that includes the wavelength characteristics of the objective lens 6, the crab light side monochromator 9, and the detector 10 in addition to the ST input and E input. This can cause erroneous results to be obtained, especially when identifying samples with similar crab light peak values (for example, 5-HT and catechol amino). An object of the present invention is to provide a transmission type crab light side light microscope capable of removing an apparatus function included in a signal from a detector and extracting only the crab light wavelength spectrum of a sample when obtaining a wavelength spectrum.

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

Light from the light source 21 enters the excitation side monochrome end 22.

The excitation side monochromator 22 selects and emits only the light having a preset wavelength from among the incident light from the light source 21 . The light emitted from the excitation side monochromator 22 passes through the quartz partially transmitting mirror 28' which is a part of the photon meter 35.
Therefore, it is divided into two regions. The light transmitted through the quartz partial transmission mirror 28 is reflected by the quartz total reflection mirror 23, and then transmitted through a quartz type condenser 24 to illuminate the sample 25. Light from the sample 25 is focused onto an aperture 27 by an objective lens 26. The light that passes through the pinhole provided in the aperture 27 enters the halo side monochrome end 29.The crab light side monochrome end 29 transmits only the light of the set wavelength among the incident light lights. The light beams of a single wavelength incident on the detector 30 are converted into electricity.On the other hand, the light beams reflected by the quartz partially transmitting mirror 281 are guided to the detector 30, which is composed of a photomultiplier or the like. triangular cell 32
Irradiate the rhodamine B3g' ethylene IJ Cole solution contained therein. As is well known, Rhodamine B is a luminous substance with constant luminous efficiency regardless of the excitation wavelength (approximately 30 to 60 meters). Quartz triangular cell 3
The light beam (halo) emitted from Rhodamine B is filtered by a cut filter 33 except for the crab light emitted by Rhodamine B, and converted into an electric signal by a photoelectric converter 34 composed of a photomultiplier or the like. The electrical signal from the photoelectric converter 34, which is the output of the photon meter 35, is applied to one input terminal of the divider circuit 36. The changeover switch SW divides the output of the detector 30 into the dividing circuit 3.
6 or one input terminal (second terminal) b of the arithmetic circuit 38 is selected. Divide circuit 36 outputs the ratio between the voltages applied to both input terminals. A storage circuit 37 stores the output of the division circuit 36. The arithmetic circuit 38 receives the stored contents of the memory circuit 37 to the other input terminal, and performs multiplication or division with the output of the detector 30. At this time, the memory circuit 37
The stored contents of the wavelength synchronized with the set wavelength of the crab light side monochromator 29 are read out by the signal from the synchronization means 39 and applied to the arithmetic circuit 38. Calculations are performed between the corresponding wavelengths of the circuit 37, and the results are output.In order to obtain the crab light wavelength spectrum in such a honey-light side light microscope, the following procedure is performed.First, the sample 25 is placed in the optical path of the microscope. , and synchronize the excitation side monochromator 22 and the crab light side monochromator 29 to change the set wavelength over a certain wavelength change range.At this time, the crab light side monochromator 29
When the set wavelength of is on, the spectral intensity obtained from the photon meter 35 is S,'X, and the spectral intensity obtained from the detector 38 is S2', the following equation Holds true.S,'in=L^・M,in・k
""" (2) S2' input: L input .MI input.

〇Enter/M2 Entry. Enter D “
Kawakawa [3} However, the wavelength characteristics M, input of the three L-input light sources 21;
Wavelength characteristic k of excitation side monochromator 22: Rhodamine B
Spectral intensity of the ethylene glycol solution (constant value) ○ ON: Wavelength characteristics of the objective lens 26 M2 ON: Wavelength characteristics of the monochromator 29 on the crab light side D ON: Wavelength characteristics of the detector 30 Switch SW converts the sample 25 into a microscope When removed from the optical path of
Connected to terminal a.

Therefore, the spectral intensity given by equation (2) and equation (2) is input to the division circuit 36. Therefore, the output from the divider circuit 36 is the ratio of the two, and is given by the following equation (2). 91
k...[4'921.

Input/M2^/D Input This value is input to the memory circuit 37.

Next, the sample 25 is inserted into the optical path of the microscope, and the excitation side monochromator 22 is set to one wavelength.

At this time, the switch SW is switched to the second terminal b. In this state, the crab light side monochromator 29 is scanned over a certain wavelength change range, and the spectrum intensity S2 obtained from the detector 30 at this time is calculated by the following formula, assuming that the crab light spectrum of the sample is ST input and E input. ■It is given by. S2
Enter=.

＾・M2 entry ・D^・ST^E entry ‐・‐・・‐＾
51 Since the synchronizing means 39 inputs the stored contents of the storage circuit 37 having a wavelength equal to the currently set wavelength of the crab light side monochromator 29 to the arithmetic circuit 38, the arithmetic circuit 38 calculates the product between both inputs.

Therefore, the output So of the arithmetic circuit 38 is given by the following equation '6) which is the product of equation {4- and equation (2). S.

=k・ST input...[6} Since k' is a constant considering equation (6), the crab light wavelength spectrum ST input of sample 25 is
You can easily take out the contents.

Since the Wakiran actual value side chest passage 36 also calculates the value, the arithmetic circuit 38 calculates the product between the two inputs and calculates the Shiyanagi division circuit 3, the arithmetic circuit. 38 is set to take the ratio between both inputs.

Furthermore, in the embodiment, equation (5) is measured after equation (41) is measured over the period of change of the monochromator 29 on the crab light side.

This has the advantage that since the value given by equation (2) is stored in the memory circuit 37 as the correction amount, there is no need to measure the correction amount one by one even when measuring different samples. However, the order of measurement may be to obtain equation (5), store it in the memory circuit, measure equation {4}, and then perform calculations between the two.Furthermore, in the embodiment, first, the crab light side monochrome Although the formula {4) is obtained over the wavelength variation of the meter 29, the calculation may be performed by measuring the formula (character 4 and formula 2) for each set wavelength of the monochromator 29 on the light side.

Furthermore, this microscope can also be used to measure the excitation wavelength spectrum of the sample 25.

The spectral intensity obtained from the photon meter 35 is given by equation {2}. Regarding the signal S3 from the detector 30, since the crab light side monochromator 29 is set to a certain wavelength, it is necessary to consider the wavelength characteristics of the objective lens 26, the crab light side monochromator 29, and the detector 30. Not signal S
3 can be expressed by the following formula (7}. S3; L input, MI^, ST
Input E input... (71 However, ST input E^: Excitation wavelength spectrum of sample 25. Here, if the ratio of equation [2' and equation [7] is calculated by the division circuit 36, Mikieni is obtained, and from this, the sample It is easy to extract only the excitation wavelength spectrum ST^E^.As described above, according to the present invention, the device function included in the signal from the detector can be removed, and the
A transmission type photometric microscope capable of determining only the light wavelength spectrum is obtained.

[Brief explanation of the drawing]

FIG. 1 is an explanatory view of a transmission-type side light microscope used in the conventional head light side light technique, and FIG. 2 is an explanatory view of an embodiment of the present invention. Explanation of symbols of main parts, 25... Sample, 29...
...Crab light side monochrome makeup, 30...Detector, 3
4... Light exposure converter, 35... Photon meter, S
W...... functional switch, 36...... division circuit,
38... Arithmetic circuit o circuit, 37... Memory circuit, 39
....Synchronization means, a...first terminal, b...second terminal. Three figures: Figure 2

Claims (1)

[Claims] 1. A light source means capable of emitting excitation light of a predetermined wavelength, a detection means capable of successively setting and detecting light of different wavelengths and photoelectrically converting the detected light, and a sample measuring means using the excitation light from the light source means. In a transmission-type fluorescence photometric microscope having an optical means for transmitting illumination and allowing fluorescence from the sample to enter the detection means, the excitation light is branched and inputted, so that the light intensity of the excitation light can be adjusted. a correction signal detection means for outputting a proportional electric signal; a selection means for selectively generating a photoelectric conversion signal outputted by the detection means at a first terminal and a second terminal; and an output terminal of the correction signal detection means; a dividing means connected to the first terminal and outputting a signal according to the ratio of signals generated at both terminals; connected to either one of the output terminal of the dividing means and the second terminal; storage means for storing the input signal in correspondence with the wavelength of the detection light;
a synchronizing means for reading out a stored value corresponding to the set wavelength in synchronization with the wavelength setting of the detecting means from the stored value of the storing means; and the other of the output terminal of the dividing means and the second terminal; calculation means connected to the output terminal of the storage means, for calculating a ratio or product between the signals generated at both terminals and removing an apparatus function, and calculating the wavelength of the detection means as an output of the calculation means. A transmission type fluorescence photometric microscope characterized by obtaining a fluorescence wavelength spectrum from which characteristics have been removed.