JPH04107418A - Automatic dimming method of microscope capable of simultaneous transmission and downward lighting observation - Google Patents

Abstract

PURPOSE:To eliminate the need to finely adjust the quantity of transmitted illumination light at the time of microscope switching by observing the brightness of a sample image with downward fluorescent light through a photodetecting element when a simultaneous observation mode is specified, and controlling a dimming member in an optical path of transmission lighting in a shutter closed state and adjusting brightness balance. CONSTITUTION:The mode is switched to the simultaneous observation mode with a mode indicating switch 28. When the signal of the simultaneous observation mode is inputted to a CPU 30, the measured value of the brightness of the downward fluorescent image photodetected by the photodetecting element 12 is read in the CPU 30 and data which are stored in a storage part 29 previously is read out to the CPU 30, which performs comparative arithmetic operation so that the brightness balance between the downward fluorescent light image and a transmitted observation image becomes proper. Then a controlled variable for dimming corresponding to the measured value of the photodetecting element 12 is determined and the dimming member 24 is driven with a control signal according to the controlled variable to reduce the quantity of transmission lighting cut off by a shutter 23 for transmitted light by a necessary quantity. Consequently, the need to adjust transmitted illumination light, etc., every time the observation mode is switched is eliminated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an epi-fluorescence microscope capable of observing a fluorescent specimen by switching between transmitted illumination light and epi-illumination light and simultaneously observing the fluorescent specimen.

[Conventional technology]

Traditionally, observation of cells as fluorescent specimens using a microscope is
This is done using epifluorescence observation. However, by observing fluorescence alone, it is difficult to determine where in the entire specimen the cells emitting fluorescence are located. Therefore, in order to observe the overall shape that is difficult to recognize with fluorescence observation and recognize the part of the cell that is emitting fluorescence, phase contrast observation using transmitted illumination light and differential interference observation are used in conjunction with transmitted light observation and fluorescence observation. Switching observation or simultaneous observation in which an epifluorescent image and a transmitted light image are observed in a superimposed manner has come to be performed.

In simultaneous observation of an epifluorescent image and a transmitted light image, since the epifluorescent image is darker, it is necessary to adjust the brightness balance with the phase contrast image or differential interference image of the transmitted light. This requires very delicate adjustments.

By the way, conventionally, the brightness of the observed image was adjusted using the specimen.

This has been done by adjusting the light source voltage of the transmitted illumination system or inserting an ND filter each time the microscopy method or the magnification of the objective lens is changed.

An example of a microscope equipped with such a light control means is described in Japanese Patent Laid-Open No. 71652/1983. The basic optical system of this microscope will be explained based on FIG. 3. In the figure, illumination light emitted from a transmitted illumination light source 1 such as a halogen lamp or a tungsten lamp is condensed by a condenser lens 2, and then is condensed by a relay lens. 3 and is reflected upward by a reflecting mirror 4, the specimen 6 is illuminated by a condenser lens 5. The transmitted specimen image formed by the illumination light is magnified by an objective lens 7, bent to an angle that is easy to observe by a spectroscopic tilting prism 8, and observed through an eyepiece lens 9. In addition, the light that passes straight through the tilted prism 8 and is divided is imaged on the photographing film surface IO, but the film surface 1
A part of the light that is split by the half mirror 11 on the way to zero is guided to the light receiving element 12 which detects the amount of light.

The amount of light detected by the light receiving element 12 is compared with a preset level, and the light amount of the light source 1 is changed by the light control device 13.

This allows the specimen image to be observed with the optimum amount of observation light depending on the switching of the objective lens or the like.

Furthermore, devices that control the light source of illumination light according to switching of the objective lens without depending on the light receiving element are described in Japanese Patent Laid-Open No. 59-172618 and Japanese Utility Model Application No. 61-185025. There is something.

Also, Utility Model Publication No. 60-37538 and Utility Model Application No. 6319810
The light adjustment means described in the publication detects the image plane illuminance of illumination light using a light receiving element such as a photoelectric converter, and adjusts the light intensity using a light amount attenuator such as a filter in the transmitted illumination optical path according to the output of the light receiving element. I'm trying to get it done. Furthermore, JP-A-51
The device disclosed in Japanese Patent No. 172,617 similarly uses a light attenuator to perform light adjustment in accordance with switching of the objective lens.

However, in all of these methods, the light intensity of the transmitted light image is measured while the specimen is illuminated, or the transmitted illumination light intensity is changed according to the switching of the objective lens, or the light intensity of the epifluorescent image is changed. This is an automatic light control means that measures and changes the amount of epi-illumination light. In any case, the amount of illumination light can be adjusted by measuring the amount of light of the specimen image caused by only one illumination light when observing with one of the illumination lights, and observation can be performed with the optimum light amount.

Therefore, when simultaneously observing an epifluorescent image and a transmitted light image,
The brightness balance has been adjusted by an observer adjusting the light source voltage of the transmitted illumination system while observing the sample image, or by inserting an ND filter into the transmitted illumination optical path.

[Problem to be solved by the invention]

However, during observation, it is necessary to frequently change the observation method from epifluorescence observation to simultaneous observation, or from transmitted illumination observation (phase contrast observation, differential interference observation) to simultaneous observation, in order to position the specimen and identify the fluorescent site. There are many cases where the observer has to adjust the brightness while observing, which is cumbersome.

Moreover, during epifluorescence observation, excitation light tends to cause fading of the fluorescent staining of the specimen, so it is important to shorten the irradiation time of the excitation light to the specimen, but adjusting the brightness balance is extremely delicate and time-consuming. There is a problem.

In view of these problems, the present invention has been developed to enable brightness balance adjustment during simultaneous observation of transmitted illumination observation and epifluorescence observation to be performed in a short time and with a simple operation, and to suppress fading of fluorescence of a specimen. An object of the present invention is to provide an automatic light control method in a microscope that enables simultaneous transmission and epi-illumination observation.

[Means and effects for solving the problem] The automatic light adjustment method in a microscope capable of simultaneous transmission and epi-fluorescence observation according to the present invention enables simultaneous observation by superimposing a specimen image obtained by transmitted illumination and a specimen image obtained by epi-fluorescence. In the microscope, the simultaneous observation designation signal closes the shutter in the transmitted illumination optical path and opens the shutter in the epi-illumination optical path at the same time. Based on the data measured by the element and read out from the storage unit in accordance with the measured values, the transmitted illumination is adjusted so that the brightness of the specimen image by transmitted illumination during simultaneous observation is balanced with the brightness of the specimen image by epifluorescence. The present invention is characterized in that the shutter in the transmitted illumination optical path is opened after controlling the light control unit disposed in the optical path.

Therefore, during simultaneous observation, the epifluorescence observation state is first entered, and the brightness of the specimen image due to epifluorescence is measured by the light receiving element.
This measured value and the data in the storage unit are compared and calculated, and the light control member is controlled so that the brightness of the specimen image by transmitted illumination is balanced with the brightness of the specimen image by epi-illumination. Transmitted illumination with the shutter in the closed state The brightness of the specimen image in the optical path is adjusted, and the shutter is opened, so that the epifluorescence specimen image and the transmitted illumination specimen image can be observed simultaneously with well-balanced brightness.

〔Example〕

Hereinafter, a preferred embodiment of the present invention will be described based on FIG. 1 and FIG. 2, but the same reference numerals will be used for the same parts as in the prior art shown in FIG. 3 above, and the explanation thereof will be omitted. do.

In the figure, 15 is a light source for epi-illumination such as a mercury lamp for irradiating the specimen 6, 16 is a condensing lens for epi-illumination light, and 17.18
19 is a relay lens, 19 is an excitation filter, and 20 is a dichroic mirror disposed between the objective lens 7 and the tilted prism 8 in the observation optical system, and these constitute a part of the epi-illumination system. 21 is an absorption filter that separates the illumination light for epifluorescence observation from the fluorescence emitted from the specimen and transmits only the fluorescence; the excitation filter 19;
Dichroic mirror 20. The absorption filter 21 is located in the optical path during epifluorescence observation, but is removed from the optical path during transmitted illumination observation.

22 is a shutter for epi-illumination light disposed in the epi-illumination optical path, for example between the condenser lens 16 and the relay lens 17;
Reference numeral 23 denotes a shutter for transmitted illumination light disposed in the transmitted illumination optical path, for example, between the condenser lens 2 and the relay lens 3, and these shutters 22 and 23 are respectively opened and closed by control signals to be described later to release the illumination light. can be passed through or blocked. Reference numeral 24 denotes a light control member which is arranged adjacent to, for example, the transmitted light shutter 23 in the transmitted illumination optical path and adjusts the amount of transmitted light by rotating or sliding a continuous ND filter, for example, and is connected to the transmitted light shutter 23. The light member 24 can also be constructed as a single mechanism. 25.
Reference numerals 26 indicate the positions of ring slits and Wollaston prisms, which are arranged in the transmission illumination optical path for transmission phase contrast observation or transmission differential interference observation.

Reference numeral 28 denotes a mode designation switch for specifying any of the transmitted illumination observation mode, epi-fluorescence observation mode, and simultaneous observation mode of transmitted illumination observation and epi-fluorescence observation, and 29 stores data to be compared with the measured value of the light receiving element 12. This data is data that is arbitrarily set by the observer according to the observation method and the magnification of the objective lens and indicates the control amount to be controlled by the light control member 24, or -172
As shown in No. 617, data regarding nine types of objective lens magnification, aperture stop of transmitted illumination system, condenser lens,
Data regarding filters, etc. may also be input. In the latter case, the optimal brightness for transmitted illumination observation is set according to the objective lens based on these data. Reference numeral 30 receives the measured value of the light receiving element 12 and the designated mode of the mote designation switch 28, and both shutters 22 and 23.
It is a CPU that calculates and outputs control signals for driving the light control member 24 and the measured values of the light receiving element 12 and the storage section 29.
The control amount is determined so that the brightness of the transmitted light image is appropriately balanced with respect to the epifluorescence image by comparing and calculating the data of
The amount of transmitted illumination light is adjusted by driving the light control member 24.

This embodiment is constructed as described above, and its operation will be explained with reference to the flowchart shown in FIG.

First, when the power is turned on, the transmitted illumination light source 1 and epi-illumination light source 15 are turned on (step 101). Furthermore, when the power is turned on, the CPU 30 automatically sets the mode to the transmitted illumination observation mode, and controls the transmitted illumination light shutter 23 to open and the epi-illumination light shutter 22 to close (step 1).
o2). In this transmitted illumination observation mode, the specimen 6 is focused and the observation position is determined. The reason why the epi-illumination optical path is closed is to prevent fading of the fluorescence of the specimen.

Next, in order to perform epifluorescence observation, the mode designation switch 28 is used to switch to the epifluorescence observation mode (step 103), and the excitation filter 19.degree. dichroic mirror 20. The absorption filter 21 is moved into the optical path from outside the optical path. The CPU 30, to which the epifluorescence observation mode signal is input, closes the transmitted illumination light shutter 23 and switches the epi-illumination light shutter 22 to open (step 104), thereby blocking the transmitted illumination light and Epi-illumination light is guided to the specimen 6. Epifluorescence observation is thereby performed (step 1o5).

Furthermore, in order to confirm the position of the cell emitting fluorescence recognized by epifluorescence observation in the entire specimen 6, the mode is switched to the simultaneous observation mode using the mote designation switch 28 (step 106). Simultaneous observation mode signal is CPU
30, the measured value of the brightness of the epi-fluorescent image received by the light receiving element 12 is read into the CPU 30 (
In step 107), the data previously stored in the storage unit 29 is read out to the CPU 30, and comparison calculations are performed so that the brightness balance between the epifluorescence image and the transmission observation image is appropriate. Then, the control amount to be dimmed corresponding to the measured value of the light receiving element 12 is determined (step 108), and based on this, the dimming member 24 controls the transmitted illumination optical path, which is blocked by the transmitted light shack 23, based on the control signal. is driven to reduce the amount of light by the required amount (step 109).
. After that, the transmitted light shutter 23 is opened (step 1) 0) and simultaneous observation is performed (step 111).
.

In this way, the brightness of the transmitted light image is automatically adjusted to a level that matches the brightness of the epifluorescent image, and the two images are superimposed to show the entire specimen 6 and the cells emitting fluorescence. Parts, etc. can be observed simultaneously.

Note that the two-part explanation describes the flow when selecting transmission illumination observation, epifluorescence observation, and simultaneous observation, but in reality, there are various observation flows such as transmission illumination observation to simultaneous observation. Regardless of which flow is selected, before simultaneous observation, close the transmitted illumination light shutter 23 and open the epi-illumination light shutter 22 in order to measure the observation light amount of the epi-fluorescent image with the light receiving element 12. The epi-fluorescence observation mode selected is now selected.

As described above, according to the present invention, when simultaneous observation of transmitted illumination and epi-fluorescence is selected, the brightness of the specimen image due to transmitted illumination is automatically adjusted to balance the brightness of the specimen image due to epi-fluorescence. Therefore, there is no need for the observer to make delicate adjustments to the amount of light such as transmitted illumination light every time the observation mode is switched, and the operation becomes easy. Moreover, the brightness balance adjustment at the time of switching can be performed in a short time, and fading of the fluorescence of the specimen can be reduced.

The switching operation between the epi-fluorescence mode and the transmitted-light illumination mode using the mode designation switch 28 is performed using the excitation filter 19. This may be done simultaneously by a switch that electrically inserts and removes the dichroic mirror 20 and the absorption filter 21 into and out of the optical path.

〔Effect of the invention〕

As described above, the automatic light control method in the microscope capable of simultaneous transmission and epi-illumination observation according to the present invention is to observe the brightness of the specimen image due to epi-fluorescence using the light receiving element when simultaneous observation is specified, and adjust the transmission illumination optical path with the shutter closed. Since the brightness balance is adjusted by controlling the light control member in the microscope, there is no need for the observer to delicately adjust the amount of transmitted illumination light every time the speculum is switched, and the operation becomes simple. Moreover, the brightness balance adjustment at the time of switching will be done in a short time,
Fading of fluorescence of the specimen can be suppressed.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an optical system of an embodiment of a microscope according to the present invention, FIG. 2 is a diagram showing a flow chart of the embodiment, and FIG. 3 is a diagram showing a basic optical system of a conventional microscope. ]...Light source for transmitted illumination, 6...Specimen, 12...
...Light receiving element, 15...Light source for epi-illumination, 22...
...Nyatta for epi-illumination light, 23...Shutter for transmitted illumination light, 24...Dimmer side, 28...Mode designation switch, 29...Storage unit, 30...・CP
U. ■

Claims (1)

[Claims] In a microscope capable of simultaneous observation by superimposing a specimen image obtained by transmitted illumination and a specimen image obtained by epifluorescence, a shutter in the transmitted illumination optical path is closed in response to a simultaneous observation designation signal, and simultaneously the epi-illumination is performed. The shutter in the optical path is opened, the brightness of the specimen image due to epifluorescence is measured with a light receiving element placed in the observation optical path system, and based on the data read out from the storage unit in accordance with the measured value, After controlling the light control member placed in the transmitted illumination optical path, the shutter in the transmitted illumination optical path is opened so that the brightness of the specimen image due to transmitted illumination during simultaneous observation is balanced with the brightness of the specimen image due to epifluorescence. An automatic light control method characterized by: