JPH0618415A - Spectral microscope - Google Patents

Abstract

PURPOSE:To cast a specified wavelength light to a subject of bacteria or cells as well as to observe a structure of the subject from an excitation light thereby. CONSTITUTION:A light out of a light source is separated into two-wavelength ones, irradiating a subject, and ion concentration is measured from the intensity of fluorescence excited. Two wavelength lights are incessantly switched at a fixed time interval for irradiation, synchronizing a rising signal for the switch, and a video signal is inputted and photographed by a CCD camera 2. The intensity of the fluorescence excited by the two-wavelength lights is measured by a photomultiplier tube 3. In addition, this microscope is provided with a concentration operation storage circuit 22 which operates and stores the extent of ion concentration from the ratio of this intensity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a spectroscopic microscope for irradiating a specimen such as bacteria or cells with light of a specific wavelength and observing the tissue of the specimen from the excitation light (fluorescence) produced by the irradiation.

[0002]

2. Description of the Related Art Usually, when observing bacteria or cells with a microscope, there is a method of administering specific ions such as calcium ions to the living body and observing the migration of the ions over time. In this case, since the ions cannot be observed as they are, a fluorescent reagent is taken into cells and the like, and when the cells are irradiated with light having an excitation wavelength peculiar to the reagent, they are combined with or dissociated from the ions to emit light having a peculiar wavelength different from the irradiation light. (Hereinafter simply referred to as fluorescence)
Give out. In order to observe the ion concentration and the distribution change state from the intensity of the generated fluorescence, a means of irradiating light of a required wavelength with a spectroscopic lamp, observing with a spectroscopic microscope, and photographing with a CCD camera is generally tried.

However, the above-mentioned fluorescence is usually weak, and CC
In order to take a picture with a D camera, the spectroscopic lamp needs to be considerably powerful, and the S / N ratio is still poor, and noise is large and it is not practical. Moreover, the wavelength setting is manual, and it is difficult to switch the wavelength quickly and accurately. Irradiation light with different wavelengths is used for spectroscopic analysis,
It is preferable to analyze the fluorescence generated by this, which has different properties, but it is difficult to switch the wavelength of the spectroscope at high speed in the conventional method.

In order to solve the above problems, the present inventor has
We proposed a spectroscopic microscope that enables automatic measurement of minute amounts of fluorescence (see Japanese Patent Publication No. 1-214521).
The outline is shown in FIG. This spectroscopic microscope 50 is a table X
A first light source 51 for irradiating a specimen W on Y from above,
The second light source 52 that illuminates from the lower surface and the eyepiece 5 for visual observation
3, a CCD camera 54 for photographing, and a photomultiplier tube 55.

The first light source 51 is for visual observation with an objective lens. The light from the second light source 52 is dispersed by the diffraction grating G1, and the light having a predetermined wavelength is reflected by the dichroic mirror DM via the lens group 56 and the solenoid shutter 57, passes through the objective lens 58, and passes through the sample W.
Is irradiated from below. The reflected light from the specimen W passes through the objective lens 58 and the dichroic mirror DM, and reaches the first solenoid rotary half mirror 59 arranged on the optical axis. When the mirror 59 is OFF (chain line position), the reflected light is the second solenoid rotary half mirror 6
When the mirror 60 reaches 0, the reflected light is reflected by the lens group 61 and the light amount amplifier (image indensifier).
The CCD camera 54 is reached via 62.

A total reflection mirror 63 is arranged below the rotary half mirror 59, and when the solenoid of the mirror 60 is OFF, the reflected light reaches the total reflection mirror 63, is reflected and passes through a lens group 64, and a filter 65, The light is projected onto the photomultiplier tube 55 via the light quantity amplifier 66. At the time of measurement, the light from the second light source 52 is split into light of a specific wavelength by the analysis grating G1, and the objective lens 58 is passed through the shutter 57.
And the specimen W is irradiated. In this case, when a reagent is administered to the sample, excitation light (fluorescence) is emitted from the sample W, reflected by the half mirror 60, enlarged by the lens group 61, amplified by the optical amplifier 62, and amplified by the CCD camera 5.
Up to 4. As a result, the S / N ratio is improved, and the CCD camera can be used for photographing. At the time of measurement by the photomultiplier tube 55, the solenoid rotary half mirror 60 is set to the OFF position, whereby the excitation light is filtered by the filter 65. After the wavelength is selected, it is amplified by the optical amplifier 66 and enters the photomultiplier tube 55.

[0008]

However, even in this system, there is a drawback in that it is difficult to set the timing of projecting the VD signal and the light of a plurality of wavelengths when photographing with the CCD camera.
In addition, the ion concentration cannot be calculated. Also, the timing of the video recorder image and the calculated ion concentration value cannot be matched. Furthermore, there is a problem that the contour of the specimen (cell) is not clear in the video image.
The present invention aims to solve these problems.

[0009]

The spectroscopic microscope of the present invention for achieving the above-mentioned object splits the light from the light source into light of two wavelengths and irradiates the sample with the intensity of the excited fluorescence. In a spectroscopic microscope that measures ion concentration, two wavelengths of light are constantly switched and irradiated at fixed time intervals, and a VD signal is input in synchronization with a rising signal for switching C
It is equipped with a concentration calculation memory circuit that captures an image with a CD camera, measures the intensity of fluorescence excited by two wavelengths of light with a photomultiplier, and calculates and stores the ion concentration from the ratio of these intensities. is there

According to a second aspect of the present invention, when a captured image is imprinted on a video recorder and then reproduced, the ion concentration is output in synchronization with the image output time, and the ion concentration is displayed on the display circuit together with the image. It is provided with a display circuit.

Further, in the third aspect of the present invention, light having a wavelength different from the wavelength to be excited is irradiated, and a differential interference filter that interferes with the wavelength light is placed in front of the CCD camera to emphasize the contour of the image. It is a thing.

[0012]

[Function] The fluorescence excited by the light of two wavelengths is C
An image is picked up by a CD camera, and the intensity of the light is measured by a photomultiplier tube in synchronism with it, and the ratio of the outputs is obtained. These can be known over time.

[0013]

1 to 5 show an embodiment of the present invention. Figure 1
Shows an outline of a spectroscopic microscope of the present invention. The spectroscopic microscope 1 includes a first light source LP1 for illumination that irradiates a sample W on a sample table XY from above, and a second light source LP1 that illuminates from below. A light source LP2, a third light source LP3 for irradiating light with a specific wavelength, an eyepiece L8 for visual observation, a CCD camera 2 for photography, a photomultiplier tube 3, and an image processing circuit 20 are provided. A halogen lamp, for example, is used for the first light source LP1 and the second light source LP2. However, the conventional tube type of each of these light sources has a drawback that the optical axis is deviated when the lamp is replaced. Therefore, the spherical type of which the discharge part a is formed at the center position is preferable.

The light from the first light source LP1 is a total reflection mirror A.
The light is reflected by TM1, becomes parallel rays by the condenser lens L6, and illuminates the sample W. The second light source LP2
The light from is dispersed by the diffraction grating G1, and the light of a predetermined wavelength is reflected by the dichroic mirror DM through the lens group L5 and the solenoid shutter RS1, and the objective lens L
After passing through 7, the sample W is irradiated from below. The reflected light from the sample W passes through the objective lens L7 and the dichroic mirror DM and reaches the first solenoid rotary half mirror RS2 arranged on the optical axis. This mirror RS2 is OFF
At the (chain line position), the reflected light is the fixed half mirror S.
Upon reaching the HM, part of the light is a lens group L2, a light amount amplifier (image indensifier) Int1, a filter F.
2 (the action of this filter will be described later) to reach the CCD camera 2.

A total reflection mirror ATM3 is disposed below the fixed half mirror SHM, and the fixed half mirror SH is provided.
The rest of the light reflected by M reaches the total reflection mirror ATM3, is reflected, passes through the lens group L3, and is transmitted through the total reflection mirror ATM.
4, the light is reflected, passes through the light amount amplifier Int2, and is projected onto the photomultiplier tube 3.

2 and 3 show an example of a lens barrel to which the diffraction grating G1 on the second light source LP2 side is attached. In this lens barrel 4, a connecting tube 5 connected to the second light source LP2, a condenser lens 6, and a total reflection mirror 7 are arranged on the same optical axis, and the light reflected by the total reflection mirror 7 irradiates a diffraction grating G1. . The diffraction grating G1 is of a reflection type and includes a grating 8, a stepping motor M1 for rotating the grating 8 and a photo sensor S1. Although only one photosensor S1 is shown in the figure, two photosensors are provided to select and emit two types of excitation wavelength light as described later.

The above-mentioned solenoid shutter RS1 and lens group L5 are provided on the reflection optical axis of this diffraction grating G1 (however, in this lens barrel, the arrangement of the solenoid shutter and the lens is reversed). The solenoid shutter RS1 includes a drum 9, a driving rotary solenoid 10, and a connecting rod 11 that connects the two, and a through hole 12 is formed in the drum 9 in a cross shape. Reference numeral 13 is a UV filter attached to one of the through holes as necessary. This will set the drum 9 to 45
The light from the diffraction grating G1 is turned on and off each time it rotates. In the figure, V1 is a light amount diaphragm, and V11 is a field diaphragm. Note that V12 in FIG. 1 is also a field stop. Further, as the material of each of the above-mentioned lenses and half mirror, it is preferable to use glass which has a small absorption of the light having the used wavelength. For example, synthetic fused silica is preferred for ultraviolet light.

The image measuring circuit 20 is the CCD camera 2
And a density calculation storage circuit 22 connected to the photomultiplier tube 3, and a display circuit for synchronizing the output signals from the video recorder 21 and the density calculation storage circuit 22 and displaying the density together with the image. And 23.

The video recorder 21 stores the output from the CCD camera 2 over time. Further, the density calculation storage circuit 22 converts the light amount (density) of the image from the photomultiplier tube 3 into a numerical value, and similarly stores it over time. The display circuit 23 includes a video signal from the video recorder 21 and a density storage circuit 22.
It receives the concentration signal from and displays it.

Next, prior to the measurement, a reagent for generating excitation light (fluorescence) for the measurement is added to the sample W. This example shows an example of detecting uptake of Ca ions in cells. It should be noted that upon spectroscopic analysis, the fluorescence emitted from the sample is weak, and therefore two types of excitation wavelength light having different characteristics peculiar to the reagent (for example, one is an ion and the other is a dissociation) are irradiated, and the two wavelengths differ. The fluorescence is measured, and the ion concentration is measured by comparing the two. This example is an example of a reagent
Ca at 340 nM wavelength light (hereinafter referred to as first wavelength light)
A reagent that binds to ions emits excitation light of 510 nM, for example, and a reagent that dissociates from Ca ions emits the same excitation light of 510 nM with light having a wavelength of 380 nM (hereinafter referred to as second wavelength light). By comparing the two, the ion concentration change and distribution state are measured. However, the structure of the cell itself cannot be confirmed only with light of both wavelengths, and in order to know the structure of the cell, light of a wavelength at which the reagent and Ca ions do not emit excitation light is simultaneously irradiated. The third light source LP3 is provided for this purpose and projects light of a specific wavelength by the filter F1. As this wavelength, a wavelength suitable for enhancing the contour of the cell by Hoffman interference in cooperation with the filter F2 provided in front of the CCD camera 2, for example, 640.
Light having a wavelength of nM (hereinafter referred to as third wavelength light) is used.

Next, the procedure of measurement by the above-mentioned spectroscopic microscope will be described. First, the first light source LP1 is turned on, and it is converged by the condenser lens L6 to irradiate the sample W on the sample table XY.
The sample image obtained by the objective lens L7 passes through the dichroic mirror DM, is reflected by turning on the half mirror RS2, is enlarged by the lens group L1, and is reflected by the total reflection mirror ATM2 to reach the eyepiece L8 after reflection. As a result, the measurement object (eg, cell) in the sample W is set at a predetermined position.

Next, the measurement is started by irradiating the excitation wavelength light peculiar to the previously administered reagent. First, the light from the second light source LP2 is alternately split into the first wavelength light and the second wavelength light by the diffraction grating G1 at regular intervals, passes through the light quantity diaphragm V1 and the field diaphragm V11, and is converged by the lens L5, and the shutter RS1. Then, the light is reflected by the dichroic mirror DM and further converged by the objective lens L7 to irradiate the sample W. As a result, the fluorescence excited by each of the first wavelength light and the second wavelength light reflected from the sample W is alternately magnified by the objective lens L7, and the half mirror RS2 is turned off.
Approximately 1/2 of the amount of light is reached by the mirror L2.
And expanded and amplified by the light amount amplifier Int1 to C
Enter the CD camera 2.

In this case, with the above-mentioned first-wavelength light, only the Ca ions, which combine with the reagent and emit the excitation light, are reflected in the CCD camera 2, and with the second-wavelength light, the excitation light is emitted from the reagent, which is separated from the Ca ions. . However, for measurement, it is necessary to know at which position in the cell the Ca ions are taken up, and therefore it is necessary to switch the wavelength at which the excitation light is not emitted. Therefore, since the third light source LP3 is provided, the procedure for switching between the first and second light sources and the procedure for irradiation by the third light source are shown in FIG.

In the figure, (A) shows a rising signal for switching between the first wavelength light and the second wavelength light in the analysis grating G1, that is, for switching the wavelength of the analysis grating G1. The figure shows a state in which switching is performed every 5 mS. Further, (B) shows the outline of switching between the first wavelength light and the second wavelength light by this, and shows the ratio between the irradiation for 5 mS and the switching time (shown by hatching in the figure). FIG. 6C shows the irradiation time of each wavelength light. (D) shows the VD signal applied to the CCD camera, and as is well known, the ODD field and E
One frame is formed by the VN field. In addition, since the third wavelength light (640 nM) knows the whole volume of the cell, the third solenoid rotary mirror RS3 is moved to the ON position so as to be constantly irradiated during the measurement, and the first,
The specimen is constantly illuminated during the measurement with the second wavelength light. As a result, the entire volume of cells, the position or distribution state of Ca ions, and the reagent that dissociates with Ca ions are displayed on the CCD camera 2, which is recorded on the video tape recorder 21.

In the spectroscopic analysis, the fluorescence emitted from the sample is weak, and it is difficult to directly judge the strength of the ion concentration from the strength of the light. For this reason, the first wavelength light combined with Ca ions and the two kinds of excitation wavelength light by the second wavelength light by the dissociated reagent are measured by the photomultiplier tube 3 and the change in ion concentration is measured by comparing the two. About half of the light amount passes through the half mirror SHM, reaches the total reflection mirror ATM3, the reflected light is converged by the lens group L3, reflected by the total reflection mirror ATM4, and is reflected by the light amount amplifier Int2. It is amplified and enters the photomultiplier tube 3. The photomultiplier tube 3 alternately detects the amount of excitation light (for example, 510 nM) by each of the first wavelength light and the second wavelength light, and this amount of light is converted into a numerical value by the concentration storage circuit 3 and both wavelengths are detected. The ratio of light (380/340) is calculated and stored.

The outputs from the video recorder 21 and the density storage circuit 22 are applied in synchronization with the display circuit 23 and displayed on the screen. The state is shown in FIG. In the figure, A is a screen, b is a cell membrane, c is its nucleus, d is Ca ion, and e is a concentration display number. In this case, Ca ion causes a difference between the concentration before being absorbed into the cell and the concentration after being absorbed. By using the video recorder 2 and the density storage circuit 3, the Ca
It is possible to know the change of the position of the ion and the change of the absorbed position and the concentration.

[0027]

According to the present invention, light of two wavelengths is radiated to a specimen while being switched at fixed time intervals, the excited light is imaged by a CCD camera, and the amount of light is measured by a photomultiplier tube. Since the ratio of the excitation lights of both wavelengths is measured by the concentration calculation storage circuit, the CC
With the D camera, the entire image can be known and the ion concentration can be known. Further, the image taken by the CCD camera is impressed on the video tape recorder, and at the same time, the ion concentration detected by the photomultiplier is stored in the storage circuit over time, so that the output time of the former video tape recorder is synchronized. Then, the latter stored ion concentration is output and simultaneously displayed on the display circuit, so that the state change of the sample and the accompanying ion concentration change can be observed with time. Further, by arranging a differential interference filter in front of the CCD camera and irradiating with light of a predetermined wavelength, it is possible to emphasize the contour of, for example, a cell of the sample by Hoffman interference. Therefore, by superimposing with the excitation light generated by the light of the two kinds of wavelengths, it is possible to clearly observe, for example, the movement of Ca ions or where the Ca ions are concentrated.

[Brief description of drawings]

FIG. 1 is a schematic explanatory view of a spectroscopic microscope of the present invention.

FIG. 2 is a vertical sectional view of a lens barrel to which a diffraction grating is attached.

FIG. 3 is a right side view of FIG.

FIG. 4 shows a relationship graph of VD signal, switching timing of two-wavelength light, timing of signal application to the CCD camera and photomultiplier tube.

FIG. 5 is a schematic explanatory view of a conventional spectroscopic microscope.

[Explanation of symbols]

 1 spectroscopic microscope, 2 CCD camera, 3 photomultiplier tube 20 image measurement circuit 21 video tape recorder 22 density calculation memory circuit 23 display circuit LP1 light source LP2 light source LP3 light source G1 diffraction grating

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[Procedure amendment]

[Submission date] February 19, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is a schematic explanatory view of a spectroscopic microscope of the present invention.

FIG. 2 is a vertical sectional view of a lens barrel to which a diffraction grating is attached.

FIG. 3 is a right side view of FIG.

FIG. 4 shows a relationship graph of VD signal, switching timing of two-wavelength light, timing of signal application to the CCD camera and photomultiplier tube.

FIG. 5 is an explanatory diagram of a state displayed on the screen.

FIG. 6 is a schematic explanatory view of a conventional spectroscopic microscope.

[Explanation of reference numerals] 1 spectroscopic microscope, 2 CCD camera, 3 photomultiplier tube 20 image measurement circuit 21 video tape recorder 22 concentration calculation memory circuit 23 display circuit LP1 light source LP2 light source LP3 light source G1 diffraction grating

Claims (3)

[Claims] 1. A spectroscopic microscope for measuring the ion concentration from the intensity of excited fluorescence by irradiating a specimen by splitting light from a light source into light of two wavelengths, and dividing light of two wavelengths at fixed time intervals. , The VD signal is input in synchronism with the rising signal for switching, the CCD camera takes an image, and the fluorescence intensity excited by the light of two wavelengths is measured by the photomultiplier tube. A spectroscopic microscope characterized by comprising a concentration calculation storage circuit for calculating and storing the ion concentration from the strength ratio. 2. A display circuit is provided which outputs the ion concentration in synchronization with the output time of the image and reproduces the ion concentration together with the image in the case where the imaged image is replayed after being inserted into a video recorder. The spectroscopic microscope according to claim 1, wherein: 3. The outline of an image is enhanced by irradiating a light having a wavelength different from that of the excited wavelength and placing a differential interference filter which interferes with the light having the wavelength in front of the CCD camera. The spectroscopic microscope described.