JPH09179037A - Method for imaging of specific material by transmission type laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce operation labor, to pick up an image in a short time, and to obtain an image of a specific material excellently without requiring laser light of large output or using a light receiving element which has specially high sensitivity. SOLUTION: This transmission type laser microscope makes a scan by irradiation with the laser spot light, obtained by converging the laser light from a light source 1 through an irradiation-side objective 4 from a material surface side, passes the transmitted light of the laser spot light to the reverse side of the material through a transmission-side objective 6 similar to the irradiation-side objective 4 and reflects it to a concave mirror 8 provided where the transmitted light is imaged by the lens so that the light travels backward, and photodetects the backward traveling light by the light receiving element. Then the light source 1 emits a 1st laser light with wavelength in an absorption band of spectral transmission characteristics of the specific material to be measured and a 2nd laser light with wavelength which is outside the said absorption band and close to that of the 1st laser light at the same time, and the backward traveling lights of both the laser lights are photodetected by different light receiving elements 11 and 12 to pick up an image of the specific material by making use of differences in variation between both the laser lights.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a specific substance imaging method for imaging a specific substance in a material by using a transmission laser microscope which images a transmitted light image from the material.

[0002]

2. Description of the Related Art Conventionally, when a specific substance such as calcium present in a material is imaged by a laser microscope, a fluorescent laser microscope is used instead of a transmission type.

In this conventional imaging method, a material is dyed with a fluorescent dye (reagent) that acts only on a specific substance to be imaged. This dye emits fluorescence with excitation light of a certain wavelength, but when it interacts with a specific substance, it emits fluorescence with excitation light of a different wavelength. After dyeing the material, the material is scanned by using laser light of a wavelength that emits fluorescence when it interacts with a specific substance as excitation light, the location of the fluorescence emitting portion is made in pseudo color, and the wavelength is switched to a wavelength unrelated to the fluorescence and scanning is performed again. Then, the pseudo-color image is superimposed on the obtained black and white (grayscale) image so that the position, shape, distribution and the like are displayed on the display.

[0004]

In such a conventional method, there is a problem that it is necessary to dye the material with a fluorescent dye, which requires labor and time.

Further, in the case of a fluorescence type laser microscope, the emitted fluorescence has only a little less than 10% of the excitation light intensity. Therefore, it is necessary to increase the output of the laser light and the light receiving element is also one having high sensitivity. It is necessary and expensive.

Further, excitation light having a wavelength for emitting fluorescence,
There is a problem in that the scanning is required twice with a laser beam having an irrelevant wavelength, a system for synthesizing two obtained images is also required, and the system is complicated.

The present invention has been made for the purpose of solving these various problems.

[0008]

The features of the method of the present invention for solving the above-mentioned conventional problems and achieving the intended object are as follows. Firstly, laser light from a light source is passed through an irradiation side objective lens. The condensed laser spot light is irradiated from the front side of the material to be scanned, and the transmitted light of the laser spot light to the back side of the material is passed through a transmission side objective lens equivalent to the irradiation side objective lens, and the lens is passed through the lens. Using a transmission laser microscope that reflects the light on a concave mirror provided at the position where the image was formed and makes it go backwards, and makes the retrograde light be received by a light receiving element. A first laser beam having a wavelength in the absorption band in the transmittance characteristic and a second laser beam having a wavelength outside the absorption band and close to the first laser beam are simultaneously emitted, and the two laser beams are reversed. Was received in separate receiving element, for imaging a specific substance by the difference in the change of both the laser beam that,
Secondly, the change of the retrograde light of the first laser light received by each of the light receiving elements and the part having no difference in the change of the retrograde light of the second laser light are different from each other, For example, displaying a part having no difference in black and white and a part having difference in color and displaying a specific substance part in a similar color in a black and white image. Thirdly, an indicator for a specific substance having a wavelength in the absorption band is displayed. The wavelength obtained by the action is used and the material previously treated with the indicator is used.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Next, an embodiment of the present invention will be described with reference to the drawings.

FIG. 1 shows an optical path system of an apparatus for carrying out the present invention. In the figure, 1 is λ ₁ , λ having different wavelengths.
A light source that simultaneously emits two types of laser light of _two wavelengths coaxially, and 2 is a polarization beam splitter that transmits the irradiation light a from the light source 1 and reflects the reflected light b that travels in the opposite direction of the optical axis. Is a scanning system for scanning the irradiation light from the light source in the X and Y directions, 4 is an irradiation side objective lens, 5 is a material supported by a data base, and 6 is a transmission side objective lens equivalent to the irradiation side objective lens. , 7 is a quarter-wave plate, and 8 is a concave mirror.

Irradiation light a emitted from the light source 1 passes through the polarization beam splitter 2 and reaches an X, Y scanning system 3, where it is operated in the X and Y scanning directions. Next, the laser light is focused by the irradiation side objective lens 4, and an image is formed on the material 5, and the material is scanned by the spot light.
The image forming position of the material 5 in the thickness direction can be adjusted by operating the material table. The laser light transmitted through the sample table 5 passes through the transmission-side objective lens 6, where it is focused again to form an image on the surface of the concave mirror 8 and is reflected there.

The reflected light (indicated by a dotted line in the figure) travels backward along the same path as that of the illuminating light (indicated by a solid line in the figure), and forms an image at the same position as the image forming position of the illuminating light from the back side of the material, and the material 5 It passes through again and reaches the polarization beam splitter 2.

In the polarization beam splitter 2, only the reflected light that has passed through the quarter-wave plate 7 twice is reflected and bent in the perpendicular direction. A dichroic mirror 9 and a mirror 10 are placed on the reflection optical axis from the polarization beam splitter 2, the laser light of λ ₁ wavelength is reflected by the dichroic mirror 9 and bent in a right angle direction, and the λ transmitted through the dichroic mirror 9 is transmitted. The laser light of _two wavelengths is reflected by the mirror 10 and bent in a right angle direction.
Two. Each of the light receiving elements 11 and 12 is adapted to photoelectrically convert and send an electric signal to the memory. In the figure, 13 is an adjuster (amplifier), and 14 is a data creation unit (CPU).

Next, an image pickup method using the above-mentioned apparatus and using raw material which has not been subjected to drug treatment will be described.

As the material, raw materials which are not subjected to dyeing work or indicator treatment are used. For the two types of laser light emitted from the light source 1, λ ₁ is selected from the material in the absorption band of the spectral transmittance characteristic of the specific substance to be imaged, and λ ₂ is outside the absorption band. Select a wavelength close to λ ₁ .

The absorption band of the spectral transmittance characteristic is peculiar to the substance, and as shown in FIG. 2, when light is transmitted by changing the wavelength of the substance, there is a portion where the transmittance decreases in a certain band. The wavelength band at that time is the absorption band c, and the wavelength band within this range is λ ₁ . Although there are multiple absorption bands for each substance, use a part that is not confused with other substances that are likely to be included in the material.

For example, when the specific substance is potassium, λ ₁ is 583 mμ, λ ₂ is 633 mμ, and when it is sodium, λ ₁ is 589 mμ and λ ₂ is 633 mμ.

In this way, when the material is scanned with both λ ₁ and λ ₂ laser beams, the transmittance curve for one scanning line is as shown in FIG. That is, the transmittance of λ ₁ is reduced in the portion where the specific substance is present, and the degree of reduction is changed according to the density and thickness of the specific substance. On the other hand, the transmittance of λ ₂ changes depending on the factors of the whole material, and λ ₁ becomes substantially parallel to λ _{2 at the} position where no specific substance exists.

The transmittance of λ ₁ and λ ₂ at each position is T
(Λ ₁ , X), T (λ ₂ , X), and the difference ΔT =
T (λ ₂ , X) −T (λ ₁ , X), and adjuster 13 (variable amplifier) so that ΔT = 0 in a portion without a specific substance
Adjust according to. Therefore, it is shown that the specific substance exists at the position of ΔT> 0, and ΔT is determined by its concentration, thickness, etc.
Changes. On the other hand, at the position of ΔT = 0, λ ₁ and λ ₂
Both show the same change, and change depending on the concentration and thickness of the entire material.

The electric signal obtained by the change of the laser light of λ ₁ and λ ₂ thus obtained is used as a data preparation unit 14 (CP
Send to U). In the data preparation section, the transmittance T by λ ₂
The intensity (black and white) corresponding to (λ ₂ , X) is used as the data at that position, but at a position where ΔT> 0, a pseudo color (for example, red) having a darkness proportional to ΔT is used as the data instead. For example, the black and white bright and dark lines are used as the background for each scanning line,
It becomes pseudo color at the specific substance position. This is shown in Table 1 by applying a temporary real number to the graph of FIG. In one scanning plane in which a predetermined number of scanning lines are arranged, a pseudo color specific substance image is obtained in the background of the black and white image.

Next, the case of using the material treated with the indicator as the data will be explained. In this case, it was found that the light absorption band of the medicine is changed by the action of the specific substance on the medicine. Existing drug is used as an indicator, for example, “PDA developed by the US fluorescent research center, which is provided as an indicator of calcium.
A "(PURPRATE-1, 1'-DIACETI
C ACID TRIPOTASSIUM SALT) (ORGANIC PREPARATIONS A
ND PROCEDURES INT. 21 (4) 4
93-500 (1989)) has a normal absorption band wavelength of 4
It is 86 mμ, and when this acts with calcium (combines with calcium ion), the absorption band changes to 524 mμ.

Then, by using the data on which "PDAA" is actuated and using the laser light having the wavelength of 524 mμ as λ _{1 and} the laser light having the wavelength of 556 mμ as λ ₂ , the same scanning as described above is performed. , Based on T (λ ₁ , X) and T (λ ₂ , X) as described above, T
An image can be obtained using (λ ₂ , X) and ΔT as data.

[0023]

As described above, in the present invention, the transmission laser microscope is used, and the light receiving element senses the change of the laser light that has transmitted the material twice in the reciprocating process of the irradiation side and the transmission side. Therefore, the rate of change is large, and therefore, a good image can be obtained without using a light source with low output and a light receiving element with high sensitivity.

Laser light having a wavelength in the absorption band in the spectral transmittance characteristic of the specific substance or absorption band when the specific substance of the indicator acts in one scan, and laser light of other wavelengths other than this , And the signal for the image is obtained by these changes in the absorptance, the background and the specific substance can be simultaneously displayed by one scanning.

Further, when the indicator is not used, the work such as dyeing is not necessary, so that the work is unnecessary, the labor and time are saved, and the raw material can be viewed immediately as it is.

[Brief description of the drawings]

FIG. 1 is a diagram showing an embodiment of the device according to the invention.

FIG. 2 is a grab showing the spectral transmission characteristics of a substance.

FIG. 3 is a graph showing the light transmittance of one scanning line in an embodiment according to the method of the present invention.

[Explanation of symbols]

 a irradiation light b reflected light c absorption band 1 light source 2 beam splitter 3 scanning system 4 irradiation side objective lens 5 material 6 transmission side objective lens 7 wavelength plate 8 concave mirror 9 dichroic mirror 10 mirror 11, 12 light receiving element 13 adjuster 14 data creation Department

Claims (3)

[Claims] 1. A laser spot light obtained by condensing laser light from a light source through an irradiation-side objective lens is irradiated from the front surface side of the material to be scanned, and the transmitted light of the laser spot light to the back side of the material is A transmission laser microscope that passes through a transmission-side objective lens equivalent to the irradiation-side objective lens, reflects the light on a concave mirror provided at the position where the image is formed by the lens to make it go backward, and causes the light-receiving element to receive the backward light. From the light source, the first laser light having a wavelength in the absorption band in the spectral transmittance characteristic of the specific substance to be measured, and the first laser light having a wavelength outside the absorption band and close to the first laser light are used. The two laser beams are emitted at the same time, the retrograde light of the two laser beams is received by different light receiving elements, and a specific substance is imaged by the difference in the changes of the two laser beams. Imaging method for specific substances with a laser microscope. 2. A change in the retrograde light of the first laser light received by each light receiving element and a part in which there is no difference in the change in the retrograde light of the second laser light are different in color. The method for imaging a specific substance using the transmission laser microscope according to claim 1, wherein the image is displayed. 3. The method according to claim 1 or 2, wherein a wavelength in the absorption band is set to a wavelength obtained by causing an indicator to act on a specific substance, and a material previously treated with the indicator is used.
The method for imaging a specific substance using the transmission laser microscope according to [4].