JPH06138118A - Optical pathologic inspection apparatus - Google Patents

Abstract

PURPOSE:To detect the change in structure and composition at the molecule level of a sample under inspection by casting a light having the narrow frequency width matching the optical absorbing band of the sample under inspection, generating hole burning in the sample under inspection, and detecting the optical characteristics of the sample. CONSTITUTION:A sample under inspection 21 is dyed with rhodamine 640. The sample 21 is set in a sample chamber 20 through an opening door 2Oa. The sample is quickly cooled with liquid helium to about 4.2K. The sample 21 can be quickly cooled under the state, wherein grains of water crystal are not contained, by the quick cooling. Mutual action is performed with the molecules constituting the sample 21 by dyeing. The state of the molecules constituting the sample 21 is made to reflect on the characteristic amount of holes. When the pathologic part of the sample 21 is detected, at first, the hole forming light is emitted from a light source part 10, and the hole burning is generated in the dyed sample 21. Then, the sample 21 is scanned with the detecting light, whose amount is weakened in order to prevent the formation of the new hole. The transmitted light is detected 40. The depth, the half-value width and the peak wavelength of the hole are analyzed with an analyzing and controlling part 50. Thus the normal state/abnormal state of the sample are judged.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an optical pathological inspection apparatus for optically inspecting a pathological state of a biological sample such as human tissue or cells.

[0002]

2. Description of the Related Art Conventionally, as a method for optically inspecting a pathological state of a biological sample such as a human tissue / cell, an optical pathological tissue / cell diagnostic method using an optical microscope or a laser scanning microscope is known. . In these conventional methods, the information in the sample can be observed by dyeing the sample mainly with a dye to obtain the morphological information of the sample including the distribution of the dye,
Pathological diagnosis was performed based on this morphological information.

[0003]

However, in the conventional inspection method, since it is based on the morphological information of the sample, it is not possible to obtain information on the structure and dynamics of the test sample at the molecular level, and at the molecular level. Pathological diagnosis was almost impossible. In particular, the conventional method may be insufficient for those that require a very subtle change seen at the early stage of disease onset-structure / composition change at the molecular level to be diagnosed. This is considered to be because the microscopic diversity of biological tissues and cells in the early stages of disease has a great influence on normal and pathological states, but it does not appear as morphological information.

In view of the above points, the present invention has an object to provide an optical pathological examination apparatus capable of detecting structural / compositional changes at the molecular level of a test sample.

[0005]

The optical pathological examination apparatus according to the present invention uses a light beam having a sufficiently narrow frequency width that matches the optical absorption band of the test sample in order to cause hole burning in the test sample. A hole forming light source for irradiating, and a detection means for detecting the optical characteristics of the test sample to detect the characteristics of the holes generated in the test sample were provided.

The detecting means may be one that detects at least one of an absorption spectrum and a photoluminescence spectrum as the optical characteristic.

In order to obtain the characteristics of the hole from the shape of the spectrum, it is possible to use the detecting means having a resolution with a frequency width similar to that of the light emitted by the hole forming light source.

Further, the detecting means detects the zero phonon hole, the side hole and the satellite hole from the absorption spectrum or the photoluminescence spectrum, and performs a very sensitive pathological examination / diagnosis at the molecular level based on the information. can do. Here, the test sample is stained with an appropriate dye,
It has optical absorption at an appropriate frequency.

[0009]

In general, the individual absorption spectra of dye molecules introduced into a heterogeneous solid medium and having a relatively small interaction with the medium are much narrower than the absorption spectra of the entire sample at extremely low temperatures. It In this state, when light with a frequency width sufficiently narrower than the absorption spectrum of each dye molecule is emitted from the hole-forming light source, only the dye molecules that have the same frequency as the emitted light among the dye molecules in the irradiation spot. Absorb light and form holes. When the excited dye molecule has a relatively long lifetime or transitions to a metastable state, the number of ground states of the dye molecule corresponding to the frequency of the irradiation light decreases, so absorption of the sample in the irradiation spot A reduced absorption portion (hole) is formed in a portion of the spectrum corresponding to the frequency of the irradiation light. This phenomenon is called hole burning, and the case where excited dye molecules undergo photochemical metastable transition is called photochemical hole burning (Photochemi
cal hole burning (PHB).

The shape of the holes reflects the state of the solid medium around the dye molecules at the molecular level. For example, it is known that its width corresponds to twice the reciprocal of the optical phase relaxation time (T2). A so-called resonance hole (zero-phonon hole) is formed in the hole at the frequency of the irradiation light.
In addition, there are those that are considered to be derived from the vibration of the solid medium called side holes and those that are derived from the vibration of dye molecules called satellite holes, which give information about the micro state of the solid medium and dye molecules, respectively.

In the optical pathological examination apparatus of the present invention, structural / compositional changes at the molecular level are detected as information of zero phonon holes, side holes, and satellite holes. Therefore, if a pathological examination is performed based on the detection result of this hole, an extremely highly sensitive optical pathological examination at the molecular level becomes possible.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the optical pathological examination apparatus according to the present invention will be described below. FIG. 1 shows a schematic configuration of the optical pathological examination apparatus of this embodiment. As shown in FIG. 1, the apparatus system of this embodiment is roughly classified into four parts surrounded by dotted lines.
Two parts, namely, the light source section 10, the illumination optical system section 30, the detection section 40 for detecting transmitted light, and the data analysis / control section 5.
It consists of zero. The light source unit 10 of this embodiment has the laser light source 1, and has a wavelength of 60 in order to cause hole burning in the test sample 21 dyed with rhodamine 640.
Irradiation with hole forming light having a frequency width of about 10 MHz centered on 0 nm. Further, the light source unit 10 irradiates the wavelength-scanned light over a frequency width of several tens GHz centering on a wavelength of 600 nm in order to detect the shape of a hole generated in the test sample 21. In this example, a dye laser excited by an argon ion laser was used as the laser light source 1. Rhodamine 6G was used as the laser dye. The frequency width of the hole forming light of the laser light source 1 is about 10
The reason why the frequency is set to MHz is to make the band sufficiently narrower than the uniform spread of the absorption band near 600 nm of Rhodamine 640 used as the dye for the test sample 21.

The sample chamber 20 has a hermetically sealed refrigerant tank provided with an opening / closing door portion 20a for loading and unloading the sample 21 to be tested and an opening / closing valve 20b for introducing liquid helium. It can be cooled down to liquid helium temperature (4.2K). The sample chamber 20 is the light source 1
In order to irradiate the test sample 21 with light from 0 and transmit the light, the material is transparent to the irradiated light and the transmitted light.

The illumination optical system section 30 is composed of a microscope. In FIG. 1, the hole forming light and the detection light from the light source unit 10 are incident on the illumination optical system unit 30 to form an appropriate spot, and the cooled test sample 21 in the sample chamber 20 is irradiated with the spot.

The detector 40 detects the transmitted light of the light detected by the test sample 21. The detection unit 40 is composed of a photodetector 11 such as a photomultiplier tube or a photodiode, a combination of an optical chopper 12 and a lock-in amplifier 13. In order to eliminate the influence of intensity fluctuation of the detection light at the time of detecting the hole, the detection unit 40 monitors the hole detection light from the light source unit 10, and includes a beam splitter 14, a photomultiplier tube, a photodiode, and the like. A photo detector 15 is provided. The detection unit 40 has a resolution for detecting the transmitted light of the detection light with a frequency width narrower than the frequency width of the hole.

The data analysis / control section 50 includes a photodetector 15
From the photodetector 11 to obtain a quotient, and normalize the transmitted light intensity.
Further, the absorption spectrum change is calculated from the data of the frequency scanning of the laser light source 1 before and after the irradiation of the hole forming light and the signals of the photodetectors 11 and 15, and the characteristics of the holes are detected as the characteristics of the absorption spectrum. The detected holes are zero phonon holes, side holes, and satellite holes. The depth, the half width, and the peak wavelength of each hole are detected as the feature quantity of the hole. Further, the data analysis / control unit 50 stores the depth of each hole, the half-value width, and the peak wavelength of a normal standard sample measured in advance. Then, the detected hole data of the test sample 21 and the data of the standard sample are compared with each other to analyze whether or not they match each other to determine whether the test sample 21 is normal or abnormal. Output.

Using the optical pathological examination apparatus of this embodiment,
The operation of detecting the pathological part of the test sample 21 will be described. First, the test sample 21 is stained with rhodamine 640. The test sample 21 is set inside the sample chamber 20 through the opening / closing door part 20a, the air inside is replaced with helium gas, and then the test sample 21 is heated to a liquid helium temperature (4.2 K) by liquid helium. , Quench quickly. By rapid cooling, the water in the test sample 21 cannot grow into large crystals, so the test sample 21 can be cooled in a state in which the grains of the water crystals are not included.
When ideally cooled, the water in the test sample 20a is considered to be amorphous. When the water crystal grains are generated, the biological cells of the test sample 21 are destroyed or deteriorated.
In this embodiment, the rapid cooling is performed to prevent the growth of water crystal grains and to accurately detect the pathological site.

Test Sample 2 Stained with Rhodamine 640
When detecting the pathological part No. 1, first, the light source unit 10 irradiates the hole forming light having a frequency width of about 10 MHz with a wavelength of 600 nm as the center to cause hole burning in the stained test sample 21. The molecule of Rhodamine 640 is
Known as a substance that causes hole burning,
In this embodiment, the sample 21 to be tested is dyed to interact with the molecules constituting the sample 21 to be tested. Therefore,
The feature amount of the detected hole reflects the state of the molecules forming the test sample 21.

After irradiating the hole forming light, in order to detect the shape of the hole, the detection light which is wavelength-scanned over a frequency width of about several tens GHz around 600 nm is irradiated. The amount of detection light is about 1/1000 or less of the amount of hole formation light. The light quantity is weakened to prevent the formation of new holes during the wavelength scanning for hole detection.

The detector 40 detects the transmitted light of the detection light from the sample 21 to be tested. As described above, the analysis / control unit 50 detects the depth of the hole, the full width at half maximum, and the peak wavelength from the transmitted light spectrum, and compares it with the data of the standard sample stored in advance so as to detect the sample 21. Judge normal / abnormal and output the result.

With the optical pathological examination apparatus of the present embodiment as described above, the examination was performed using the tumor tissue of human liver tissue stained with Rhodamine 640 as the test sample 21. As a standard sample, a normal tissue of human liver tissue stained with Rhodamine 640 was used, and the hole data obtained in advance was stored in the analysis / control unit. As a result, the two could be clearly distinguished. That is, it was possible to distinguish between normal and abnormal human liver tissue, and a very sensitive pathological examination was possible. Therefore, by performing an examination using the optical pathological examination apparatus of this embodiment,
It is possible to detect an abnormality in a test sample at an early stage of disease, which cannot be detected by a conventional test method, and it is possible to perform early detection and early treatment.

The test sample was not limited to a tissue sample, and could be a cell sample. In addition, very subtle changes such as antigen-antibody reactions that occur in tissues / cells could be detected.

In this example, Rhodamine 640 was used as the dye, but similar results were obtained using Texas Red and its derivatives. Further, it goes without saying that the pathological examination by the apparatus of the present embodiment can be performed by using the in vivo dye of the test sample 21.

As the laser light source 1, a semiconductor laser, a solid-state laser or the like can be used in addition to the dye laser. Further, it is also possible to dispose a spectroscopic means such as a filter or a spectroscope in the light source unit 10 and use a mercury lamp, a xenon lamp or the like as a light source to disperse and use these lights. Especially in the case of hole detection, it is not always necessary to use the same light source in which holes are formed, and in addition to the laser light source 1, light from a halogen lamp, a mercury lamp, or a xenon lamp may be selected and irradiated by a spectroscope or the like. is there.
The same result can be obtained by arranging a spectroscope in the detection unit 40 and irradiating the light from a halogen lamp, a mercury lamp, a xenon lamp, or the like as the hole detection light without selecting it with the spectroscope or the like.

Further, in the above-mentioned embodiment, the frequency scanning range of the detection light is set to several tens GHz, but it is not limited to this. Usually, when only the zero phonon hole is detected, it may be detected in the range of about 100 MHz. Further, when detecting side holes and satellite holes, a range of about several THz may be necessary. Therefore, by disposing the means for changing the frequency scanning range of the detection light in the light source 10, the operability for the user can be further improved.

Further, the sample chamber 20 may be provided with a spatial scanning means so that a laser scanning microscope can be configured together with the irradiation optical system. In this case, the data analysis / control unit 50 also controls the spatial scanning of the laser scanning microscope.
By configuring the laser scanning microscope in this way, it is possible to simultaneously obtain molecular level information based on the hole shape and morphological information data for the test sample 21. If the illumination optical system section 30 is used as an objective optical system and an eyepiece lens system is added to the objective optical system, an optical microscope can be constructed and morphological observation can be performed.

Two-dimensional imaging is also possible by mapping the hole shape data obtained by the inspection apparatus of this embodiment.

Further, in the above-mentioned embodiment, the transmission spectrum of the detection light by the test sample 21 is detected, but the present invention is not limited to this, and the fluorescence or phosphorescence spectrum of the test sample 21 is detected. Similarly, the pathological part of the test sample 21 can be detected. In this case, the position of the photodetector 11 does not need to be arranged on the optical axis of the detection light as shown in FIG. 1, but may be arranged at a position where fluorescence or phosphorescence from the test sample 21 can be detected. The sample chamber 20 is made of a material transparent to the light from the light source 10 and fluorescence or phosphorescence.

Further, in the present embodiment, the illumination optical system section 30 is constituted by a microscope, but it may be constituted simply by a lens system.

[0030]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, an extremely highly sensitive optical pathological examination apparatus capable of obtaining information on a sample to be examined at a molecular level is provided. Therefore, by using the optical pathological examination apparatus of the present invention, it is possible to detect a subtle change in the tissue / cell which cannot be detected by the conventional pathological examination apparatus, and it becomes possible to carry out a very sensitive pathological examination.

[Brief description of drawings]

FIG. 1 is a block diagram showing the configuration of an embodiment of an optical pathological examination apparatus according to the present invention.

[Explanation of symbols]

1 ... Light source, 10 ... Light source part, 11 ... Photodetector, 12 ... Optical chopper, 13 ... Lock-in amplifier, 14 ...
Beam splitter, 15 ... Photodetector, 20 ... Sample chamber,
21 ... Test sample, 30 ... Irradiation optical system section, 40 ... Detection section,
50 ... Data analysis / control unit.

Claims (6)

[Claims] 1. A sample stage for holding a test sample, a hole forming light source for irradiating the test sample with light to cause hole burning, and detecting optical characteristics of the test sample. 2. An optical pathological examination apparatus, comprising: a detection unit that detects the characteristics of the holes generated in the test sample. 2. The optical pathological examination apparatus according to claim 1, wherein the detection means detects at least one of an absorption spectrum and a photoluminescence spectrum as the optical characteristic. 3. The optical system according to claim 1, wherein the detection means includes a detection light source for irradiating the test sample with light and a light receiving means for detecting light from the test sample. Pathological examination device. 4. The optical pathological examination apparatus according to claim 1, wherein the detection means detects at least one of a zero phonon hole, a side hole, and a satellite hole. 5. The optical pathological examination apparatus according to claim 2, wherein the detecting means obtains the characteristic of the hole from the shape of the spectrum. 6. The storage device according to claim 1, which stores the feature amount of the hole of the standard sample detected in advance, the feature amount of the hole of the test sample detected by the detecting device, and the standard sample of the storage device. An optical pathological examination apparatus further comprising: a judgment unit that judges normality / abnormality of the test sample by comparing with a hall feature amount.