JPH0721451B2 - Microscopic absorption distribution measuring device for opaque samples - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring a microscopic absorption distribution of an opaque sample such as a biological sample and an apparatus therefor, and more particularly, to accurately measure the absorption of a microscopic region in a sample. The present invention relates to a microscopic absorption distribution measuring device that removes unnecessary scattered light and improves resolution.

[Conventional technology]

Since the discovery of X-rays, a technique for observing the inside of a living body (human body) without external damage (pessimistic blood or non-invasive measurement method) has been strongly demanded and developed in the field of biology, particularly in medicine. . This technology uses gamma rays and X-rays having the shortest wavelengths when viewed as electromagnetic waves, and radio waves having the longest wavelength. The former is X-ray CT and the latter is NMR-CT (Magneti
c Resonance Imaging, MRI) has been put to practical use.

On the other hand, spectroscopy in the ultraviolet-visible-near-infrared-infrared region, which is widely used in the fields of physics and chemistry, is called "whole body".
There are relatively few attempts to apply it to (vivo). This is because many problems regarding the most basic "quantitativeness" remain unsolved in biometrics using light, particularly those that utilize absorption and luminescence processes. At present, the measurement of the reflection spectrum using a solid-state element and the measurement with a high-sensitivity TV camera are attempted, but this is the reason why the reproducibility and the absolute value obtained are less reliable.

When a scatterer such as a biological tissue is irradiated with light, it can extract straight-ahead light to some extent if it is received at 180 ° facing each other, but at present, its spatial resolution is not so good.

The difference in spatial resolution between X-ray and light cannot be filled up so far. However, the use of light, especially near infrared light, should allow imaging of tissue oxygen concentration from hemoglobin in blood. These are other NMR-CT and X-ray C
It will give different information than T.

With 3-5 cm thick tissue, we can detect the transmitted light. This means that "light-radiography" can be used for diagnosis. Women's breasts have relatively uniform tissue and light is easily transmitted, and the transmitted light can be easily detected (thickness: about 3 cm) due to its shape, and Diaphanography (Lights) has long been used to diagnose breast cancer.
canning) has been used.

Under these circumstances, the present inventor has filed a patent application No. 1-62898.
In Japanese Patent Application No. 1-250034 and Japanese Patent Application No. 2-77690, plane waves mixed in scattered light are separated and taken out.
For observation, it is sufficient to observe only the 0th-order spectrum of the plane wave Franforfer diffraction image (Airy disc) (the portion in the first dark ring of the Airy disc corresponds). It was shown that almost all the scattered components can be removed by doing so. Then, as one of the highly directional optical systems for realizing such an observation, an optical system including two pinholes P ₁ and P ₂ spaced apart from each other as shown in FIG. 16 was proposed. This optical system is 0 through the pinhole P _2.
The next light is detected by the detector 23. Further, as shown in FIG. 17, a highly directional optical system which is composed of a linear elongated hollow glass fiber 35, and an inner wall surface of which is coated with a light absorbing material 35 such as carbon. Proposed. Further, as shown in FIG. 18 to FIG. 25, a high directivity consisting of an objective lens Ob and a pinhole P for passing only the 0th-order diffraction image of Franforfer diffraction by the objective lens Ob arranged in the focal plane thereof. Directional optical system (Fig. 18), a highly directional optical system (Fig. 19) consisting of a gradient index lens GL and a similar pinhole P arranged at the focal plane at one end thereof, instead of the pinhole P. Optical fiber SM
High-directivity optical system (Figs. 20 and 21) in which is arranged, and an objective lens O similar to the objective lens Ob1 on the incident side on the exit side of the pinhole P or the optical fiber SM of these high-directivity optical systems.
Highly directional optical system with b2 (Figs. 22 and 24)), refractive index distribution lens G similar to the incident side refractive index distribution lens GL1
We have proposed a highly directional optical system (Figs. 23 and 25) in which L2 is arranged.

By the way, conventionally, in order to measure the microscopic absorption distribution of a sample with scattering, for example, as shown in FIG. 26, light from a light source that emits light of a wide range of spectral components is transmitted through an interferometer for Fourier spectroscopy and And a reflection type switching mirror to switch to the transmitted light path or the incident light path. In the case of a transmissive type, the lower cassegrain system that acts as a condenser lens,
The illumination light is focused on a small point of the sample on the sample table, and the light transmitted through that point and the light scattered forward at that point are imaged on the aperture by the upper cassegrain system acting as an objective lens. The light passing through the aperture is made incident on the detector, and the absorption characteristic at that point is measured. Then, the transmission microscope absorption distribution of the sample can be measured by scanning the sample table in the XY directions and repeating the same measurement. The switching mirror is switched to the epi-illumination path, the illumination light is focused by the upper cassegrain system and applied to a point on the sample, and the light reflected and scattered backward from that point is imaged on the aperture by the same upper cassegrain system, Similarly, the reflection microscopic absorption distribution of the sample can be measured.

Further, as a method for investigating the absorption spectrum of a minute portion, there is a method shown in FIG. 27 as another conventional microspectroscopic method in which an optical microscope and a spectrophotometer are combined. The light from the light source 1 is converted into monochromatic light by the spectroscope m ₀ , and the diaphragm (pinhole) p is proved. This diaphragm p is used as the light source of the microscope system,
When light is passed through the illumination microscope m ₁ , a reduced image of the diaphragm p is formed on the sample surface s. This is magnified by another microscope m ₂ and guided to the detector d. At this time, if the sample is placed at the position s of the reduced image of the diaphragm, the absorbance of only a local portion of the sample can be measured.

By the way, conventionally, when the absorption spectrum of a sample with scattering is obtained by the same measurement method as that of a sample without scattering, the influence of scattering becomes large and an accurate absorption spectrum cannot be obtained. As a method for measuring the absorption of these opaque samples with scattering, there is known a method of measuring the transmission integrated extinction using an opal glass or an integrating sphere (for example, Kazuo Shibata, "Introduction to Photobiology Series Spectroscopy" 62-82, Sho 51, 6, 20, published by Kyoritsu Shuppan Co., Ltd.)).

[Problems to be Solved by the Invention]

By the way, in the infrared spectroscopic measurement method using the Frier spectrometer described above, also in the visible spectroscopic measurement method using the diffractive folding spectroscope, since there is no measure against opaque samples with scattering, The measurement of these samples has a large error and reliable data cannot be obtained. That is, since unnecessary scattered light is mixed from the surroundings including before and after the measurement point, the accurate absorption characteristics cannot be measured, and the Franforfer diffraction image component other than the 0th order enters the objective lens. Therefore, the resolution is limited. A measurement method developed as an absorption measurement method for an opaque sample and simply combining the measurement method using an opal glass or an integrating sphere and the microspectroscopic measurement method is not practically used because the detection signal light is weak and measurement is difficult. Even if the detection sensitivity is dramatically improved, the opal glass method and the method using the integrating sphere are only approximation methods.
A sample that cannot be approximated to have a large reflected light flux or the same wavelength change of the scattered transmitted light flux and the scattered reflected light flux cannot be used with a large error. As described above, at present, there is no suitable measuring method for the absorption spectrum of a minute portion of an opaque sample accompanied by scattering.

The present invention has been made in view of such a situation,
It is an object of the present invention to provide a microscopic absorption distribution measuring device in which unnecessary scattered light is removed and resolution is improved so that the absorption of a minute region of an opaque sample such as a biological tissue can be accurately measured.

[Means for Solving the Problems]

A first embodiment of the apparatus for measuring a microscopic absorption distribution of an opaque sample of the present invention which achieves the above object divides light from a wavelength-changeable monochromatic light source into two and shifts the frequency of incident light into one optical path. A frequency shift means is provided, a confocal optical system composed of two converging optical systems is arranged in the other optical path, and a relatively scannable sample is arranged at the condensing position of the confocal optical system. The beam combining means is provided for combining the highly directional light emitted from the sample and the light diverged from the measurement point of the sample and converted into parallel light by the confocal optical system and emitting in the same direction. It is characterized in that a detecting means for converting the emitted light into an electric signal and detecting the intensity of only the AC component equal to the shift frequency is provided.

A second device for measuring a microscopic absorption distribution of an opaque sample divides light from a monochromatic light source whose wavelength can be changed into two, and provides an optical path length changing means for changing the optical path length at a predetermined speed in one optical path and the other. A confocal optical system composed of two converging optical systems is arranged in the optical path, a relatively scannable sample is arranged at the condensing position of the confocal optical system, and the directionality of the light emitted from the optical path length changing means is high. A beam combining means for combining the light and the light diverging from the measurement point of the sample and converted into parallel light by the confocal optical system and emitting in the same direction is provided, and the light combined by the beam combining means is converted into an electric signal. Then, a detecting means for detecting the intensity of only the AC component of the frequency according to the optical path length changing speed is provided.

A third opaque sample microscopic absorption distribution measuring device, which is a modification of the first device, is a frequency shift means for dividing light from a wavelength-changeable monochromatic light source into two and shifting the frequency of incident light into one optical path. Is provided, and the first converging optical system that condenses the highly directional light on the other optical path and irradiates it to the minute measurement point of the sample is arranged, and the first converging optical system is relatively arranged to the converging position. A sample that can be scanned is arranged in the second converging optical system, and a second converging optical system that produces the same converging spherical wave as the first converging optical system that irradiates the sample with highly directional light emitted from the frequency shift means is arranged. Beam combining means for synthesizing and emitting in the same direction, and detecting means for detecting the intensity of only the AC component equal to the shift frequency by converting the light combined by the beam combining means into an electric signal. It is what

In any of these devices, the light from the scattering sample is blocked, the intensity of the luminous flux emitted from the frequency shift means or the optical path length changing means is detected as the reference light intensity, and the light synthesized by the beam synthesizing means is electrically converted. The signal intensity from the sample is converted into an AC component equal to the shift frequency or an AC component with a frequency according to the optical path length changing speed, and the transmission integrated extinction is obtained using these reference intensities and signal light intensities. Is desirable.

[Action]

According to the microscopic absorption distribution measuring device for an opaque sample of the present invention,
Concentrate and irradiate a small measuring point of the sample with highly directional light, convert the light diverging from the measuring point into parallel light, or
Since the intensity of the spherical wave is detected using the heterodyne light receiving system and the Michelson light receiving system, it is possible to accurately absorb the small area of the sample with high resolution without picking up scattered light from the measurement point surroundings and other noise light. It is a device that can measure and is suitable for measuring the microscopic absorption distribution of opaque samples such as biological tissues.

〔Example〕

Conventionally, a heterodyne light receiving system is known as a means for detecting coherent light with high sensitivity. As shown in FIG. 1 for example, the light receiving system 3 divides the light having a specific frequency ω ₁ emitted from the laser 1 into two by a beam splitter BS, and inserts a sample S into one straight light. The other reflected light passes through the mirrors M1 and M2, and is combined with the above-described straight traveling light by the half mirror HM, and the combined light is photoelectrically converted by the detector 2. When a frequency shifter AO such as an ultrasonic optical modulator that shifts the frequency to ω ₁ is inserted in the reflected light, a beat signal with an observable frequency, which is the difference between the frequencies ω ₁ and ω ₂ , appears from the detector 2. , The strength of the AC component is proportional to the transmittance of the sample S. Therefore, a weak signal transmitted through the sample S can be detected. By the way, such a heterodyne light receiving system 3 can not only detect the weak signal as described above, but also can reflect the reflected light (the seed is several ω) by the sample S.
Light of _two . Hereinafter, also referred to as reference light. Since the component scattered in the direction different from the direction (1) does not overlap with the reference light on the detection surface of the detector 2, it does not generate a beat signal and is simply detected as a DC component. It has a property as a highly directional detection system that can easily remove components and detect only a light advancing component in the same direction as the reference light.

The Michelson light receiving system is well known as a means for detecting a minute change in refractive index. This light receiving system 4
As shown in FIG. 2, the light emitted from the laser 1 is divided into two by a beam splitter BS, the sample S is inserted into the reflected light that has passed through one of the mirrors M1 and M2, and the transmitted light is used as a straight-forward light described later. Combined by half mirror HM. Beam splitter
The straight-ahead light (hereinafter also referred to as reference light) that has passed through the BS passes through the half mirror HM and strikes the moving mirror M that is moved as shown by the double-headed arrow in the figure, and is reflected in the opposite direction, and the half mirror HM Is combined with the light transmitted through the sample, and the combined light is photoelectrically converted by the detector 2. From the detector 2, a signal on which an interference signal having a frequency corresponding to the speed of the movable mirror M is superimposed is obtained. The intensity of the AC component is proportional to the transmittance of the sample S, and the phase depends on the thickness or the refractive index of the sample S. This Michelson light receiving system 4 is also the heterodyne light receiving system 3 described above.
Similar to, it can detect not only minute changes in the refractive index,
The component scattered by the sample S in a direction different from that of the reference light does not overlap the reference light on the detection surface of the detector 2 and thus does not generate an interference signal and is simply detected as a DC component. It has a property as a highly directional detection system capable of easily removing such a scattered component and detecting only a light component traveling in the same direction as the reference light.

By the way, it can be said that both the heterodyne light receiving system 3 and the Michelson light receiving system 4 detect the intensity of the light transmitted through the sample S or the light scattered by the sample S based on the same principle. This point will be briefly described. The reference light to be combined is V ₂ , and the light transmitted through the sample S or the light scattered by the sample S (hereinafter, also referred to as sample light) is V _1, and they are respectively expressed as follows.

V ₁ = A ₁ exp [−i (ω ₁ t + φ ₁ )], V ₂ = A ₂ exp [−i (ω ₂ t + φ ₂ )] These two light waves V ₁ and V ₂ are observed (detected by superposition). )
Then, the detection signal S becomes as follows.

S = | V ₁ + V ₂ | ² = V ₁ · V ₁ ^* + V ₂ · V ₂ ^* + V ₁ · V ₂ ^* + V ₁ ^* · V ₂ By the way, V ₁ · V ₁ ^* = A ₁ ² , V ₂ · V ₂ ^* = A ₂ ² and V ₁ · V ₂ ^* = A ₁ A ₂ exp [−i (ω ₁ −ω ₂ ) t−i (φ ₁ −
φ ₂ )], V ₁ ^* · V ₂ = A ₁ A ₂ exp [+ i (ω ₁ −ω ₂ ) t + i (φ ₁ −
φ ₂ )] V ₁ · V ₂ ^* + V ₁ ^* · V ₂ = 2A ₁ A ₂ cos [(ω ₁ −ω ₂ ) t + (φ ₁ −
φ ₂ )], S = A ₁ ² + A ₂ ² + 2A ₁ A ₂ cos [(ω ₁ −ω ₂ ) t + (φ ₁ −
φ ₂ )].

By the way, in the heterodyne light receiving system 3, since ω ₂ = ω ₁ −Δω and φ ₁ = φ ₂ can be written, S = A ₁ ² + A ₂ ² + 2A ₁ A ₂ cos Δωt, and the AC component of the detected signal The amplitude A ₁ of the sample light V ₁ can be known from the magnitude of.

Similarly, according to the Michelson light receiving system 4, ω ₁ = ω ₂ , φ
Since it can be written that ₂ = φ ₁ + kt, the detection signal S is S = A ₁ ² + A ₂ ² + 2A ₁ A ₂ coskt, and the heterodyne photodetection system 3
A signal similar to that of is obtained. That is, similarly to the heterodyne light receiving system 3 and the Michelson light receiving system 4, the amplitude A ₁ of the sample light V ₁ can be known from the magnitude of the AC component of the detection signal.

Furthermore, in addition to the above-mentioned heterodyne light receiving system 3 and Michelson light receiving system 4 as a highly directional detection system, the system shown in FIG.
There is a highly directional optical system illustrated in Fig. 25. As a representative of these high directivity optical systems, the objective lens Ob on the incident side
1 and a pinhole P which is arranged on the focal plane of the objective lens Ob1 and passes only the 0th-order diffraction image of Franforfer diffraction by the objective lens Ob, and a similar objective lens which is arranged so that the front focal point coincides with the pinhole P. A highly directional optical system 5 composed of Ob2 is shown in FIG. (The highly directional optical system 5 in the figure is the same as the optical system shown in FIG. 22.). However, in the following description, the high directivity optical system 5 is not limited to that shown in FIG.

By the way, in the high directivity detection systems 3, 4, and 5 as shown in FIGS. 1 to 3, the relationship between the sample S and the parallel light flux is
By changing to the high-resolution detection system 30, 40, 50 having the configuration as shown in FIGS. 4A to 6, a small amount of the sample S corresponding to the 0th-order component of the Franforfer diffraction image component of the lens is changed. It is possible to apply incident light to a point region and collect and detect scattered light only from that point. That is, in the incident direction of the sample S, a condenser lens L1 having a large numerical aperture (NA) is interposed so that the rear focal point matches the measurement point of the sample S,
Further, the objective lens L2 having the same large NA is arranged so that the front focus coincides with the rear focus of the condenser lens L1, and the light from the laser 1 is focused on a minute point of the sample S by the condenser lens L1. The light emitted from that point is collected by the objective lens L2 and converted into parallel light directed in a predetermined direction, and only the light directed in this direction is detected by the principle of the high directional detection system 3, 4, 5 described above. By using the detector 2, it is possible to collect and detect scattered light only from the fine point region corresponding to the 0th-order component of the Franforfer diffraction image component of the lens of the sample S. Therefore, by using the high-resolution detection system 30, 40, 50 as described above, it is possible to avoid mixing of unnecessary scattered light from the surroundings including before and after the measurement point, and the absorption characteristics of the sample with extremely high resolution. Can be measured.

A modification of FIG. 4A is shown in FIG. 4B. In FIG. 4A, light is applied to a minute point of the sample S by the condenser lens L1, and the light emitted from the point is converted into parallel light (substantially plane wave) directed in a predetermined direction by the objective lens L2 to be heterodyne. Although it receives light, in FIG. 4B, the objective lens L2 is arranged on the light flux side of the frequency shifter AO, and the divergent spherical wave from the focal point of the condenser lens L1 and the divergent spherical wave from the focal point of the objective lens L2 coincide with each other. And the detector 2 performs heterodyne detection. This is because a beat component obtained by converting the light into a plane wave by the lens and performing the heterodyne detection is equal to that obtained by performing the heterodyne detection with the spherical wave, and the conditions required for the wavefront matching are equal.

Now, first, the opaque sample that is the target of the spectral absorption measurement in the present invention is not a sample that completely blocks incident light and does not allow it to pass forward, but it does not depend on the sample such as a biological sample. There is almost no light that is directly transmitted without being scattered, but a sample in which light scattered forward due to multiple scattering due to scattering fine particles in the sample emerges from the sample. Of course, a sample in which light that directly transmits exists can also be a measurement target. The second measurement target of the present invention is a sample, such as a powder, which reflects and scatters only the rear portion of the incident light that is substantially completely blocked. The former sample is called a transmission sample, and the latter sample is called a reflection sample.

The opaque sample microscopic absorption distribution measuring device according to the present invention shown in FIG. 7 measures the microscopic absorption distribution of the transmitted sample 20 using the high resolution detection system 30 by the heterodyne light receiving system shown in FIG. 4A. is there. For such measurement, a variable wavelength laser 10 capable of continuously sweeping and emitting monochromatic light in a wide spectrum range is used as a monochromatic light source. The light beam emitted from the laser 10 is divided into two by the beam splitter BS, the straight light is condensed at the measurement point of the transmission sample 20 by the condenser lens L1, and the transmitted scattered light is converted into parallel light by the objective lens L2, and the half light is obtained. It is combined with the reference light by the mirror HM. The reference light reflected by the beam splitter BS is
Before the above synthesis, frequency shifter AO such as ultrasonic optical modulator
The frequency is slightly changed by, and when it is combined with the sample light and photoelectrically converted, a signal including an AC signal having a frequency corresponding to the frequency difference between the reference light and the sample light is output from the detector 2. The magnitude of the AC component of the signal obtained from the detector 2 is
Since it is proportional to the amplitude of the sample light, the AC component of the output of the detector 2 is separated, and the absorption characteristic at the measurement point can be obtained from its magnitude. Since the intensity of the sample light changes according to the absorption characteristic of the measurement point of the sample 20, the absorption distribution on the scanning surface of the sample 20 is measured by scanning the sample 20 with the XY scanning device XY. can do. By measuring the absorption at each measurement point while sweeping the wavelength of the laser 10,
The spectral absorption distribution of the sample 20 can also be measured. By the way, in order to make the measurement resolution smaller than the size of the 0th-order image of the Franforfer diffraction image component of the lenses L1 and L2, a pinhole smaller than the size of the image is located in close proximity to the measurement point (focus of the lens L1). It is also possible to arrange the plate PH to limit the illumination area. In this case, the resolution is improved, but the amount of transmitted light is reduced. Note that the pinhole plate PH can be similarly arranged in the following examples.

Now, FIG. 8 is a modification of the one using the high resolution detection system 30 of FIG. 7 to measure the reflection sample 21 accompanied by scattering. In this case, in order to convert the light beam emitted from the laser 10 into a parallel light beam having an appropriate diameter, the beam converter 11
Is placed in front of the laser 10 and this beam converter 11
Is not always necessary. In this arrangement,
The direction of the half mirror HM in FIG. 7 is changed, the light from the laser 10 is transmitted through the beam splitter BS, and the condenser L and the lens L acting as the objective lens are arranged at the position where the light is transmitted through the half mirror HM, and thereafter, The reflection sample 21 is arranged on the side focal plane, and the reflected and scattered light is combined with the reference light by the half mirror HM. For example, when the scatterer NZ is schematically written so as to exist in front of the sample 21 as shown in the figure, the scattered light by the scatterer NZ becomes the DC component of the detector 2 and the high resolution detection system 30 is set. When used, it is not detected as an AC component by the detector 2.

Next, the apparatus shown in FIG. 9 is applied to the high resolution detection system 40 based on the Michelson light receiving system shown in FIG.
20 is used to measure the microscopic absorption distribution, the light emitted from the variable wavelength laser 10 is converted into a parallel light flux having an appropriate diameter by the beam converter 11, and the light is divided into two by the beam splitter BS.
The condenser lens L1 and the objective lens L2 are arranged confocal in the reflected light passing through one of the mirrors M1 and M2, the transmission sample 20 is inserted at the common focal point position, and the scattered transmission light is used as the reference light and the half mirror. Synthesized by HM. The reference light that has passed through the beam splitter BS hits the movable mirror M that is transmitted through the half mirror HM and is moved as shown by the double-headed arrow in the figure, is reflected in the opposite direction, and is combined with the sample light by the half mirror HM. The combined light is photoelectrically converted by the detector 2. From the detector 2, a signal on which an interference signal having a frequency corresponding to the speed of the movable mirror M is superimposed is obtained. Since the intensity of the AC component is proportional to the intensity of the scattered light of the transmitted sample 20, the absorption of the sample 20 is obtained by determining the magnitude of the AC component at each measurement point while scanning the sample 20 with the XY scanning device XY. The distribution can be measured. The spectral absorption distribution can also be measured by obtaining the absorption distribution while sweeping the wavelength of the variable wavelength laser 10.

FIG. 10 shows a modification of the high-resolution detection system 40 utilizing the Michelson light receiving system so as to obtain the absorption distribution of the reflective sample 21. In this case, a lens L acting as a condenser lens and an objective lens is provided at a position where the beam splitter BS is reflected.
Is arranged, and then the reflection sample 21 is arranged on the focal plane, and the reflected and scattered light is combined with the reference light reflected from the movable mirror M by the beam splitter BS.

By the way, FIGS. 11 and 12 show an application of the high resolution detection system 50 using the highly directional optical system 5 of FIG. 6 to measure the absorption distribution of the transmission sample 20 or the reflection sample 21. is there. FIG. 11 shows an arrangement in which an excitation light cut filter 12 that passes only the fluorescent light generated from the measurement point of the sample 20 is inserted to the rear side of the objective lens L2 and the microscopic fluorescence distribution measurement of the sample 20 is performed. However, in the case of absorption distribution measurement, this excitation light cut filter 12 is removed. In the microscopic absorption distribution measurement of FIG. 11, the light from the laser 10 which is converted from the beam converter 11 into a parallel light beam of an appropriate diameter and emerges is a condensing lens.
The light focused by the L1 at the measurement point of the transmitted sample 20, transmitted through that point, and scattered forward from that point is converted into parallel light by the objective lens L2 arranged so that the front focus matches the measurement point. Then, the high-directivity optical system 5 separates the light from other directions and the detector 2 detects its intensity. Therefore, while scanning the sample 20 with the XY scanning device XY, the intensity of the light from each measurement point is obtained,
20 absorption distributions can be measured. Also in this case, the spectral absorption distribution can also be measured by obtaining the absorption distribution while sweeping the wavelength of the variable wavelength laser 10. However, when the excitation light cut filter 12 is inserted to measure the fluorescence distribution, the wavelength sweep of the laser 10 is not performed, or the laser 10 having a fixed wavelength instead of the variable wavelength is used. In the case of the apparatus for measuring the absorption distribution of the reflection sample 21 in FIG. 12, the condenser lens and the lens L acting as the objective lens are arranged at the position where the half mirror HM is transmitted, and the reflection sample 21 is provided on the rear focal plane. Place the reflection sample
The reflected and scattered light from 21 is converted into parallel light by the lens L, reflected by the half mirror HM in a direction different from the direction of the incident light, and only the light from the measurement point of the reflective sample 21 is reflected by the high directional optical system 5. It is designed to be extracted. Others are the same as in FIG.

By the way, when the wavelength of the laser 10 is swept, the light intensity generally changes. Therefore, when using a highly directional detection system,
A part of the irradiation light flux is taken out, and in FIG. 3, for example, a half mirror is inserted between the laser 10 and the sample S to detect the output laser light intensity. When the heterodyne detection system is used, in FIG. 4A, a light blocking element is inserted behind the sample S, and the output intensity of the laser is monitored by the detector 2. Alternatively, a light intensity monitor detector may be installed behind the half mirror HM in FIG. 4A for detection (not shown). These light intensities are used as reference light intensities, and the light intensities transmitted through the sample S or the light intensities reflected in a specific direction are detected by the highly directional detection system or the heterodyne detection system, and these values are used as signal light intensities. Then, the transmission integrated extinction is obtained to calculate the absorption spectrum of the sample. This is a method already used in the conventional method for obtaining the absorption spectrum of an opaque sample or in the method for obtaining the absorbance of a sample without scattering.

Next, some examples of the apparatus for specifically measuring the microscopic absorption distribution characteristics of the sample in the micro size region will be briefly described. FIG. 13 shows a schematic configuration of an apparatus capable of measuring the transmission absorption distribution characteristic and the reflection absorption distribution characteristic of the sample 20 placed on the sample table 13 capable of XY scanning, and shows a single color from the variable wavelength laser 10. The light is switched by the switching mirror MS to a transmission optical path indicated by a solid line and a reflection optical path indicated by a dotted line. When the solid line optical path is selected, the light passing through the mirrors M3 and M4 is condensed at the measurement point of the sample 20 by the Cassegrain reflection optical system K1 which acts as the condenser lens L1, and the light transmitted through the sample 20 passes through the objective lens L2. It is converted into parallel light by the Cassegrain reflection optical system K2 that operates, passes through the mirror M6, and the objective lens shown in FIG.
It is incident on the highly directional optical system 5 consisting of Ob and a pinhole P arranged at its focal point. Light emitted from other than the measurement point is removed by the highly directional optical system 5, and the intensity of light showing the absorption characteristic of the sample 20 is detected by the detector 2. Therefore, the transmission absorption distribution of the sample 20 can be measured by obtaining the intensity of light from each measurement point while scanning the sample 20 in XY scanning. When the switching mirror MS is switched to the reflected light path of the dotted line,
Monochromatic light from the variable wavelength laser 10 is reflected downward by the half mirror HM via the mirror M5, and the sample is produced by the Cassegrain reflection optical system K2 that acts as a condenser lens and an objective lens.
Focused on 20 measurement points. The light scattered backward from the measurement point is converted into parallel light by the Cassegrain reflection optical system K2, is incident on the high directivity optical system 5 via the mirror M6, and the light emitted from other than the measurement point is removed, and the sample 20 The detector 2 detects the intensity of the light having the reflection absorption characteristic. Sample as well
The reflection absorption distribution of the sample 20 can be measured by obtaining the intensity of light from each measurement point while XY scanning the 20. An excitation light cut filter 12 can be inserted after the cassegrain reflection optical system K2 to perform microscopic fluorescence distribution measurement. In the figure, reference numeral 15 denotes a chopper, which is for modulating the laser light incident on the sample at a predetermined frequency and performing synchronous detection to remove noise. Also, the code
14 is an aperture and 16 is an eyepiece.

The apparatus shown in FIGS. 14 and 15 is an apparatus using the high resolution detection system 30 based on the heterodyne light receiving system shown in FIG. 4A, and FIG.
The apparatus using the high-resolution detection system 40 based on the Michelson light receiving system shown in the figure is merely a vertical modification, and no special explanation is necessary. The drive system shown in FIG.
Reference numeral 17 is for moving the movable mirror M in the optical axis direction.

In the above description, the variable wavelength laser 10 is based on the assumption that it continuously oscillates, but a variable wavelength laser that performs pulse operation may be used. In particular, in the case of a sample whose characteristics change rapidly when continuously irradiated with laser light, it is preferable to use a variable wavelength laser that performs pulse operation. Further, the detector 2 is not particularly described, but any known means can be used. Also, as a processing method of the detection signal, for example, intensity modulation of incident light by a chopper or the like and phase-locked detection of the detection signal can be considered. In this case, since the time constant of infrared light detection is long, it is necessary to reduce the synchronous modulation frequency. Also, photon counting method (visible light), charge storage method (infrared light)
A sync signal integration detection method, a heterodyne beat signal detection method, or the like may be used. Further, as the frequency shifter, not only the one using ultrasonic light diffraction such as an ultrasonic modulator but also the combination of the wave plate and the diffraction grating, and the electro-optic effect of the crystal can be used. Further, not only the Michelson interferometer which moves the reflecting mirror at a constant speed but also the reflecting mirror may be vibrated by a sawtooth wave. In addition, for the heterodyne reception, not only the laser of the monochromatic light source is divided into two light fluxes to generate local light, but also two lasers may be used.

〔The invention's effect〕

In the microscopic absorption distribution measuring device for an opaque sample of the present invention, light having high directivity is condensed and irradiated to a minute measuring point of the sample, and light diverging from the measuring point is converted into parallel light, or Since the intensity of the spherical wave is detected using the heterodyne light receiving system and the Michelson light receiving system, it is possible to accurately absorb the small area of the sample with high resolution without picking up scattered light from the measurement point surroundings and other noise light. It is a device that can measure and is suitable for measuring the microscopic absorption distribution of opaque samples such as biological tissues.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram for explaining the configuration and operation of a heterodyne light receiving system, which is the premise of the present invention, and FIG. 2 is a diagram for explaining the configuration and operation of a similar Michelson light receiving system. FIGS. 3A and 3B are views for showing the constitution of a typical one of the same high directivity optical system, and FIG. 4A uses the heterodyne light receiving system used in the microscopic absorption distribution measuring apparatus for an opaque sample of the present invention. FIG. 4 is a diagram for explaining the structure and operation of the high resolution detection system, FIG. 4B is a diagram for showing a modification of the measuring device of FIG. 4A, and FIG. 5 is a high resolution detection system using a Michelson light receiving system. 6 is a diagram for explaining the configuration and action of the above, FIG. 6 is a diagram for explaining the configuration and action of a high resolution detection system using a highly directional optical system, and FIG. 7 is a diagram for explaining the present invention applied to a transmission sample. The figure which shows the configuration of the embodiment of the microscopic absorption distribution measurement using the heterodyne light receiving system, FIG. 8 is a diagram showing a configuration of an embodiment of measuring the microscopic absorption distribution by modifying the apparatus of FIG. 7 for a reflective sample, and FIG. 9 is a microscopic absorption distribution using the Michelson light receiving system of the present invention applied to a transmissive sample. FIG. 10 is a diagram showing a configuration of an embodiment of measurement, FIG. 10 is a diagram showing a configuration of an embodiment of microscopic absorption distribution measurement in which the apparatus of FIG. 9 is modified for a reflection sample, and FIGS. 11 and 12 show the present invention. The figure which shows the constitution of the example of the microscopic absorption distribution measurement which uses the high directivity optical system, Figure 13-Figure 15 shows some concrete examples of the device which measures the microscopic absorption distribution characteristic of the sample by this invention Figures 16 to 25 show the configuration of the previously proposed highly directional optical system, and Figure 26 shows the configuration of a device for measuring the microscopic absorption distribution using a conventional Fourier spectrometer. Fig. 27 shows a device for measuring the microscopic absorption spectral distribution of a micro sample using a conventional diffraction grating spectrometer. It is a diagram showing a configuration. 1 ... Laser, 2 ... Detector, 3 ... Heterodyne light receiving system, 4 ... Michelson light receiving system, 5 ... High directional optical system, 10 ... Variable wavelength laser, 11 ... Beam converter, 12 …
… Excitation light cut filter, 13 …… Sample stand, 14 …… Aperture, 15 …… Chopper, 16 …… Eyepiece, 17…
… Driving system, 20 …… Transmissive sample, 21 …… Reflective sample, 30,40,
50 …… High resolution detection system, S …… Sample, BS …… Beam splitter, HM …… Half mirror, M1 to M7 …… Mirror, AO
…… Frequency shifter, M …… Movable mirror, Ob, Ob1, Ob2 ……
Objective lens, P ... Pinhole, L1 ... Condensing lens, L2
...... Objective lens, XY ... XY scanning device, PH ... Pinhole plate, NZ ... Scatterer, L ... Lens, MS ... Switching mirror, K1, K2 ... Cassegrain reflection optical system

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-63-274848 (JP, A) JP-A-63-243839 (JP, A)

Claims (4)

[Claims] 1. A frequency shift means for dividing the light from a wavelength-changeable monochromatic light source into two and shifting the frequency of incident light in one optical path, and using two converging optical systems in the other optical path. The confocal optical system is arranged, and the sample which can be relatively scanned is arranged at the condensing position of the confocal optical system. The light having high directivity emitted from the frequency shift means and the confocal point diverged from the measurement point of the sample The beam combining means for combining the light converted into parallel light by the optical system and emitting in the same direction is provided, and the light combined by the beam combining means is converted into an electric signal and the intensity of only the AC component equal to the shift frequency. An apparatus for measuring a microscopic absorption distribution of an opaque sample, which is provided with a detection means for detecting. 2. An optical path length changing means for dividing the light from a wavelength-changeable monochromatic light source into two and changing the optical path length at a predetermined speed in one optical path, and two converging optics in the other optical path. A confocal optical system consisting of a system is arranged, a sample that can be relatively scanned is arranged at the condensing position of the confocal optical system, and the highly directional light emitted from the optical path length changing means and the divergence from the measurement point of the sample A beam combining means for combining the light converted into parallel light by the confocal optical system and emitting in the same direction is provided, and the light combined by the beam combining means is converted into an electric signal and is adjusted according to the optical path length changing speed. An apparatus for measuring a microscopic absorption distribution of an opaque sample, which is provided with a detection means for detecting the intensity of only the AC component of the frequency. 3. A frequency shift means for dividing the light from a wavelength-changeable monochromatic light source into two and shifting the frequency of the incident light in one optical path, and concentrating light with high directivity in the other optical path. Then, a first converging optical system for irradiating a minute measuring point of the sample is arranged, a relatively scannable sample is arranged at the converging position of the first converging optical system, and the direction is emitted from the frequency shift means. The second converging optical system that produces the same converging spherical wave as the first converging optical system that irradiates the sample with highly stable light is provided, and a beam combining means that combines both light beams and emits them in the same direction is provided. An apparatus for measuring a microscopic absorption distribution of an opaque sample, which is provided with a detecting means for converting the light combined by the combining means into an electric signal and detecting the intensity of only an AC component equal to the shift frequency. 4. The light from the scattering sample is blocked, the intensity of the luminous flux emitted from the frequency shift means or the optical path length changing means is detected as the reference light intensity, and the light combined by the beam combining means is converted into an electric signal. It is characterized in that an AC component equal to the shift frequency or an AC component having a frequency corresponding to the optical path length changing speed is converted into the signal intensity from the sample, and the transmission integral extinction degree is obtained using these reference intensity and signal light intensity. The microscopic absorption distribution measuring device for an opaque sample according to any one of claims 1, 2 and 3.