JPH0427844A - Method and apparatus for measuring microscopic absorption distribution of opaque sample - Google Patents

Abstract

PURPOSE:To measure the absorption of a sample with high resolving power without picking up the scattering beam and noise from the circumference of a measuring point by a method wherein beam having high directionality is condensed and applied to the minute measuring point of the sample and the diffused beam is converted to parallel beam to be detected by a highly directional detection system. CONSTITUTION:The beam from a laser 1 is applied to the minute point of a sample S by a condensing lens L1 and the beam emitted from said point is condensed by an objective lens L2 to be converted to parallel beam in a predetermined direction and, by detecting only the beam turning toward a highly directional detection system by a detector 2, the scattering beam only from the minute point region corresponding to the zero order component of the diffraction image component of the lens of the sample S can be condensed to be detected. Therefore, when a resolving power detection system 30 is used, the mixing of the unnecessary scattering beam from the circumference of the measuring point can be avoided and the absorption characteristics of the sample can be measured with extremely high resolving power.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method and apparatus for measuring N minute absorption distribution of an opaque sample such as a biological sample, and in particular, to a method and apparatus for measuring the N minute absorption distribution of an opaque sample such as a biological sample, and in particular to a method that can accurately measure the absorption of a minute region of the sample. The present invention relates to a method for measuring a microscopic absorption distribution, which removes unnecessary scattered light and improves resolution, and an apparatus therefor.

[Conventional technology]

Since the discovery of X-rays, technology for observing the inside of living organisms (human bodies) from the outside without causing damage (non-invasive or non-invasive measurement methods) has been strongly sought after and developed in the field of biology, especially medicine. Ta. This technology uses gamma rays and X-rays, which have the shortest wavelengths of electromagnetic waves, and radio waves, which have the longest wavelengths. The former is an X-ray CT, the latter is an NMR-CT
(Magnetic Re5onance rma
ginglMRr).

On the other hand, spectroscopy in the ultraviolet-visible, near-infrared, and infrared regions, which is widely used in the fields of physics and chemistry, can be applied to the entire living body (i.e.
There have been relatively few attempts to apply it to n vivo. This is because in biological measurements using light, especially those that utilize absorption and luminescence processes, many problems remain unsolved regarding basic "quantitativeness." At present, attempts are being made to measure reflection spectra using solid-state devices, high-sensitivity TV cameras, etc., but this is the reason why the reproducibility and reliability of the obtained absolute values is low.

When light is irradiated onto a scattering object such as biological tissue, it is possible to extract a certain amount of straight light if the light is received 180 degrees facing the object, but at present the spatial resolution is not very good.

The difference in spatial resolution between X-rays and light cannot currently be bridged. However, using light, particularly near-infrared light, it should be possible to image tissue oxygen levels from hemoglobin in the blood. These are other NMR-CT and
It will give different information than line CT.

If the tissue is 3 to 5 cm thick, we can detect the light that passes through it. This means that "optical radiography" can be used for diagnosis. The tissue of the female breast is relatively uniform, allowing light to easily pass through it, and its shape makes it easy to detect transmitted light (approximately 2 to 3 cm thick).
It has been used under the name aphy (Lightscanning).

Under these circumstances, the present inventor filed Japanese Patent Application No. 1-628.
No. 98, Japanese Patent Application No. 1-250034, Japanese Patent Application No. 2-776
No. 90, etc., in order to separate and extract the plane wave mixed in the scattered light and observe it, the 00th order spectrum of the Framphoffer diffraction image (Airy disk) of the plane wave (inside the first dark ring of the Airy disk) is used. It is sufficient to observe only the corresponding parts), and it was shown that by doing this, it is possible to almost eliminate the scattered components.

One of the highly directional optical systems that realizes this type of observation is
As one example, we proposed an optical system consisting of two pinholes P l and P x separated from each other as shown in Fig. 16. This optical system passes the zero-order light through the pinhole P to the detector 23.
It is detected by Additionally, as shown in Figure 17,
It is made of straight, elongated, hollow glass fiber 35,
The inner wall surface has a light absorbing material, for example an absorbing material 3 such as carbon.
We proposed a highly directional optical system coated with 5. Furthermore, as shown in FIGS. 18 to 25, an objective lens Ob
A highly directional optical system (Fig. 18) consisting of a pinhole P that allows only the zero-order diffraction image of Franphofer diffraction by an objective lens ob placed on its focal plane to pass through, a gradient index lens GL, and one end thereof. A highly directional optical system (Fig. 19) consisting of a similar pinhole P placed in the focal plane of the pinhole P, and a highly directional optical system (Fig. 21), a highly directional optical system (second
Figure 2, Figure 24)) Gradient index lens GLI on the entrance side
We have proposed a highly directional optical system (Figs. 23 and 25) in which a gradient index lens GL2 similar to the above is arranged.

By the way, conventionally, in order to measure the microscopic absorption distribution of a sample that involves scattering, for example, as shown in Figure 26, light from a light source that emits light with a wide range of spectral components is transmitted through an interferometer for Fourier spectroscopy. The optical path is switched to a transmitted optical path or an incident optical path using a switching mirror between a type and a reflective type. In the case of a transmission type, the lower Cassegrain system, which acts as a condenser lens, focuses the illumination light onto a minute point on the sample on the sample stage, and collects the light that passes through that point and the light that is forward scattered at that point. is imaged onto an aperture by an upper Cassegrain system that acts as an objective lens, and the light that passes through the aperture is incident on a detector to measure the absorption characteristics at that point. Then, by scanning the sample stage in the X-Y direction and repeating similar measurements, the transmission microscopic absorption distribution of the sample can be measured.

The switching mirror is switched to the incident light path, and the upper Cassegrain system focuses the illumination light to a point on the sample, and the light reflected and scattered behind that point is imaged onto 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 examining the absorption spectrum of a minute portion, there is another conventional method of microspectroscopy that combines an optical microscope and a spectrophotometer, as shown in FIG. 27. Light from a light source l is made into monochromatic light by a spectroscope mo, and a diaphragm (pinhole) p is illuminated.

When this aperture p is used as the light source of the microscope system and light is passed through the illumination microscope m+, a reduced image of the aperture p is formed on the sample surface S. This is magnified by another microscope m2 and guided to the detector d. At this time, if the sample is placed at the position S of the reduced image of the aperture, the absorbance of only a local part of the sample can be measured.

By the way, conventionally, when the absorption spectrum of a sample with scattering is determined 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 an absorption measurement method for opaque samples that involves scattering, there is a known measurement method that measures the transmission integrated attenuation using an opal glass or integrating sphere (for example, Kazuo Shibata, "Introduction to Spectroscopic Measurement Series", Vol. pp. 62-82, 1985, 16.20
, Kyoritsu Shuppan ■) Published)).

[Problems to be Solved by the Invention] By the way, both the above-mentioned microspectrometry method in the infrared region using a Frie spectrometer and the microspectrometry method in the visible region using a diffraction refractometer spectrometer, both involve the use of opaque samples that involve scattering. Because no measures have been taken to prevent this, measurements of these samples have large errors, making it impossible to obtain reliable data. In other words, unnecessary scattered light from the surrounding area including before and after the measurement point mixes in, making it impossible to measure t1 accurate absorption characteristics, and in addition, non-zero-order Franphofer diffraction image components enter the objective lens. Therefore, resolution is limited. Measurement methods that simply combine measurement methods using opal glasses or integrating spheres, which were developed to measure absorption of opaque samples, with microspectrometry methods are not put into practical use because the detection signal light is weak and measurements are difficult. In addition, even if the detection sensitivity is dramatically improved, the opal glass method and the method using an integrating sphere are only approximate methods, so the reflected light flux may increase, and the wavelength of the scattered transmitted light flux may change and the wavelength of the scattered reflected light flux may change. Samples whose changes cannot be approximated as being the same have a large error and cannot be used. As described above, there is currently no suitable method for measuring absorption spectra in minute portions of opaque samples that involve scattering.

The present invention was made in view of this situation, and
The purpose is to provide a microscopic absorption distribution measurement method that removes unnecessary scattered light and improves resolution so that the absorption of minute regions of opaque samples such as biological tissue can be accurately measured, and an apparatus therefor. .

[Means to solve the problem]

The method for measuring the microscopic absorption distribution of an opaque sample according to the present invention, which achieves the above object, focuses and irradiates highly directional light onto a minute measurement point on the sample, and converts the light diverging from the measurement point into parallel light. Then, by detecting the intensity of only the parallel light using a highly directional detection system and repeating the detection operation while scanning the sample relative to the focused light, absorption separation of the sample is performed. This method is characterized by measuring a specific method.

In this case, if you limit the illumination area by placing a pinhole that is smaller than the zero-order image of the Franphofer diffraction image component of the condensed light very close to the measurement point, you can obtain a higher-resolution microscopic absorption distribution. Measurement becomes possible.

Furthermore, by using variable wavelength light as highly directional light, microspectral absorption distribution measurement becomes possible.

Further, by inserting an excitation light cut filter that blocks light at the wavelength of the irradiation light in the optical path from the measurement point to the detector, it becomes possible to measure the microscopic fluorescence distribution of the sample.

In addition, a part of the irradiation light beam irradiating the sample is extracted and the intensity of the light beam is detected as a reference light intensity, and the light intensity detected using a highly directional detection system is used as the signal light intensity from the sample. It is also possible to ask for

In addition, the first lid for microscopic absorption distribution measurement of opaque samples of the present invention, which implements such a method, divides light from a monochromatic light source whose wavelength can be changed into two, and directs the incident light into one optical path. A confocal optical system consisting of two converging optical systems is arranged in the other optical path, and a sample that can be scanned relatively is arranged at the focusing position of the confocal optical system. A beam combining means is provided to combine the highly directional light emitted from the frequency shifting means and the light diverged from the measurement point of the sample and converted into parallel light by the confocal optical system and emit the same in the same direction. The present invention is characterized in that it includes a detection means that converts the light synthesized by the synthesis means into an electrical signal and detects the intensity of only the alternating current component equal to the shift frequency.

The second microscopic absorption distribution measuring device for opaque samples divides the light from the wavelength-changeable monochromatic light source into two parts, is provided with an optical path length changing means for changing the optical path length at a predetermined speed in one optical path, and is provided with an optical path length changing means for changing the optical path length at a predetermined speed in one optical path, A confocal optical system consisting of two converging optical systems is arranged in the optical path, a sample that can be scanned relatively is arranged at the converging position of the confocal optical system, and a highly directional light beam is emitted from the optical path length changing means. A beam combining means is provided to combine the light and the light diverged from the measurement point of the sample and converted into parallel light by the confocal optical system and emit it in the same direction, and the beam combining means converts the combined light into an electrical signal. The present invention is characterized in that a detection means is provided for detecting the intensity of only the alternating current component of a frequency corresponding to the speed of changing the optical path length.

Furthermore, the third apparatus for measuring the microscopic absorption distribution of an opaque sample according to the present invention has a confocal optical system consisting of two converging optical systems disposed in the optical path of the light emitted from a wavelength-changeable monochromatic light source. A highly directional optical system that places a sample that can be scanned relative to the focusing position of the system and extracts only the light in the traveling direction of the light that diverges from the measurement point of the sample and is converted into parallel light by the confocal optical system. The device is characterized in that it is provided with a detection means for detecting the intensity of the light extracted by the highly directional optical system.

In the case of the third device, two optical path switching means are provided in the optical path of the light emitted from the wavelength-changeable monochromatic light source, and the one optical path and the other optical path are configured to be coaxial and the traveling direction of the light is opposite. , placing the confocal optical system in a coaxial optical path;
By providing a light extraction means for extracting the light transmitted through the sample or the light reflected and scattered by the sample on one side of the forward-bending confocal optical system, and arranging the highly directional optical system behind the light extraction means, One device can measure the absorption distribution characteristics of both transmitted light and reflected scattered light of a sample.

A fourth opaque sample microscopic absorption distribution measuring device, which is a modification of the first device, has a frequency shifting means that divides light from a wavelength-changeable monochromatic light source into two and shifts the frequency of the incident light into one optical path. A first converging optical system that condenses highly directional light and irradiates it onto a minute measurement point on the sample is placed in the other optical path, and a A second converging optical system that produces the same convergent spherical wave as the first convergent optical system that irradiates the sample with highly directional light emitted from the frequency shift means is disposed, and both light beams A beam combining means is provided to combine the lights and emit them in the same direction, and a detection means is provided to convert the light combined by the beam combining means into an electrical signal and detect the intensity of only the alternating current component equal to the shift frequency. That is.

In both of these devices, the light intensity transmitted through the sample or the light intensity reflected in a specific direction is used as the signal light intensity, and all or a part of the irradiated light beam is taken out and used as the reference light intensity to determine the transmitted integral attenuation degree. You can do it like this. When using a highly directional detection system, a part of the irradiated light beam is taken out and used as a reference light intensity, and the transmitted integral attenuation degree is determined using the light intensity detected using the highly directional detection system as a signal intensity.

When using a heterodyne detection system or a Michelson detection system, the light from the scattering sample is blocked and only the highly directional light emitted from the frequency shift means or optical path length change means is detected and used as the reference light intensity, and the heterodyne The transmitted integral attenuation degree is determined by using the beat component of the detection system or the Michelson detection system as the signal light intensity.

[Effect]

According to the method and apparatus for measuring the microscopic absorption distribution of an opaque sample of the present invention, highly directional light is focused and irradiated onto a minute measurement point on the sample, and the light diverging from the measurement point is converted into parallel light. ,
Alternatively, by detecting the intensity of the spherical wave as it is using a highly directional detection system such as a heterodyne light receiving system, Michelson light receiving system, or highly directional optical system, it is possible to detect scattered light and other noise light from around the measurement point. This method and apparatus can accurately measure the absorption in a microscopic region of tJ=) with high resolution without picking up the rays, and are 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. This light receiving system 3, as briefly shown in FIG.
The sample S is inserted into one of the straight beams, and the reflected light of the other passes through mirrors M1 and M2 to form the straight beam and the half mirror H.
The combined light is photoelectrically converted by the detector 2. When a frequency shifter AO such as an ultrasonic optical modulator is inserted into the reflected light to shift the frequency to ω2, a beat signal with an observable frequency difference between frequencies ω1 and ω3 appears from the detector 2, and the alternating current The strength of the 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 above-mentioned weak signals, but also detect the above-mentioned reflected light (light with frequency ω2; hereinafter also referred to as reference light) by the sample S.

) does not overlap with the reference light on the detection surface of the detector 2, so it does not generate a beat signal and is simply detected as a DC component.
It has the property of being a highly directional detection system that can easily remove such scattered components and detect only light components traveling in the same direction as the reference light.

In addition, a Michelson light receiving system is well known as a means for detecting minute changes in refractive index, etc. This light receiving system 4 is
As shown in Fig. 2, the light emitted from the laser 1 is split into two by a beam splitter BS, a sample S is inserted into the reflected light that has passed through one mirror ML M2, and the transmitted light is divided into straight light and the straight light, which will be described later. Synthesize by half mirror HM. The straight light that has passed through the beam splitter BS (hereinafter also referred to as reference light).

) passes through the half mirror HM and hits the movable mirror M, which is moved as shown by the double arrow mark, is reflected in the opposite direction and is combined with the light that has passed through the sample by the half mirror HM, and the combined light is The detector 2 performs photoelectric conversion. The detector 2 obtains a signal on which an interference signal having a frequency corresponding to the speed of the movable mirror M is superimposed. The strength of the AC component is proportional to the transmittance of the sample S, and the phase depends on the thickness or refractive index of the sample S. Like the heterodyne light receiving system 3 described above, this Michelson light receiving system 4 is also capable of detecting minute changes in refractive index, etc., and the components scattered by the sample S in a direction different from the reference light are detected by the detector 2. Since it does not overlap with the reference light on the surface, it does not generate an interference signal and is simply detected as a DC component, so such scattered components can be easily removed and the light component traveling in the same direction as the reference light can be detected. It has the property of being a highly directional detection system that can detect only

By the way, it can be said that the heterogain light receiving system 3 and the Michelson light receiving system 4 detect the intensity of light transmitted through the sample S or light scattered by the sample S based on the same principle. This point will be briefly explained. The reference light to be synthesized is V.

, the light transmitted through the sample S or the light scattered by the sample S (hereinafter also referred to as sample light) is defined as VI, and each is expressed as follows.

V + = A + exp [-+ (rn +
t + φ 1)], V 2 = A 2 eXP
[-i ((11at+φ2)] These two light waves V
When 1 and 2 are observed (detected) in a superimposed manner, the detection signal S is as follows.

S= V+ +V2 =V+ ・V+ ” +V2 ・V
2+V, ・V2 ” +v,” ・V2 By the way, V, ・V, ”=A, ,V, ・V2”=A.

and V+ 'V2'' = A+ A28XIll[-1
((ll+-a+2)j-i(φ1-φ,)ko, v 1 umbrella ・V2 =A+ Aa eXp
[+1(ω t-QJa)t+i(φ1-φ2)] V+ ・V2 ” +V+ ” ・Vz= 2
A + A 2cos [(ω1-al z) as 10(
φ 1−φ, )], so S=A+ 2+A2 12 A + A zcos[(a+I w 2)
It becomes t+(φdoφ2)ko.

By the way, in the heterodyne light receiving system 3, ω2=ω
Since it can be written as 1-Δω, φ plate = φ2, S=A+ '+
A2'+2AI A2C09Δact, and the amplitude A1 of the sample light V1 can be determined from the magnitude of the AC component of the detected signal.

Similarly, according to the Michelson light receiving system 4, ω.

Since it can be written as =ω2, φ2=φI+kt, the detection signal S becomes S=A+ 2+A22+2AI Atcoskt, and the same signal as in the case of the heterodyne light receiving system 3 is obtained. That is, in both the heterodyne light receiving system 3 and the Michelson light receiving system 4, the amplitude A1 of the sample light V1 can be determined from the magnitude of the AC component of the detection signal.

Furthermore, in addition to the heterogain light receiving system 3 and the Michelson light receiving system 4 described above as high directivity detection systems, there are high directivity optical systems illustrated in FIGS. 16 to 25. These highly directional optical systems are representative of an objective lens Obl on the incident side, a pinhole P that is placed on its focal plane and allows only the 0th-order diffraction image of Franhofer diffraction by the objective lens Obl to pass; FIG. 3 shows a highly directional optical system 5 consisting of a similar objective lens Ob2 arranged so that its front focus coincides with the pinhole P (highly directional optical system 5 in the figure).
is similar to the optical system shown in FIG. ). however,
In the following description, the highly directional optical system 5 is not limited to the one shown in FIG.

By the way, in the highly directional detection system 3.4.5 shown in Figs. 1 to 3, the relationship between the sample S and the parallel light flux is as follows:
By changing to the high-resolution detection system 30.40.50 having the configuration shown in FIG. 4A to FIG.
It is possible to irradiate incident light onto a minute black area of the object and collect and detect the scattered light from only that point. That is, a condensing lens L1 with a large numerical aperture (NA) is interposed in the incident direction of the sample S so that the rear focal point coincides with the measurement point of the sample S, and the front focal point is aligned with the rear focal point of the condensing lens L1. An objective lens L2 having a similarly large NA is arranged so that the focus coincides, and the light from the laser 1 is transferred to the condenser lens L.
1 to a minute point on the sample S, and the light emitted from that point is collected by the objective lens L2 and converted into parallel light directed in a predetermined direction, and according to the principle of the high-directivity detection system 3.4.5 described above, By detecting only the light directed in this direction with the detector 2, the scattered light from only the minute region corresponding to the 0 component of the Franphofer diffraction image component of the lens of the sample S is collected and detected. You can do as you like. Therefore, by using the high-resolution detection system 30.40.50 as described above, it is possible to avoid the contamination of unnecessary scattered light from the surroundings, including before and after the measurement point, and also to detect the absorption characteristics of the sample with extremely high resolution. can be measured.

A modification to Fig. 4 is shown in Fig. 4-8. In Fig. 4A, a condensing lens L1 irradiates light onto a minute point on the test US, and an objective lens L2 converts the light emitted from that point into parallel light (approximately a plane wave) directed in a predetermined direction and converts it into a heterodyne. In 4-8, the objective lens L2 is placed on the light flux side of the frequency shifter A○, and the diverging spherical wave from the focus of the condenser lens L1 and the divergent spherical wave from the focus of the objective lens L2 match. Detector 2 performs heterodyne detection. This is because the same beat component is obtained by converting into a plane wave using a lens and performing heterogain detection, and by heterodyne detection using a spherical wave, and the conditions required for wavefront matching are the same.

First of all, the opaque sample that is the subject of spectral absorption measurement in the present invention is not a sample that completely blocks incident light and does not transmit it forward; for example, it is not a sample that completely blocks incident light and does not transmit it forward. Although there is almost no light that directly passes through the sample without being scattered, the sample is one in which light is subjected to multiple scattering by scattering particles in the sample, and light that is scattered forward comes out of the sample. Of course, samples in which directly transmitted light exists can also be measured. Furthermore, the second measurement object of the present invention is a sample, such as a powder, which substantially completely blocks incident light and reflects and scatters it only backwards. The former sample will be called a transmission sample, and the latter sample will be called a reflection sample.

The method for measuring the microscopic absorption distribution of a transparent sample 20 according to the present invention shown in FIG. It is.

For such measurements, a tunable wavelength laser "Q" that can continuously sweep and emit monochromatic light in a wide spectral range is used as a monochromatic light source. The light flux emitted from the laser 10 is split into two by the beam splitter BS, the straight light is focused on the measurement point of the transmission sample 20 by the condenser lens Ll, and the transmitted scattered light is converted into parallel light by the objective lens L2. It is combined with the reference light by the half mirror HM. The frequency of the reference light reflected by the beam splitter BS is slightly changed by a frequency shifter AO such as an ultrasonic optical modulator before the above synthesis, and when it is combined with the sample light and photoelectrically converted, it becomes the reference light and The detector 2 outputs a signal including an alternating current signal with a frequency corresponding to the frequency difference between the sample lights.

Since the magnitude of the AC component of the signal obtained from the detector 2 is proportional to the amplitude of the sample light, the AC component of the output of the detector 2 is separated and the absorption characteristics of the measurement point can be obtained from its magnitude. The intensity of the sample light changes depending on the absorption characteristics of the measurement point on the sample 20, so by scanning the sample 20 with the X-Y scanning device XY, the absorption distribution on the scanning surface of the sample 20 can be measured. can do. In addition, laser 10
The spectral absorption distribution of the sample 20 can also be measured by measuring the absorption at each measurement point while sweeping the wavelength. By the way, in order to make the measurement resolution smaller than the size of the O& image of the Franphofer diffraction image components of lenses L1 and L2, a pinhole that is located very close to the measurement point (focal point of lens L1) and smaller than the size of the image is used. It is also possible to limit the illumination area by arranging a plate PH.

In this case, although the resolution is improved, there is a drawback that the amount of transmitted light is reduced. Note that pinhole plates PH can be similarly arranged in the following embodiments as well.

Now, FIG. 8 shows a modification of the system using the high-resolution detection system 30 of FIG. 7 to measure a reflection sample 21 accompanied by scattering. In this case, a beam converter 11 is placed in front of the laser 10 in order to convert the beam emitted from the laser 10 into a parallel beam of appropriate diameter, but this beam converter 11 is not necessarily necessary. do not have. In this arrangement, change the direction of the half mirror-HM in Fig. 7,
A lens L functioning as a condensing lens and an objective lens is placed at the position where the light from the laser 10 passes through the beam splitter BS and passes through the half mirror HM, and a reflection sample 21 is placed on the focal plane on the rear side. The reflected and scattered light is combined with the reference light by a half mirror HM.

For example, if the scatterer NZ is schematically drawn as being present in front of the sample 21 as shown in the figure, the light scattered by the scatterer NZ becomes a DC component of the detector 2, and the high-resolution detection system 30 is If used, it will not be detected by the detector 2 as an alternating current component.

Next, the apparatus shown in FIG. 9 measures the microscopic absorption distribution of the transmission sample 20 by applying the high-resolution detection system 40 using the Michelson light receiving system shown in FIG. The beam converter 11 converts the light emitted from the beam into a parallel beam of an appropriate diameter, the beam splitter BS splits the light into two, and the reflected light passes through one mirror ML M2, and the condensing lens L1 and objective lens L2 are inserted into the reflected light. A transmission sample 20 is inserted at the common focal point of the confocal arrangement, and the scattered and transmitted light is combined with the reference light and the half mirror HM. The reference light transmitted through the beam splitter BS is transferred to the half mirror HM.
The light passes through the mirror M and is reflected in the opposite direction, and is combined with the sample light by the half mirror HM, and the combined light is photoelectrically converted by the detector 2. The detector 2 obtains a signal on which an interference signal having a frequency corresponding to the speed of the movable mirror M is superimposed. The strength of the alternating current component is proportional to the strength of the scattered light of the transmitted sample 20, so the sample 20 can be scanned with an XY scanning device! By determining the magnitude of the AC component at each measurement point while scanning with tXY, sample 2
0 absorption distribution can be measured. Note that the spectral absorption distribution can also be measured by determining 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 using a Michelson light receiving system so as to determine the absorption distribution of the reflection sample 21. In this case, a lens L functioning as a condenser lens and an objective lens is placed at the position reflected by the beam splitter BS, a reflection sample 21 is placed at the focal plane on the rear side, and the reflected and scattered light is reflected by the beam splitter BS. It is designed to be combined with the reference light reflected from the movable mirror M.

Now, FIGS. 11 and 12 show cases in which the high-resolution detection system 50 using the highly directional optical system 5 of FIG. 6 is applied to measure the absorption distribution of the transmission sample 20 or the reflection sample 21. be. The one in FIG. 11 shows the arrangement when the excitation light cut filter 12 that passes only the fluorescent light generated from the measurement point of the sample 20 is inserted behind 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 shown in FIG.
The light that is focused on the measurement point, transmitted through that point, and scattered forward from that point is converted into parallel light by the objective lens L2, which is arranged so that the front focus coincides with the measurement point. The optical system 5 separates the light from the light going in other directions, and the detector 2 detects its intensity. therefore,
The absorption distribution of the sample 20 can be measured by determining the intensity of light from each measurement point while scanning the sample 20 with the xY scanning device xY. In this case as well, the spectral absorption distribution can also be measured by finding the absorption distribution while sweeping the wavelength of the variable wavelength laser 10. However, when measuring the fluorescence distribution by inserting the excitation light cut filter 12, the wavelength sweep of the laser 10 is not performed, or a laser 10 with a fixed wavelength is used instead of one with a variable wavelength. In the case of the apparatus for measuring the absorption distribution of the reflection sample 21 shown in FIG. Place the
The reflected and scattered light from the reflective sample 21 is converted into parallel light by the lens L, and reflected in a direction different from the direction of the incident light by the half mirror HM, and only the light from the measurement point of the reflective sample 21 is converted into a highly directional optical system. 5. Others are the same as in FIG. 11.

By the way, when a laser sweeps 100 wavelengths, the light intensity generally changes. Therefore, when using a highly directional detection system, a part of the irradiated light beam is taken out and, for example, in FIG. 3, a half mirror is inserted between the laser 10 and the sample S to detect the output laser light intensity. When using a heterodyne detection system, as shown 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 monitoring 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 sample S
The intensity of the transmitted light or the intensity of light reflected in a specific direction is detected by a high-directivity detection system or a heterodyne detection system, and these values are used to determine the transmission integral attenuation and determine the absorption spectrum of the sample. Calculate. This is a method that has already been used in the conventional method of determining the absorption spectrum of an opaque sample, or in the method of determining the absorbance of a sample without scattering.

Next, some examples of devices for specifically measuring the microscopic absorption distribution of a microscopic region of a sample will be briefly described. FIG. 13 shows a schematic configuration of an apparatus capable of measuring the transmission absorption distribution characteristics and reflection absorption distribution characteristics of a sample 20 placed on a sample stage 13 capable of X-Y scanning. The light is switched by a switching mirror MS into a transmitted optical path indicated by a solid line and a reflected optical path indicated by a dotted line. When the solid optical path is selected, the light that has passed through mirrors M3 and M4 is focused by 1 on the measurement point of the sample 20 by the Cassegrain reflective optical system that acts as a condenser lens L1, and the light that has passed through the sample 2a is focused on the measurement point of the sample 20. It is converted into parallel light by the Cassegrain reflective optical system 2, which has the function of incident on .

The highly directional optical system 5 removes light emitted from areas other than the measurement point, and the detector 2 detects the intensity of the light indicating the absorption characteristics of the sample 20. Therefore, the transmission absorption distribution of the sample 20 can be measured by determining the intensity of light from each measurement point while constantly scanning the sample 20 in the XY direction. When the switching mirror MS is switched to the reflection optical path indicated by the dotted line, the monochromatic light from the variable wavelength laser 10 passes through the mirror M5 and reaches the half mirror H.
The sample 20 is reflected downward by M, and the sample 20
The light is focused on the measurement point. The light scattered backward from the measurement point is converted into parallel light by the Cassegrain reflection optical system 2, and enters the highly directional optical system 5 via the mirror M6, where the light emitted from other than the measurement point is removed and the sample 20 The detector 2 detects the intensity of light exhibiting reflection/absorption characteristics. Similarly, the reflection/absorption distribution of the sample 20 can be measured by determining the intensity of light from each measurement point while scanning the sample #J20 at a constant XY rate. Microscopic fluorescence distribution measurement can be performed by inserting an excitation light cut filter 12 after 2 into the Cassegrain reflective optical system. In the figure, reference numeral 15 indicates a chopper, which modulates the laser beam incident on the sample at a predetermined frequency and performs synchronous detection to remove noise. Further, the reference numeral 14 indicates an aperture, and the reference numeral 16 indicates an eyepiece.

The devices shown in FIGS. 14 and 15 are the devices using the high-resolution detection system 30 using the heterodyne light receiving system shown in FIG. 4, and the high-resolution detection system 4 using the Michelson light receiving system shown in FIG.
It is simply a vertical version of the device using 0, and it is exceptional! ! A gate may not be necessary. In addition, in the one shown in FIG. 15, the drive system 17 is for moving the movable mirror M in the optical axis direction.

In the above description, it is assumed that the tunable wavelength laser 10 oscillates continuously, but a tunable wavelength laser that operates in pulses may also 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 operates in pulses. Moreover, although the detector 2 was not specifically explained, any known means can be used. Further, as a method of processing the detection signal, for example, intensity modulation of incident light may be performed using a chopper or the like, and phase synchronization detection of the detection signal may be considered. In this case, since the time constant of infrared light detection is long, it is necessary to reduce the synchronous modulation frequency. Furthermore, a synchronous signal integral detection method such as a photon counting method (visible light) or a charge accumulation method (infrared light), a heterodyne beat signal detection method, or the like may be used. Further, as a frequency shifter, not only one using ultrasonic light diffraction such as ultrasonic modulation Y, but also a combination of wave plates, a diffraction grating, and the electro-optic effect of a crystal can be used.

In addition to a Michelson interferometer that moves a reflecting mirror at a constant speed, it is also possible to use a sawtooth wave to vibrate the reflecting mirror.

Furthermore, for heterodyne reception, it is of course possible to use two lasers instead of just dividing a monochromatic light source laser into two beams to create local light.

C Effects of the Invention] In the method and apparatus for measuring the microscopic absorption distribution of an opaque sample of the present invention, highly directional light is focused and irradiated onto a minute measurement point on the sample, and the light diverging from the measurement point is converted into parallel light. or convert the intensity of the spherical wave into a heterogain light receiving system,
Since detection is performed using a highly directional detection system such as a Michelson light receiving system or a highly directional optical system, it is possible to detect absorption in minute areas of the sample with high resolution without picking up scattered light or other noise light from around the measurement point. This method and device allow accurate measurement and are suitable for measuring microscopic absorption distribution of opaque samples such as biological tissues.

[Brief explanation of drawings]

Figure 1 is a diagram for explaining the configuration and operation of a heterodyne light receiving system, which is the premise of the present invention, Figure 2 is a diagram for explaining the configuration and operation of a similar Michelson light receiving system, and Figure 3 is a similar diagram. Figure 4A is a diagram showing the configuration of a typical highly directional optical system. Diagram for explaining the configuration and operation, No. 4
Figure B is a diagram showing a modification of the measurement method and apparatus to Figure 4, Figure 5 is a diagram explaining the configuration and operation of a high-resolution detection system using a Michelson light receiving system, and Figure 6 is a diagram to explain the configuration and operation of a high-resolution detection system using a Michelson light receiving system. A diagram for explaining the configuration and operation of a high-resolution detection system using a highly directional optical system. Figure 7 shows the configuration of an example of microscopic absorption distribution measurement using the heterogain light receiving system of the present invention applied to a transmitted sample. 8 is a diagram showing the configuration of an embodiment of the microscopic absorption distribution measurement by modifying the apparatus shown in FIG. 7 for use with reflective samples. FIG. Figure 10 showing the configuration of the microscopic absorption distribution measurement using
The figure shows the configuration of an embodiment of the microscopic absorption distribution measurement by modifying the apparatus shown in Fig. 9 for reflection samples, and Figs. 11 and 12 show the configuration of the microscopic absorption distribution measurement using the highly directional optical system of the present invention. Figures 13 to 15 are diagrams showing some specific examples of the apparatus for measuring the microscopic absorption distribution characteristics of a sample according to the present invention, and Figures 16 to 25 are diagrams showing the configuration of an embodiment of the present invention. Figure 25 is a diagram showing the configuration of the highly directional optical system proposed earlier, Figure 25 is a diagram showing the configuration of an apparatus for measuring microscopic absorption distribution using a conventional Fourier spectrometer, and Figure 27 is a diagram showing the configuration of a conventional diffraction grating. FIG. 2 is a diagram showing the configuration of an apparatus for measuring the microscopic absorption spectral distribution of a microsample using a spectrometer. DESCRIPTION OF SYMBOLS 1... Laser, 2... Detector, 3... Heterodyne light receiving system, 4... Michelson light receiving system, 5-... Highly directional optical system, 10... Tunable wavelength laser, 11... ... Beam converter 12 ... Excitation light cut filter, 13.
... Sample stage, 14... Aperture, 15... Chopper, 16... Eyepiece, 17... Drive system, 2
0...Transmission sample, 21...Reflection sample, 30.40.
50... High resolution detection system, S... Sample, BS...
Beam splitter-1HM...Half mirror-1M1~
M7...Mirror, AO...Frequency shifter, M...
・Moving mirror, Ob, Obl, Ob2...Objective lens, P
...Pinhole, Ll...Condensing lens, L2...
Objective lens, XY...X-Y scanning device, PH...pinhole plate, NZ...scatterer, L...lens, MS
...Switching mirror, K1, K2...Cassegrain reflective optical system Applicant: New Technology Corporation Agent Patent attorney Masaru Hirukawa Shindai 1 Zume 2 Figure 3 Go to Figure 4m Figure 5 Figure 6 Fig. 7 Fig. 8 Fig. M9 Fig. 10 Fig. 11 Fig. 13 Fig. 14 Fig. 15 Fig. 19 Fig. GL Fig. 20 N, A child 0.1 NA = 0.1 Fig. 21 Fig. 22 Fig. 23 Figure 26

Claims (11)

[Claims] (1) Highly directional light is focused and irradiated onto a minute measurement point on the sample, the light diverging from the measurement point is converted into parallel light, and the intensity of only the parallel light is detected using a highly directional detection system. Detect using
A method for measuring a microscopic absorption distribution of an opaque sample, characterized in that the absorption distribution characteristics of the sample are measured by repeating the detection operation while scanning the sample relative to the condensed light. (2) The opaque device according to claim 1, characterized in that a pinhole smaller than the size of the zero-order image of the Franphofer diffraction image component of the condensed light is arranged extremely close to the measurement point to limit the illumination area. A method for measuring the microscopic absorption distribution of a sample. (3) The method for measuring microscopic absorption distribution of an opaque sample according to claim 1 or 2, wherein the highly directional light is light with a variable wavelength. (4) The microfluorescence distribution measurement of the sample is performed by inserting an excitation light cut filter that blocks light at the wavelength of the irradiation light into the optical path from the measurement point to the detector. A method for measuring the microscopic absorption distribution of an opaque sample as described above. (5) Take out a part of the irradiation light flux that irradiates the sample, detect the intensity of the light flux, and use it as the reference light intensity, and use the light intensity detected using the highly directional detection system as the signal light intensity from the sample, and reduce the transmission integral. Claims 1 to 3 characterized in that the luminous intensity is determined.
A method for measuring microscopic absorption distribution of an opaque sample according to any one of the above. (6) The light from the wavelength-changeable monochromatic light source is divided into two, a frequency shifting means for shifting the frequency of the incident light is provided in one optical path, and a common system consisting of two converging optical systems is provided in the other optical path. A focusing optical system is arranged, a sample that can be scanned relatively is arranged at the focusing position of the confocal optical system, and the highly directional light emitted from the frequency shift means and the confocal optical system are separated from the measurement point of the sample. A beam combining means is provided to combine the light converted into parallel light by the converter and emit it in the same direction, and the beam combining means converts the combined light into an electrical signal and detects the intensity of only the alternating current component equal to the shift frequency. 1. A microscopic absorption distribution measuring device for an opaque sample, characterized in that it is provided with a detection means for detecting. (7) Light from a wavelength-changeable monochromatic light source is divided into two, an optical path length changing means for changing the optical path length at a predetermined speed is provided in one optical path, and two converging optical systems are provided in the other optical path. A confocal optical system is arranged, and a sample that can be scanned relatively is arranged at the focusing position of the confocal optical system. A beam combining means is provided to combine the light converted into parallel light by the focusing optical system and emit it in the same direction, and the beam combining means converts the combined light into an electrical signal and generates a frequency corresponding to the optical path length change speed. 1. A microscopic absorption distribution measuring device for an opaque sample, characterized in that it is provided with a detection means for detecting the intensity of only the alternating current component. (8) In the optical path of the light emitted from the wavelength-changeable monochromatic light source,
A confocal optical system consisting of several converging optical systems is arranged, a sample that can be scanned relatively is arranged at the converging position of the confocal optical system,
A highly directional optical system is installed to extract only the light in the traveling direction of the light that is diverged from the measurement point of the sample and converted into parallel light by the confocal optical system, and the intensity of the light extracted by the highly directional optical system is A microscopic absorption distribution measuring device for an opaque sample, characterized in that it is provided with a detection means for detection. (9) In the optical path of the light emitted from the wavelength-changeable monochromatic light source,
An optical path switching means is provided, one optical path and the other optical path are coaxial, and the traveling directions of light are opposite to each other, and the confocal optical system is arranged in the coaxial optical path, and the confocal optical system is arranged in the coaxial optical path. 8. A light extracting means for extracting light transmitted through the sample or light reflected and scattered by the sample is provided on one side of the system, and the highly directional optical system is disposed behind the light extracting means. A microscopic absorption distribution measuring device for an opaque sample as described above. (10) A frequency shifting means is provided to split the light from a wavelength-changeable monochromatic light source into two, shift the frequency of the incident light into one optical path, and focus highly directional light into the other optical path. A first converging optical system that irradiates a minute measurement point on the sample is arranged, a sample that can be scanned relatively is arranged at the condensing position of the first converging optical system, and the directional light emitted from the frequency shift means is A first converging optical system that irradiates the sample with high-temperature light and a second converging optical system that emits the same convergent spherical wave are arranged, and a beam combining means is provided that combines both light beams and emits them in the same direction. 1. An apparatus for measuring microscopic absorption distribution of an opaque sample, comprising a detection means for converting light synthesized by the above into an electrical signal and detecting the intensity of only an alternating current component equal to a shift frequency. (11) Blocking the light from the scattering sample, detecting the intensity of the luminous flux emitted from the frequency shifting means or the optical path length changing means and using it as a reference light intensity, and converting the combined light into an electrical signal by the beam combining means. A claim characterized in that an alternating current component equal to the shift frequency or an alternating current component with a frequency corresponding to the optical path length change speed is used as the signal intensity from the sample, and the transmission integral attenuation is determined using these reference intensities and signal light intensities. Item 6
, 7 or 10. The apparatus for measuring the microscopic absorption distribution of an opaque sample according to any one of Items 7 and 10.