JPH06331537A - Optical inspection apparatus - Google Patents

Abstract

PURPOSE:To provide an optical inspection apparatus which detects an optical echo at a high S/N ratio. CONSTITUTION:An optical inspection apparatus is provided with a first light source 1 which supplies first light, with a light divider 2 which divides the first light into two luminous fluxes with an optical delay device 4 which changes the optical path length of light of one out of the two luminous fluxes, with an irradiation means 8 by which an object 20 to be inspected is irradiated with the two luminuous fluxes and with a photodetector 9 which detects and outputs light from the object 20 to be detected. The optical inspection apparatus is provided with a second light source 60 which radiates second light used to reduce the influence of the light generated in the object 20 to be detected after the object 20 to be detected has been irradiated with the first light.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an optical inspection device capable of optically observing a difference in physicochemical properties of substances.

[0002]

2. Description of the Related Art Generally, there is known a laser scanning microscope for observing a sample by using optical information obtained by scanning the sample with a laser beam. However, the information of the sample obtained by the conventional laser scanning microscope is qualitatively the same as the information obtained from the image of the usual optical microscope. On the other hand, the applicant of the present invention has disclosed in
In Japanese Patent Laid-Open No. 44-44, a light echo microscope was proposed as a microscope capable of obtaining information on the difference in physicochemical properties of a sample, which cannot be obtained by an ordinary optical microscope. The optical echo microscope irradiates a sample with a laser beam, and an optical phase relaxation time (generally, "T") due to an optical echo in a light absorber that can resonate with the laser beam contained in the sample.
_{2 This} is the time called "relaxation time" or "lateral relaxation time". ) Is a thing to measure. The phenomenon of optical echo is described in, for example, “Ultrafast optical technology” (edited by Tatsuo Yajima, Maruzen, published in March 1990).

In Japanese Unexamined Patent Publication No. 4-269644, a combination of optical echo measurement and a laser scanning microscope is used to relatively scan the laser beam and the sample two-dimensionally and three-dimensionally. It was described that the distribution state of the physicochemical properties of the sample can be clarified by mapping the phase relaxation time in a two-dimensional or three-dimensional manner. It was also stated that an image as a normal laser scanning microscope could be obtained at the same time.

For example, a dye that can resonate with laser light is
When dispersed in a polymer in which crystalline and amorphous are mixed, by utilizing the fact that the optical phase relaxation time is different between crystalline and amorphous, a crystalline phase and an amorphous phase are obtained. It can be observed by an optical echo microscope what kind of form is mixed in the polymer. In the case of a solid, its optical phase relaxation time can be easily measured by the heterodyne detection method of accumulated photon echo by intensity modulation. Further, a heterodyne detection method of accumulated photon echo by optical phase modulation can also be used. The heterodyne detection method by optical phase modulation of optical echo is disclosed in Japanese Patent Application Laid-Open No. 4-1322020 by the present applicant. Hereinafter, the measuring method will be described in JP-A-4-132020.

The measurement in JP-A-4-132020 mainly uses pulsed light, and basically utilizes a phenomenon called stimulated photon echo (stimulated optical echo) in photon echo (optical echo). As is well known, the photoexcited state of a substance is the equation of motion (Liovil) of its density matrix.
Le's equation). For convenience, the relaxation time of the diagonal component of the density matrix is called T ₁ hour (longitudinal relaxation time), and the relaxation time of the non-diagonal component is called T ₂ hour (horizontal relaxation time). Different. Longitudinal relaxation is considered to mean the process in which the photoexcited state relaxes with the release of energy, and transverse relaxation refers to the disturbance of the coherency of the oscillation of electrical polarization brought into a substance by incident light. It is believed to show the process of going.

The phenomenon called optical echo (photon echo) is considered to be a kind of third-order nonlinear optical effect, and the induced photon echo (induced optical echo) in it will be described with reference to FIG. Consider the case where a substance is excited with an appropriate pulsed light in energy resonance. First, the pulsed light of E ₁ is irradiated at t ₁ , and then the pulsed light of E ₂ is irradiated at t ₂ . Next, when irradiated with t ₃ at a third pulse _{E 3, (t 3 + t} 2 -t 1 = t 3 + τ (τ = t 2 -
At time t ₁ )), light is emitted from the substance.
This is the stimulated photon echo light. Then, the intensity of the stimulated photon echo light is attenuated in proportion to exp (-4τ / T ₂ ). Therefore, in the optical echo microscope disclosed in JP-A-4-132020, by measuring the intensity of the stimulated photon echo light (particularly the accumulated photon echo) while changing τ, the T that differs for each substance or each state can be obtained.
₂ (lateral relaxation time) was required.

When the above-mentioned Lioville equation is calculated by perturbation expansion by the rotating wave approximation and the weak excitation light approximation, a third-order nonlinear polarization wave, that is, an echo wave is obtained. The photon echo is a kind of third-order nonlinear optical phenomenon, and its effect is generally small. Regeneration excitation light (E ₃ )
On the other hand, the photon echo light intensity is usually several orders of magnitude weaker. It is known that detection of such weak light (photon echo light) is improved by utilizing interference of light.

In this case, weak light (photon echo light)
Is observed (measured) by interfering with a light wave (probe light E ₄ ) having a known phase characteristic, amplifying a change in intensity and phase of weak light (photon echo light). At this time, the intensity or frequency of the weak light (photon echo light) is modulated by some method, and only the synchronous component in the output of the combined light (the combined light of the photon echo light and the probe light) is amplified to increase the contrast. Is commonly practiced.

The signal intensity of the light obtained by the interference between the optical echo and the probe light is the delay time t ₂₁ (= t ₂₎ at the time of writing.
-T ₁ ) and delay time t ₄₃ (= t ₄ −t ₃ ) at the time of reading
It changes with a slight difference from. This is a very excellent feature of optical interference, but it is also a drawback. This is because this slight difference determines whether or not the optical echo interferes with the probe light.

In the generally known optical echo detection,
Total time t_{twenty one}And delay time t₄₃And 10 ^-16Match with second precision
I have to let you. This time is 1 when converted to the optical path length.
0^-2Equivalent to μm. Therefore, the echo light is used as the probe light.
The device that detects by the interference of 10^-2Machine of about μm
Accuracy is required, and this is
If you try to get it done,
You will need it. To this end, JP-A-4-132020
According to the report, it is as follows.

It is understood that when t ₄₃ -t ₂₁ is modulated for one light cycle or more, the intensity of the combined light of the echo light and the probe light is also modulated in synchronization with it. Here, it is considered that t _43, that is, the delay time between the excitation light and the probe light is synchronized and oscillated. This is equivalent to phase-modulating the probe light relative to the excitation light. At this time, it can be seen that only the modulation component having an even multiple of the phase modulation frequency remains.

Therefore, the excitation light and the probe light are relatively surrounded.
Phase-modulated with wave number f, and the combined light of echo light and probe light
From an electric signal obtained by photoelectric conversion of
If you extract only 4 times, 4 times, 6 times ...)
-It is possible to detect light stably and with high accuracy.
It In the experimental example described in JP-A-4-132020,
In addition, the light source serves as both the excitation light source and the probe light source.
The harmonic excitation of a mode-locked YAG laser
Kiton red dye laser with a repetition frequency of 82
Outputs pulsed light of MHz. Kitten red dye laser
All wavelength selective elements were removed from the laser. as a result,
Center wavelength 620nm, spectral width 400cm^-1(correlation
Incoherent light of time 37 femtoseconds equivalent) is obtained.
Was given. Also, in this embodiment, t ₁, T₂, T₃,
t_FourThe relationship of is shown in FIG. t₁And t₃, T₂And t_FourIs 8
Repeated at 2MHz, t₁And t₂, T₃And t_FourBetween
Phase modulation is applied by the phase modulation means.

As described above, the optical echo microscope optically observes, for example, the difference in physicochemical properties of substances.

[0014]

The conventional optical echo microscope as described above uses the laser light previously irradiated to the object to be inspected when a laser having a relatively narrow wavelength width such as a semiconductor laser is used as a light source. However, there is a problem that the S / N ratio of the optical echo detection decreases due to the influence of the above. In view of such a point, the present invention has an object to provide an optical inspection apparatus capable of detecting an optical echo with a higher S / N ratio.

[0015]

As a result of research in the present invention,
The effect of the laser light (first light) as described above is that a permanent hole is formed relatively deeply at the wavelength of the laser light irradiated within the wavelength of the light absorption band of the object to be inspected. It was found that the S / N ratio of the light echo detection is lowered because the permanent hole has an effect even if the light is irradiated.

Therefore, in order to remove the influence of the laser light (first light) previously irradiated on the object to be inspected, it is considered that the permanent hole formed by the previous irradiation should be erased, and the object to be inspected is considered. Irradiate light (second light) having a spectrum close to almost the entire light absorption band of the above, and light (second light) corresponding to the light absorption band of the photoreaction product newly generated by the permanent hole formation. However, they have found that it is only necessary to induce the reverse reaction of permanent hole formation, and to induce the re-coordination of the photoreactive substance by irradiating light having a shorter wavelength than the first light. Therefore, in the present invention, in addition to the laser light source for detecting the optical echo (first light source), an erasing light source (second light source) for reducing the influence of previous irradiation is added.

Therefore, the optical inspection apparatus according to the present invention comprises a first light source for supplying a first light, an optical splitter for splitting the light from the first light source into two light beams, and one of the two light beams. Optical inspection including an optical delay device that changes the optical path length of the light, an irradiation unit that irradiates the test object with the two light fluxes, and a photodetector that detects and outputs the light from the test object. In the device, after the object to be inspected is irradiated with the first light,
It is configured to include a second light source that emits second light for reducing the influence of light generated on the object to be inspected. A first light source for supplying a first light;
A light splitter that splits the light from the light source of
An optical delay unit that changes the optical path length of one of the light beams, an irradiation unit that irradiates the test object with the two light beams, and a photodetector that detects and outputs the light from the test object. In the optical inspection device, a second light source that emits second light for reducing the influence of light generated on the test object before the test object is irradiated with the first light. It consists of having.

[0018]

The light (second light) having a spectrum close to almost the entire light absorption band of the object to be inspected, the light corresponding to the light absorption band of the photoreaction product newly generated by the formation of the permanent hole, and the like. Irradiation with light having a shorter wavelength than the first light (second light) induces a reverse reaction of permanent hole formation. In this way, optical echoes can be detected with a higher S / N ratio, and high-precision optical inspection becomes possible.

[0019]

1 is an optical inspection apparatus according to an embodiment of the present invention. Hereinafter, description will be made with reference to FIG. FIG. 1 shows a schematic configuration of the optical inspection apparatus of this embodiment. As shown in FIG. 1, the optical system of this embodiment is roughly divided into two parts, that is, light source means surrounded by dotted lines. (A) and a light modulation optical system (B). Light source means (A) of this embodiment
Is composed of a light source 1 (first light source) and an erasing light source 60 (second light source). The light source 1 (first light source) is a dye laser excited by a mode-locked argon ion laser of 80 MHz. Besides, as the light source 1 (first light source), a multi-mode semiconductor laser, a mode-locking semiconductor laser, a light emitting diode or a solid-state laser can be used. Further, the light (first light) generated from the light source 1 (first light source) has a spatial coherence that can be regarded as a point on the image plane, and the optical absorption band of the light absorption band of the object to be examined is optical. It is desirable to have a time coherence that is shorter than the phase relaxation time. The erasing light source 60 (second light source) is
The position is not limited to the position shown in FIG. 1, and any arrangement may be used as long as it can emit the erasing light (second light) to the test object.

In FIG. 1, the laser light (first light) generated by the light source 1 (first light source) is used as a light modulation optical system (B).
The laser beam (first light) is split into two light beams by the light splitter 2 on the input side. On the output side of the light modulation optical system (B), an optical multiplexer 3 that multiplexes the two light beams thus branched is arranged. Both the optical splitter 2 and the optical multiplexer 3 are polarization beam splitters, but a semi-transparent mirror or the like having little or no polarization characteristics can be used instead. A first orthogonal reflecting surface 6a is arranged in parallel between the optical splitter 2 and the optical multiplexer 3.
6b and the second orthogonal reflecting surfaces 7a and 7b are arranged. As the optical system that divides the light from the light source means (A) into two and combines them again, a two-path interference optical system similar to a Michelson interferometer or a Mach-Zehnder interferometer is suitable.

The light beam that has passed through the split surface of the light splitter 2 is reflected by one of the first orthogonal reflecting surfaces 6a and then,
It goes to the first corner cube 4a fixed to a movable stage (not shown). The light beam reflected by the corner cube 4a is reflected by the other reflecting surface 6b of the first orthogonal reflecting surface and enters the optical multiplexer 3. Reference numeral 4b denotes a displacement device that displaces the corner cube 4a via a movable stage (not shown). The optical delay means 4 is composed of the corner cube 4a, the movable stage and the displacement device 4b. By moving the corner cube 4a using the displacement device 4b, it is possible to delay one of the light fluxes by a predetermined amount. Here, instead of the corner cube 4a, two mirrors or a right angle prism may be used.

The light beam reflected by the split surface of the light splitter 2 is
After being reflected by one of the reflecting surfaces 7 a of the second orthogonal reflecting surfaces, it enters the phase modulating means 5. As the phase modulation means 5, generally one using an electro-optic crystal is well known, but in this embodiment, a piezoelectric element 5b having one end fixed and a corner cube 5a fixed at the other end is used. . Then, by applying an AC voltage of a predetermined frequency f to the piezoelectric element 5b by the AC driving power supply 5c, a phase modulation unit of the frequency f is configured. Here, instead of the corner cube 5b, a rectangular prism having a small dispersion may be used. Corner cube 5a, piezoelectric element 5b,
The phase modulation means 5 is composed of the AC drive power source 5c. The light flux that has passed through the phase modulation means 5 has a second orthogonal reflection surface 7b.
It is reflected by and enters the optical multiplexer 3.

In this embodiment, the optical delay means 4 and the phase modulation means 5 are arranged on the transmission optical path and the reflection optical path of the optical splitter 2, but the arrangement is not limited to this, and the reverse arrangement is also possible. It is also possible to arrange both the optical delay means and the phase modulation means on one optical path. These optical splitter 2, optical multiplexer 3,
The light delay means 4 and the phase modulation means 5 constitute a light modulation optical system (B). The two light beams in the light modulation optical system (B) are combined by the optical combiner 3 and are irradiated onto the test sample 20 in the sample chamber 21 through an appropriate irradiation optical system 8. The irradiation optical system 8 can be shared with the objective optical system of the optical microscope. In that case, if an eyepiece optical system is additionally provided, the irradiation optical system 8 becomes an optical microscope, which enables visual observation. Further, by combining an irradiation optical system and another confocal optical system, arranging a photodetector with a pinhole in front, and combining the sample chamber 21 with a movable stage, a laser scanning microscope type optical echo microscope is obtained. . The sample chamber 21 can be cooled if necessary.

Light emission by fluorescence or phosphorescence in the light spot forming portion of the test sample 20, reflection of irradiation laser light,
A detector 9 composed of a photomultiplier is arranged so that diffraction or scattering can be detected well. A filter is inserted in front of the detector 9 if necessary. Here, the position of the detector 9 is not limited to the position shown in the figure, and may be an arrangement for detecting transmitted light.

The detector 9 outputs a signal corresponding to the amount of incident light by photoelectric conversion, and of the signals output from the detector 9, only the modulation component having twice the phase modulation frequency is amplified by the lock-in amplifier 10. It The amplified signal corresponds to the delay time of the optical delay device 4 and the electric signal processing device 4
It is stored in the data processor 50 via 0 and analyzed.

As the erasing light source 60 (second light source), a halogen lamp, a xenon lamp, a mercury lamp or the like is used, but in some cases, a combination of a lamp and a wavelength selecting element such as a filter or a spectroscope is used. , A light emitting diode, a dye laser, a gas laser, a solid-state laser, and a combination of these with a wavelength selection element is used depending on the case. The wavelength of the erasing light (second light) is determined according to the test sample.

The erasing light source 60 (second light source) has a wide range of wavelength components including the light absorption band of the object to be inspected, but depending on the case, the measurement laser light (first light source) is used. It may be aligned with the light absorption band of the object to be measured on the shorter wavelength side than (light). The erasing light (second light) may be applied to the object to be measured after measuring the optical echo with one delay time and before measuring the optical echo with the next delay time, or before measuring the optical echo. Alternatively, the erasing light may be previously irradiated.

In the optical inspection apparatus as described above, the inspection was carried out using normal tissues and tumor tissues of human liver tissue stained with rhodamine 640 as test samples. The wavelength width of the dye laser (first light source) was set to about 2 nm, and the wavelength was set to about 590 nm. The temperature of the sample was 5 Kelvin. As the erasing light (second light), light from a halogen lamp combined with a filter for removing light in the infrared region is used,
The irradiation intensity was 100 mW / cm 2. After measuring with one delay time and before measuring with another delay time, the erase light (second
Light) was applied to the test sample for 10 seconds. Normal tissue and tumor tissue could be clearly distinguished from the phase relaxation time T ₂ obtained from this series of measurements. That is, it was possible to distinguish between normal and abnormal human liver tissue, and a very sensitive pathological examination was possible. However, when the erasing light (second light) was not used, the S / N ratio of the obtained optical echo was very low. As the dyes, Texas red and its derivatives, Rhodamine 640 derivative, Giemsa dye, methylene blue, Azure B, eosin, etc. can be similarly used when the wavelength width of the irradiation laser (first light source) is several nm or less. The S / N ratio was very low without using the erasing light (second light). The test sample was not limited to a tissue sample and could be a cell sample. As an experimental condition, the phase modulator 5
The frequency of the phase modulation was 20 KHz. When only the 40 KHz electric signal was amplified and recorded by the lock-in amplifier 10, an optical phase relaxation time was obtained, and an objective inspection result could be obtained based on the information.

In the present embodiment, the optical inspection device for detecting the optical echo by using the optical phase modulation has been described. However, the optical inspection device for measuring the optical echo by using the intensity modulation or the like is not limited to this. Even when (second light) was used, an optical echo could be detected with a high S / N ratio.
In this embodiment, the erasing light (second light) may be applied before the measurement.

[0030]

As described above, according to the present invention, the light (first light and the light other than the first light) generated on the object to be inspected is irradiated with the erasing light (second light). Since the influence of light) can be reduced, optical echoes can be detected with a higher S / N ratio, and high-precision optical inspection can be performed.

By using the optical inspection apparatus of the present invention, it is possible to detect a difference in physicochemical properties of an object to be inspected other than morphological information with extremely high sensitivity.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an optical inspection device according to an embodiment of the present invention.

FIG. 2 is an explanatory diagram of a conventional optical echo measurement principle.

[Explanation of symbols for main parts]

DESCRIPTION OF SYMBOLS 1 ... Light source (first light source) 2 ... Optical splitter 3 ... Optical multiplexer 4 ... Optical delaying means 5 ... Optical modulating means 8 ... Irradiation optical system 9 ... Photodetector 10 ... Lock-in amplifier 30 ... Control means 40 ... Electrical signal processing device 50 ... Data processing device 60 ... Erase light source (second light source)

Claims (2)

[Claims] 1. A first light source for supplying a first light, an optical splitter for splitting the first light into two light beams, and an optical delay device for changing the optical path length of one of the two light beams. An optical inspection device that irradiates the object to be inspected with the two light fluxes and a photodetector that detects and outputs light from the object to be inspected. An optical inspection apparatus comprising: a second light source that emits a second light for reducing the influence of the light generated on the object to be inspected after being irradiated with the first light. 2. A first light source that supplies first light, an optical splitter that splits the first light into two light beams, and an optical delay device that changes the optical path length of one of the two light beams. An optical inspection device that irradiates the object to be inspected with the two light fluxes and a photodetector that detects and outputs light from the object to be inspected. An optical inspection apparatus comprising: a second light source that emits a second light for reducing the influence of the light generated on the object to be inspected before being irradiated with the first light.