JPH04269644A - Optical echo microscope - Google Patents

Abstract

PURPOSE:To make it possible to obtain the data concerning to the difference in physical and chemical properties of a sample by measuring the optical-phase alleviating time of the sample. CONSTITUTION:A light source means A supplies the light having space coherence which can be regarded as a point light source on an image plane and the specified time coherence from a light source 1. A light-modulation optical system B splits the light with a light splitter 2. One light is delayed by a specified time with a delaying means 4. The other undergoes phase modulation at a specified frequency with a light modulating means 5. These lights are synthesized with a light synthesizer 3. A cofocal scanning microscope C guides the light on a test specimen 20 with an illuminating optical system, and a spot is formed. Scanning is relatively performed with a scanning means. The light guided from the substance is detected with a photodetector 15. The modulated part which is even-number times the modulating frequency in the means 5 is extracted with an electric-signal processing part 40 from the output signal of the photodetector 15. Thus, the optical-phase alleviating time of the sample is obtained based on the change in optical echo caused by the change in difference between the lengths of the light paths of two light beams. The difference in physical properties can be made clear based on the change.

Description

Description: FIELD OF THE INVENTION The present invention relates to a microscope capable of optically observing differences in physicochemical properties of substances. 2. Description of the Related Art In contrast to general optical microscopes, laser scanning microscopes using a laser light source are known and are used in various types of microscopes. Among these, confocal laser scanning microscopes place the object to be examined at the focal point of the illumination system, form a light spot there, and detect the light by focusing the light from the object to be examined onto a pinhole. It is. Images obtained with a confocal laser scanning microscope have a very shallow depth of focus, so by scanning in the optical axis direction,
It has the advantage that an infinitely deep depth of focus can be obtained due to the so-called sectioning effect. [Problems to be Solved by the Invention] However, the information on the object to be examined obtained using a confocal laser scanning microscope is qualitatively not much different from the information obtained from images obtained using a normal optical microscope. there were. An object of the present invention is to provide an optical echo microscope that is capable of obtaining information regarding differences in the physicochemical properties of a sample, in addition to the function of a conventional microscope for simply observing a sample. Means for Solving the Problems The optical echo microscope according to the present invention includes a laser light source that supplies light having a spatial coherence that can be regarded as a point light source on an image plane and a predetermined temporal coherence, and A light splitter that splits light into two light beams, an optical delay device placed in the optical path of one of the two light beams split by the light splitter, and a light delayer that splits one of the two light beams at a predetermined frequency. a phase modulator for phase modulation, a light combiner for combining the two light beams, and an irradiation optical system for guiding the combined light beam onto a test object to form a light spot;
a scanning means for scanning a light spot formed by the irradiation optical system on the test object relative to the test object; and a condenser for guiding light from the test object onto a photodetector. It has an optical system and a signal processing means for extracting a modulation component of an even multiple of the modulation frequency of the phase modulator from the output signal of the photodetector. Here, it is desirable that the laser light source has a time coherence shorter than the optical phase relaxation time of the optical absorption band of interest for the object to be inspected. [Operation] Conventional laser scanning microscopes have used laser light sources (eg, helium neon lasers, CW argon lasers, etc.) that have long temporal coherence, that is, extremely narrow oscillation spectrum widths, as light sources. In the optical echo microscope of the present invention, a laser light source having a wide oscillation spectrum width is used as a light source in order to measure the optical phase relaxation time of a sample. In order to measure the phase relaxation time of a solid sample, in addition to a laser light source with a wide oscillation spectrum width, a light modulation optical system and a corresponding photodetection system are required. The phase relaxation time of a solid can be easily measured by heterodyne detection of accumulated photon echoes. In particular, optical phase modulation is a convenient method for measuring optical echoes at the focal point of the illumination system in a confocal laser scanning microscope. It is appropriate to employ a heterodyne detection method according to The heterodyne detection method using optical phase modulation of optical echoes is disclosed in Japanese Patent Application No. 2-25301 filed by the same applicant as the present application.
As explained in No. 1. [0007] If the object to be tested has physicochemical characteristics that can produce an optical echo, the optical echo is detected as a signal with a frequency component that is twice or an even number multiple of the phase modulated optical frequency component. The magnitude of the optical echo at the laser focal position on the sample can be detected by extracting the magnitude of the optical intensity modulation component that is twice or even multiples of the phase modulation frequency at the focal position of the light receiver. It is possible. Therefore, the phase relaxation time of the sample can be obtained by recording the change in the optical echo caused by sequentially changing the optical path length difference between the two light beams using the optical delay device. In a normal laser scanning microscope image, such a modulation operation is not necessary, and it is sufficient to detect only the light intensity of the light receiving section. As described above, the optical echo microscope according to the present invention has optical phase relaxation time (generally T2 relaxation time,
It has a new function of clarifying the transverse relaxation time (also called transverse relaxation time) by mapping it two-dimensionally or even three-dimensionally in combination with a scanning means. In other words, in addition to the scanned image of a normal laser microscope, if there is a light absorber in the sample that can resonate with the laser beam, the physicochemical information of the sample can be obtained by measuring the optical phase relaxation time by optical echo. properties can also be revealed. For example, if a dye that can resonate with laser light is dispersed in a polymer containing a mixture of crystalline and amorphous materials, it is possible to obtain amorphous by taking advantage of the fact that the optical phase relaxation time is different between crystalline and amorphous materials. It is possible to observe how the crystalline phase and the crystalline phase coexist. [Example] As an example of the present invention, first, a light source has spatial coherence that can be regarded as a point light source on the image plane,
In addition, it is appropriate to have a time coherence shorter than the optical phase relaxation time of the optical absorption band of interest in the object to be measured. An optical system having a light splitter that splits the light from the light source into two beams and a light combiner that combines the two beams is similar to a Michelson interferometer or a Mach-Zehnder interferometer.
Optical path interference optical system (2path interferome
ter ) is preferred. The optical delay device has a function of changing the length of one optical path of the interference optical system, and the optical modulator has a function of phase modulating the light passing through one path of the interference optical system at a fixed frequency f. Then, the light emitted from the interference optical system is focused on or inside the object to be measured by the light irradiation optical system to form a light spot. Light scattered, transmitted, or emitted from the object to be measured is guided onto a photodetector by a condensing optical system, and the signal processing means converts the output signal of the photodetector into a frequency that is twice the modulation frequency of the phase modulator or an arbitrary value. It has the function of selectively detecting and amplifying only AC components with even frequency multiples. The configuration of the specific embodiment shown in the drawings will be explained below. FIG. 1 is a block diagram schematically showing a first embodiment of the present invention. The configuration of the optical system in this example can be roughly divided into three parts surrounded by dotted lines in the figure: a light source means (A), a light modulation optical system (B), and a confocal laser scanning microscope (C). It has become. In this embodiment, the light source means (A) has a light source 1 consisting of an 80 MHz mode-locked argon laser-excited dye laser. In addition, as the light source 1, a multimode semiconductor laser, a mode-locked semiconductor laser, an LED, or a solid-state laser can be used. The light splitter 2 for splitting the light beam from the laser light source into two light beams and the combiner 3 for combining the two branched light beams are both polarization beam splitters. Little or no semi-transparent mirrors and the like can also be used. In the transmitted light path of the light splitter 2,
Corner cube 4 fixed on a movable stage (not shown)
a is arranged, and a displacer 4b for displacing the movable stage constitutes an optical delay means 4. This displacement device 4b is configured to move the corner cube 4a to cause an optical delay by a predetermined amount. Further, a phase modulation means 5 is arranged on the reflected optical path of the light splitter 2. As the phase modulation means 5, one using an electro-optic crystal is generally 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. . And AC drive source 5
By applying an alternating current voltage of a predetermined frequency f to the piezoelectric element 5b by c, a phase modulation means 5 of a frequency f is constructed. These optical splitter 2, multiplexer 3, optical delay means 4
and the phase modulation means 5 constitute a light modulation optical system (B). Orthogonal reflecting surface 6 arranged in the branched light beam
a, 6b and 7a, 7b are optical delay means 4, respectively.
and an optical path bending reflective surface for guiding light to the phase modulation means 5 and facilitating optical multiplexing in the multiplexer 3. In this embodiment, the optical delay means 4 is arranged on the transmitted optical path of the light splitter 2, and the phase modulation means 5 is arranged on the reflected optical path, but the arrangement is not limited to this. It is also possible to arrange both the phase modulation means and the phase modulation means on one optical path. The confocal laser scanning microscope (C) includes an irradiation optical system consisting of a semi-transmissive mirror 10, objective lenses 11 and 12, a condensing lens 13, and a light shielding plate 1 having a pinhole aperture.
4, a photomultiplier tube 15 as a photodetector, and a lock-in amplifier for signal processing. The light flux from the light modulation optical system (B) that passes through the semi-transmissive mirror 10 is focused onto the object to be measured 20 by objective lenses 11 and 12 to form a light spot there. Test object 2
The light emitted from 0 passes through the objective lenses 11 and 12 again, is reflected by the semi-transparent mirror 10, and is focused by the condensing lens 13 onto the pinhole opening 12a of the light shielding plate 12. The light passing through the pinhole aperture 12a enters a photomultiplier tube 15 as a detector, and the photomultiplier tube 15 outputs a signal according to the amount of incident light by photoelectric conversion. Only the modulation component that is twice the amount of is amplified by the lock-in amplifier 16. The light shielding plate 14 having a pinhole opening functions as a spatial filter for detecting only scattered light or light emitted from the focal position of the irradiation optical system. Note that an optical trap 17 is provided to prevent the light reflected by the semi-transmissive mirror 10 from being incident on the condensing optical system to generate stray light from the light modulation optical system. A scanning means 3 is provided between the semi-transmissive mirror 10 and the objective lenses 11 and 12 in the irradiation optical system for scanning the light spot produced by the condensing optical system on the object 20 to be inspected.
0 is placed. This scanning means 30 can be a well-known optical scanning means using optical reflection or refraction, and a light spot scans the object 20 to be inspected at a predetermined speed based on a signal from a scanning driving means 31. The scanning means 30 is a spatial modulator, and it is also possible to employ a device that moves a stage holding an object to be measured relative to the condensing optical system. Further, a test object 20 as a sample is housed in a sample chamber 21 for cooling and holding the sample as necessary. Furthermore, the optical echo microscope of this embodiment includes:
The electrical signal processing device 40 corresponds the output signal from the lock-in amplifier 16 to the position of the light spot of the irradiation optical system obtained by the monitor of the scanning means 30 and the delay time by the optical delay device 4 of the light modulation optical system. It has a recording and reproducing means that can record and reproduce as necessary. Also, data that allows analysis of the amount of change in the output signal of the electrical signal processing device 40 due to a change in the delay time of the irradiation optical system with respect to a desired point in the object to be inspected where a light spot is formed by the irradiation optical system is provided. An analysis device 50 is provided. Laser beam scanning by the scanning means 30 and scanning of the optical path length by the optical delay device 4 can be performed in any combination as required, but the relative scanning speed of the light spot on the object to be inspected is required to be sufficiently slow relative to the phase modulation by the phase modulator, and the phase modulator 5
and the control of the optical delay device 4 is performed by the control means 32,
A signal is sent to the data analysis device 50 via the electrical signal processing device 40. The data analysis device 50 analyzes the output signal and displays a desired image. When using the optical echo microscope according to the first embodiment of the present invention as described above, for example, when a biological tissue is stained and observed using a dye called Giemsa, which is a type of biological dye, the laser wavelength is set to around 640 nm. The phase modulation frequency of the phase modulator 5 was set to 20 KHz. Scanning means 30
By fixing the laser beam at one point on the sample 20 and amplifying and recording only the 40 KHz electrical signal with the lock-in amplifier 16 while scanning the stage position of the optical delay device 4, the optical phase relaxation time at the laser focal position can be obtained. Ta. Similarly, the optical phase relaxation time at each spatial point on the sample 20 was obtained, and detailed information about the living tissue was obtained. FIG. 2 is a schematic configuration diagram showing the configuration of a second embodiment according to the present invention. This embodiment differs from the optical echo microscope shown in FIG. 1 in that it includes a laser scanning microscope (C) that does not have a confocal condensing optical system, and that it is a transmission type microscope. There is. Scanning of the light spot by the irradiation optical system is performed by moving a stage (not shown) in the sample chamber 21 that accommodates the object 20 to be examined using the scanning drive means 33. Since the other configurations are the same, the same reference numerals are given to the members having the same functions as the configuration of the first embodiment. The transmitted light, scattered light, and emitted light from the test object 20 are transmitted to the photodetector 15 by the condensing lens 17.
From the output signal of the photodetector 15, only the modulation component twice the phase modulation frequency is sent to the lock-in amplifier 1.
6 and subjected to desired processing by the electrical signal processing device 40 and data analysis device 50. [0018] Even with the configuration of the second embodiment,
Optical echoes of the test object can be detected, and physicochemical information about the test object can be obtained. In each of the above embodiments, the detector 15 detects a light echo having the same wavelength as the irradiation light, but it is also possible to detect a light echo of fluorescence emitted from the object to be examined. In this case, it goes without saying that only the optical echo of the fluorescence can be detected by providing a filter that transmits only the fluorescence wavelength range on the optical path leading to the detector. [0019] The optical echo microscope of the present invention makes it possible to clarify not only the fine shape of an object to be examined, but also the differences in its physical properties by measuring the optical phase relaxation time of the fine parts. Can be done. This new function possessed by the microscope of the present invention is a function that conventional optical microscopes or laser scanning microscopes could not possess.

[Brief explanation of the drawing]

FIG. 1 is a schematic configuration diagram of an optical echo microscope according to a first embodiment of the present invention.

FIG. 2 is a schematic configuration diagram of an optical echo microscope according to a second embodiment of the present invention.

[Explanation of symbols of main parts]

1...Light source
2... Optical splitter 3... Optical multiplexer
4... Light delay means 5... Light modulation means 11, 12 ・Objective lens 13... Condensing lens
15... Photodetector 20... Test object
30... Scanning means 40... Electric signal processing device 50... Data analysis device

Claims (7)

[Claims] 1. A laser light source that supplies light having a spatial coherence and a predetermined temporal coherence that can be regarded as a point light source on an image plane, a light splitter that splits the light from the light source into two beams, and a light splitter that splits the light from the light source into two beams. an optical delay device disposed in one optical path of the two beams branched by the device, a phase modulator for phase modulating one of the two beams at a predetermined frequency, and a phase modulator for combining the two beams. an irradiation optical system for guiding the combined light flux onto a test object to form a light spot; and a light spot formed by the irradiation optical system on the test object. a scanning means for scanning relative to the test object; a condensing optical system that guides light from the test object onto a photodetector; and modulation of the phase modulator from the output signal of the photodetector. An optical echo microscope characterized by comprising: signal processing means for extracting modulation components of even multiples of frequency. 2. The optical echo microscope according to claim 1, wherein the laser light source has a time coherence shorter than an optical phase relaxation time of a light absorption band of interest for the object to be examined. 3. The optical echo according to claim 1, wherein the relative scanning speed of the light spot on the object to be inspected by the scanning means is sufficiently slow relative to the phase modulation by the phase modulator. microscope. 4. The condensing optical system includes a light shielding member having a pinhole aperture disposed near the photodetector, and the test object and the pinhole aperture of the light shielding member are formed to be conjugate. The optical echo microscope according to claim 2 or 3, characterized in that: 5. A record in which the output signal from the signal processing means is recorded in correspondence with a light spot position of the irradiation optical system by the scanning means and a delay time by the optical delay device, and can be reproduced as necessary. 5. The optical echo microscope according to claim 3, further comprising a reproduction device. 6. A data analysis device capable of analyzing the amount of change in the output signal of the electrical signal processing device due to a change in the delay time of the optical delay device with respect to a light spot irradiation position by the irradiation optical system. The optical echo microscope according to any one of claims 1 to 5, characterized in that: 7. Light source means for supplying light having a spatial coherence and a predetermined temporal coherence that can be regarded as a point light source on an image plane, the light from the light source means being divided into two light beams, and one light beam being used as the other light beam. a light modulating optical means that delays the light relative to the target object and performs phase modulation and then multiplexing; and a light beam from the light modulating optical means is guided onto a test object to detect light from the test object. 1. An optical echo microscope comprising: a scanning microscope means for detecting light from the object to be examined; and a signal processing means for detecting light from the object to be examined and extracting a modulation component having an even multiple of the phase modulation frequency.