JP6000010B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope that performs high-speed observation and measurement of the surface shape of an opaque object by scanning with laser light, and observation and measurement of the surface or internal structure of a transparent object.

  Optical heterodyne interferometry is well known for measuring minute heights with high accuracy. This is because two laser beams with different frequencies interfere with each other, create a beat signal of the difference frequency, detect the phase change of the beat signal with a resolution of about 1/500 of the wavelength, It measures changes. As such, Japanese Patent Laid-Open No. 59-214706 of the following Patent Document 1 is specifically known, but in this Patent Document 1, two beams having different wavelengths are adjacent to each other using an acoustooptic device. And generating a surface profile by detecting the phase change between these two beams and accumulating the phase change.

  However, in this Patent Document 1, scanning is performed by a DC voltage applied to an acoustooptic device that is an optical modulator, and a sine wave signal is applied to the acoustooptic device to separate two spatially different frequencies from each other. Had created a beam. Here, the Bragg diffraction grating d of the acoustooptic device is d = Va / fa, where Va is the velocity of ultrasonic waves and fa is the applied frequency. That is, the Bragg diffraction angle and the applied frequency have an inversely proportional relationship.

  On the other hand, the resolution of ordinary microscopes is limited by the limits of so-called Abbe's theory. This limit is a result of the diffraction phenomenon that the wave has, and is regarded as a theoretical limit that cannot be exceeded. For example, a laser scanning microscope not only has a lateral resolution limit due to the limit of Abbe's theory, but also the resolution in the depth direction is not sufficient due to various limitations.

JP 59-214706 A

That is, in such a laser scanning microscope, when quantifying the depth direction information of the object to be measured, the depth direction information cannot be easily obtained, and three-dimensional There is a restriction that the depth at which information can be acquired is within the depth of focus of the objective lens.
The present invention has been made in view of the background described above, and can obtain a three-dimensional image by quantifying and obtaining information in the depth direction of an object, and also in an object having a height difference equal to or greater than the depth of focus. An object of the present invention is to provide a laser scanning microscope capable of acquiring quantified three-dimensional information.

In order to achieve the above object, the present invention provides a laser light source that emits laser light,
An optical modulator that emits light in different directions while modulating the laser light into two lights having mutually different frequencies;
A scanning optical element having a scanning element surface for one-dimensional scanning or two-dimensional scanning of the two lights, and scanning the two lights based on a control signal;
An objective lens that has a pupil position and emits two lights to the object being placed;
The diffracted light exit surface of the optical modulator and the pupil position of the objective lens are positioned so as to be conjugate with each other and positioned between the optical modulator and the objective lens and emitted from the optical modulator. A pupil transfer magnifying lens system that magnifies two lights;
Moving means for moving either the objective lens or the object along an optical axis that is an optical path of two lights, and relatively changing a distance between them;
First light reception comprising two or more divided light-receiving elements that receive reflected light or transmitted light from the object and perform photoelectric conversion, and are arranged along a separation direction of two lights generated by the light modulator. Elements,
Signal comparison means for summing or subtracting each signal photoelectrically converted by the first light receiving element and obtaining phase information and intensity information of the two lights based on the sum or difference;
A first data processing unit for constructing a three-dimensional image within the depth of focus of the objective lens from the phase information and intensity information of the signal comparison unit and the control signal of the scanning optical element;
A second data processing unit that constructs three-dimensional position information from the control signal of the scanning optical element and the amount of movement by the moving unit, based on the light intensity data of the reflected return light from the object;
A third data processing unit for constructing a three-dimensional image quantified by the three-dimensional image within the focal depth of the objective lens and the three-dimensional position information based on the light intensity data;
A laser scanning microscope characterized in that

  In the present invention, the laser light that is linearly polarized light emitted from the laser light source is converted into circularly polarized light, and the reflected return light that is circularly polarized light from the object is transmitted again to be orthogonal to the laser light. A quarter-wave plate for linear polarization, a polarization beam splitter for separating reflected return light whose polarization plane is orthogonal to the laser light from the optical path, and a lens for collecting the return light separated by the polarization beam splitter And a second light receiving element that obtains light intensity data by receiving and photoelectrically converting reflected return light that has passed through the spatial filter, and a spatial filter disposed on the focal plane of the lens. is there.

  Further, in the present invention, the scanning optical element is a first scanning optical element that scans the two lights along the diffraction direction of the optical modulator, and the two lights are orthogonal to the first scanning optical element. And a second scanning optical element that scans along a direction to be separated, and is disposed between the first scanning optical element and the second scanning optical element to separate reflected return light from the object A beam splitter, a lens for focusing the return light separated by the beam splitter to form an image, and a second light receiving element positioned at the focal plane of the lens and generated by the light modulator. And a two-dimensional image sensor composed of a plurality of divided light-receiving elements arranged along the separation direction, and the second data processing unit receives reflected light by the one-dimensional image sensor and photoelectrically converts the reflected return light. Based on light intensity data Are preferred those to construct a three-dimensional position information from the shift amount by the control signal and the moving means of said scanning optical element.

  Further, in the present invention, the laser light that is linearly polarized light emitted from the laser light source is converted into circularly polarized light, and the reflected return light that is circularly polarized light from the object is made transparent again to be orthogonal to the laser light. A quarter-wave plate for linearly polarized light, a polarized beam splitter that separates reflected return light whose polarization plane is orthogonal to the laser light, and a return beam separated by the polarized beam splitter. A lens and a pinhole disposed on a focal plane of the lens, and the first light receiving element can also serve as the second light receiving element by receiving light transmitted through the pinhole. It is preferable that the first data processing unit constructs a three-dimensional image within the depth of focus of the objective lens narrowed by the confocal optical system as the first light receiving element receives light.

On the other hand, it is preferable that the first light receiving element according to the present invention receives light that has passed through the object directly under the object.
An optical modulator according to the present invention includes an acousto-optic modulation element on which laser light emitted from the laser light source is incident, a signal generator that applies a carrier AC signal and a sine wave signal to the acousto-optic modulation element, A spatial light modulator on which laser light emitted from the laser light source is incident, and writing a sinusoidal lattice pattern as amplitude or phase information to the spatial light modulator, and applying a carrier AC signal and a sine wave signal And a signal generator for moving the lattice fringes in a certain direction.

The scanning optical element according to the present invention includes a one-dimensional scanning element using a galvano mirror or a resonant mirror, an optical scanning device using a nonlinear optical crystal or a photonic crystal, two one-dimensional scanning devices, and a pupil transmission magnifying lens system. A two-dimensional scanning optical system or a one-dimensional or two-dimensional micromirror device is suitable.
Furthermore, a photoelectric conversion unit that photoelectrically converts light received by the first light receiving element according to the present invention generates a beat signal, and the generated beat signal is divided into a plurality of divisions constituting the first light receiving element. What is acquired from the sum signal of all the divided light receiving elements of the light receiving elements or the difference signal between the divided light receiving elements at the corresponding positions of the plurality of divided light receiving elements is preferable.
The movement by the moving means according to the present invention is preferably a stage movement driven by a stepping motor, a direct movement by a piezo element, or a stage movement.

The operation of the claimed invention will be described below.
The optical modulator emits light in different directions while modulating the laser light emitted from the laser light source into two lights having different frequencies. In other words, when an acousto-optic modulator, which is an optical modulator, is driven by an electrical signal of frequency fc and frequency fm, two beams having frequency fc + fm and frequency fc-fm are generated by AM modulation using frequency fc as a carrier. To do. When the frequency fc, which is the carrier frequency, is set to about several tens of MHz and a frequency fm of about several MHz is given, the Bragg diffraction angle of the acousto-optic modulation element becomes considerably large.

  The light emitted from the acousto-optic modulation element becomes two beams having a large angle difference. This angular difference is made to enter the two-dimensional scanning optical system, which is a scanning optical element, by making the overlapping degree of the two beams remarkably small by a magnifying optical system that is a pupil transmission magnifying lens system. At this time, the entrance surface of the two-dimensional scanning optical system and the exit surface of the acoustooptic modulator are arranged in a conjugate manner.

In this way, the emitted light from the two-dimensional scanning optical system becomes a beam having a small angle difference from each other, but the frequencies possessed by fc + fm and fc-fm are not changed. That is, the two beams are scanned toward the objective lens via the scanning optical element that is the above-described scanning optical element regardless of the modulation frequency, or the scanning optical element that performs two-dimensional scanning and the second pupil transfer lens system. By doing so, the object is scanned with two adjacent beams.
When the target object is a reflective object, the beat signal by the two adjacent beams can be acquired by a light receiving element disposed at a position almost conjugate with the acousto-optic element, and the target object is a transmission object. In some cases, it can be obtained by a light receiving element arranged at a position that is far field but not far from the object.

  Further, the beat signal at this time is 2 fm, but this is detected as a beat signal as high as several tens of MHz. The phase difference θ between the beat signal and the reference signal reflects the substantial height d and refractive index difference n of the object. That is, if the laser wavelength is λ, there is a relationship of θ = 2πnd / λ.

Further, the signal comparison means creates each photoelectrically converted beat signal of the first light receiving element, the signal comparison means obtains the phase difference or intensity difference of the signals obtained based on the beat signal, and compares the signals. Based on the data obtained by acquiring the phase information or intensity information of the means, a first data processing unit that constructs a three-dimensional image within the focal depth of the objective lens processes.
Then, the second data processing unit constructs three-dimensional position information from the control signal of the scanning optical element and the amount of movement by the moving means based on the light intensity data of the reflected return light from the object, The three-dimensional image quantified by the three-dimensional image within the focal depth of the objective lens and the three-dimensional position information based on the light intensity data is constructed. From this, the surface profile is measured if the object is a reflective object, and the substantial refractive index difference or thickness is measured if the object is a transmissive object.

  As described above, according to the laser scanning microscope of the present invention, the optical system can be set regardless of the distance between the two beams and the frequency of the beat signal. Thus, the resolution and scanning speed can be significantly improved.

  On the other hand, when a spatial light modulator is used as the optical modulator, a strip-like sine wave grating is written in this spatial light modulator and moved in one direction at a high speed, so that the pitch of the lattice fringes changes the beam separation distance. It becomes. Since the phase is modulated by moving the grating one after another, the ± first-order diffracted light generated by the grating stripes can be light having different frequencies by twice the modulation frequency. In this case, if the pitch of the writing grating is sufficiently large, two adjacent beams can be produced, and the enlargement ratio of the enlargement optical system can be reduced, so that the optical system can be reduced in size.

  The beat frequency generating means and the scanning means can be separated by the optical system and the optical modulator as described above. For this reason, a higher-frequency beat signal can be created and the scanning speed can be further increased, so that data acquisition can be performed at a higher speed. As a result of the above, 3D information can be acquired at video rates and higher, and the horizontal resolution can be significantly improved by significantly reducing beam separation, and the resolution in the height direction is also about 1/500 of the wavelength. Can be improved.

  Further, since the two beams almost share the optical path, a laser scanning microscope apparatus that is extremely resistant to external environmental changes, vibrations, and the like can be obtained. When two beams exist in this way, if a divided light receiving element that is divided into two or more in the direction perpendicular to the beam separating direction is used as the light receiving element, the sum signal of the outputs of all the divided light receiving elements is Effectively, since the integral value of the region corresponding to the beam diameter of the phase difference corresponding to the degree of separation of the two beams collected by the objective lens is given, the resolution almost equivalent to that of the differential interference microscope is given.

  In order to further increase the resolution, if the difference signal between the light receiving elements adjacent to the divided light receiving elements divided into two or more parts is acquired, the separation between the two beams collected by the objective lens is effectively increased. The integral value of the area corresponding to the beam diameter of the differential of the corresponding phase difference is given. In this case, as compared with the sum signal, only the portion where the phase difference occurs contributes to the phase difference, and thus the sensitivity is remarkably increased. Accordingly, it is possible to improve the lateral resolution comparable to the resolution according to the beam separation degree.

  This is a distinguishing feature that is not found in ordinary differential interference microscopes. As a result, a lateral resolution much higher than the lateral resolution dominated by the wavelength can be obtained. A similar effect is obtained for the intensity of the beat signal.

  On the other hand, the vertical resolution in such a laser scanning microscope is very high in the focal depth region of the objective lens used, but it is not easy to quantify easily, and However, when the height difference of the object is large, it is not easy to obtain a three-dimensional image. In contrast, when focusing on the fact that the basic apparatus configuration of the present invention is a laser scanning type, the configuration of the confocal laser scanning microscope and the configuration of the present invention are the same, so a confocal optical system is incorporated. It is possible to obtain the tomographic imaging capability of the confocal laser scanning microscope.

  As described above, according to the laser scanning microscope according to the present invention, it is possible to realize a microscope apparatus having a resolution substantially equal to or higher than the diffraction limit, and further to realize an optical system that does not cause a decrease in beam utilization efficiency.

In summary, it is possible to provide a laser scanning microscope with high light utilization efficiency that obtains three-dimensional information such as height and refractive index distribution at a very high speed with one two-dimensional scanning and with extremely high lateral resolution. it can. Therefore, such as a conventional laser scanning confocal microscope that obtains two-dimensional information and integrates it in a three-dimensional direction, such as three-dimensional measurement in real time of the state of living cells and micromachines. Has significant features that cannot be compared.
In addition, if the transmission type is used, living organisms and cells can be observed and measured in real time and with high resolution, which is a major feature not available in an electron microscope that inactivates and measures cells and the like.

As described above, according to the laser scanning microscope of the present invention, an acousto-optic modulation element or a spatial modulator capable of modulation is used as an optical modulator, and the pupil transfer magnification lens system and a two-dimensional scanning device are used in combination. Thus, two very close beams can be modulated with a very high modulation frequency, so that three-dimensional measurement of the video rate is possible. Further, for example, the beam is separated by applying a modulation signal to the acousto-optic modulation element, and the sum calculation of all the divided light receiving elements is performed using two or more divided light receiving elements divided along the beam separation direction. Alternatively, extremely high lateral resolution can be obtained by performing a difference calculation between corresponding light receiving elements and performing heterodyne detection.
In other words, a laser scanning microscope capable of acquiring three-dimensional information having a resolution that greatly exceeds the Abbe diffraction limit in one two-dimensional scan by intensity information and phase information obtained by orthogonally transforming the signal obtained by heterodyne detection. Is feasible.

  Further, in such a laser scanning microscope, a confocal laser scanning type is incorporated by incorporating a light receiving optical system of the confocal optical system and a moving means for moving the objective lens or the sample stage in the depth direction with high resolution. A tomographic imaging capability of an optical microscope enables quantification in the depth direction and realizes a laser scanning microscope capable of measuring an object having a height difference exceeding the depth of focus of an objective lens.

  From the above, it has a very high three-dimensional resolution originally within the depth of focus of the objective lens. Therefore, according to the present invention, the three-dimensional resolution is much higher than that of the conventional confocal laser scanning optical microscope. A very high speed laser scanning microscope can be provided. Furthermore, the present invention makes it possible to observe fine structures that could only be observed with an electron microscope or a scanning probe microscope. In the transmission type, living organisms and cells can be observed in real time with high resolution. Since it can measure, it has the big characteristic which an electron microscope which inactivates a cell etc. and measures is not.

  Therefore, it is possible to observe and measure the state change of cells and microorganisms, the transient change of the surface state, etc. at high speed. In addition, it is possible to display 3D 3D images at video rates by using 3D displays that have already been commercialized and 3D displays using polarized glasses, which is useful in education, research, and medicine. It can be a device.

  On the other hand, since two beams passing through almost the same path that are very close to each other are used, observation and measurement that are not easily affected by disturbances and the like can be performed. In addition, the light receiving element is divided, and at least two light receiving elements are used in the beam separating direction, and the sum calculation of all the light receiving elements or the difference calculation between the corresponding light receiving elements is performed to perform heterodyne detection. This makes it possible to obtain a very high lateral resolution, particularly in the difference calculation.

It is a block diagram which shows Example 1 which concerns on the laser scanning microscope of this invention. It is a figure which expands and shows the objective lens of FIG. 1, and a measurement object peripheral part. It is explanatory drawing showing the irradiation area | region in the target object by Example 1 which concerns on the laser scanning microscope of this invention. It is a figure which shows typically the light which returns to a sample shape and a division | segmentation light receiving element. It is a figure which shows the phase information and intensity | strength information about the sample shape demonstrated in FIG. It is a figure explaining the sample shape and the information of the height direction obtained by this invention. It is a block diagram which shows Example 2 which concerns on the laser scanning microscope of this invention. It is a block diagram which shows Example 3 which concerns on the laser scanning microscope of this invention. It is a block diagram which shows Example 4 which concerns on the laser scanning microscope of this invention. It is a block diagram which shows Example 5 which concerns on the laser scanning microscope of this invention. It is a block diagram which shows Example 6 which concerns on the laser scanning microscope of this invention.

  Embodiments 1 to 6 of the laser scanning microscope according to the present invention will be described below in detail with reference to the drawings.

A laser scanning microscope according to a first embodiment of the present invention will be described below with reference to the drawings.
FIG. 1 is a block diagram illustrating a configuration of a laser scanning microscope according to the present embodiment. As shown in FIG. 1, as an optical system, a collimator lens 2 is provided between a laser light source 1 from which laser light is emitted and an acousto-optic modulation element (AOM) 4 that is an optical modulator for separating the light. In addition, a beam shaping optical system 3 is arranged. The laser light source 1 and the acousto-optic modulation element 4 are connected to a control board 14, and the operation is controlled by the control board 14. A signal generator built in the control board 14 applies a frequency fc as a carrier AC signal and a modulation frequency fm as a sine wave signal to the acousto-optic modulation element 4.

  Further, the first pupil transfer magnifying lens system 5 composed of two groups of lenses, the limiting aperture 7 for optimizing the beam diameter, and the reflected return light from the object S described later with respect to the acoustooptic modulator 4. Polarizing beam splitter 8 that divides and emits light into a confocal optical system, ¼ wavelength plate 9 that rotates the polarization plane of laser light that is linearly polarized light by 45 °, and light phase and intensity information from reflected return light A beam splitter 10 for separating light is arranged in order on the light receiving element. A pinhole 6 for cutting unnecessary non-diffracted light and high-order diffracted light passing outside the optical axis L is disposed between the two groups of lenses constituting the first pupil transmission magnifying lens system 5. .

  Further, a first one-dimensional scanning device 11 for one-dimensionally scanning the input laser beam is arranged on the right side in FIG. 1 with respect to the beam splitter 10, and the first one-dimensional scanning device 11 is illustrated. 1, a second pupil transmission magnifying lens system 12 composed of two groups of lenses and a second one-dimensional scanning device 13 that deflects light in a direction orthogonal to the first one-dimensional scanning device 11 are arranged. ing.

  In the present embodiment, the beams whose emission directions are changed downward in FIG. 1 by the first one-dimensional scanning device 11 pass through these in order, and these first one-dimensional scanning device 11 and second The pupil transmission magnifying lens system 12 and the second one-dimensional scanning device 13 constitute a two-dimensional scanning optical system. In addition, a third pupil transmission magnifying lens system 15 composed of two groups of lenses and an objective lens 16 facing the object S are arranged below the second one-dimensional scanning device 13.

The light reflected by the sample S returns through the optical path opposite to that described above and enters the beam splitter 10. The optical axis L is arranged in a direction perpendicular to the direction in which the optical axis L passes and is adjacent to the beam splitter 10, and is divided into at least two parts with respect to the optical axis in the beam separation direction separated by the acoustooptic modulator 4. The incident light enters the first light receiving element 18. The first light receiving element 18 is connected to the photoelectric conversion unit 19, is connected to a signal comparator 20 that amplifies the signal to an appropriate magnitude and compares the signal from the photoelectric conversion unit 19, and the first data processing unit 21. It is connected to the.
The first data processing unit 21 controls the control signals of the acousto-optic modulation element 4 and the one-dimensional scanning devices 11 and 13 controlled by the control board 14, and signals of intensity information and phase information from the signal comparator 20. A profile of the object S is created based on the above. That is, the first data processing unit 21 obtains a three-dimensional image within the focal depth of the objective lens 16 based on the intensity information and the phase information.

  On the other hand, the light reflected by the object S and transmitted through the beam splitter 10 of the reflected return light returning from the object S is incident on the quarter-wave plate 9 again. The reflected return light beam returns to linearly polarized light by being transmitted through the quarter-wave plate 9 again. However, the polarization plane is rotated by 90 ° with respect to the illumination light beam. It is reflected and separated from the optical path of the illumination beam.

  The light beam separated by the polarization beam splitter 8 is transmitted through a converging lens 22 and a pinhole 23 that is a spatial filter having a diameter that is about the diffraction limit spot diameter of the converging lens 22 installed on the focal plane of the converging lens 22. The light receiving element 24 receives the light. Since the focal plane of the converging lens 22 is a conjugate position with the focal plane of the objective lens 16, the light beam is transmitted through the pinhole 23 only when the object S, which is the observation sample, is focused, and the light receiving element 24. Is received. For light from a location where the focal position is deviated, the luminous flux is larger than the pinhole diameter on the surface of the pinhole 23, so that the amount of light received by the light receiving element 24 is remarkably reduced. improves.

The signal received by the light receiving element 24 is amplified to an appropriate magnitude in the photoelectric conversion unit 25, and the photoelectric conversion unit 25 is connected to the second data processing unit 27.
In the present embodiment, a moving mechanism 26 that is a moving unit that moves the objective lens 16 along the optical axis L with high accuracy while being controlled by the control circuit 14 is disposed. As this moving mechanism 26, for example, a stage movement by driving a stepping motor or a direct or stage movement by a piezo element may be considered.
As the objective lens 16 is moved by the moving mechanism 26, the light receiving element 24 receives light for each pixel from the position information of the one-dimensional scanning devices 11 and 13 from the control circuit 14 and the position information of the moving mechanism 26. The second data processing unit 27 performs processing for obtaining a focused position where the light intensity is the highest, thereby constructing three-dimensional position information. However, the moving mechanism 26 may be a mechanism that moves the object S instead of moving the objective lens 16, and in this case, the same effect as described above can be obtained.

A three-dimensional image within the depth of focus of the objective lens 16 based on intensity information and phase information obtained in the first data processing unit 21, and three-dimensional position information obtained in the second data processing unit 27 Are processed in the third data processing unit 31 and quantified in the depth direction with the same resolution as that of the confocal laser scanning microscope. From this, the profile information on the surface can be easily derived, and the profile information on the surface can be displayed on the display device 32 sent from the third data processing unit 31. In this embodiment, the first data processing unit 21, the second data processing unit 27, the third data processing unit 31, and the like are separate from each other. A processing unit may be used.
As described above, the measurement can be performed even on the object S having a height difference deeper than the focal depth of the objective lens 16. Note that the third data processing unit 31 has a function of saving the obtained three-dimensional image data as electronic data.

  On the other hand, the laser light source 1 described above is a gas laser such as He—Ne, a semiconductor laser, or a solid-state laser, and generates coherent laser light. The laser light is converted into a parallel light beam by the collimator lens 2 and is incident on the acousto-optic modulation element 4 with an optimum diameter using the beam shaping optical system 3. A DSB modulation signal such as sin (2πfct) sin (2πfmt) is applied to the acousto-optic modulation element 4 as a modulation signal from the control board 14.

When such modulation is performed, the acousto-optic modulation element 4 to which two frequency modulations of fc + fm and fc-fm have been added generates acoustic dense waves corresponding to the pitch d of the Bragg diffraction grating. . That is, if the velocity of the ultrasonic wave is Va and the applied frequency is f, d = Va / f. Specifically, the beam, which is laser light incident on the acousto-optic modulator 4, is separated into ± first-order diffracted light by the rough wave, and each diffracted light is modulated at a frequency of frequency fc ± fm. However, for example, when TeO ₂ is used as the material of the acoustooptic modulator 4, the sound velocity in the crystal is 4200 m / s.

  When 80 MHz is selected as the frequency fc of the carrier frequency, the pitch is d = 52.5 μm, and when the He-Ne laser is used for the laser light source 1, the diffraction angle θ is about 1.38 °. When fm of frequency 8 MHz is added to this carrier frequency, ± 1st order diffracted light is separated into two beams of θ = 1.38 ° ± 0.138 ° with a separation angle Δθ of 0.276 ° and is modulated at 88 MHz and 72 MHz, respectively. Become.

Next, a method for reducing the separation angle Δθ on the entrance pupil plane of the objective lens 16 will be described.
The incident angle from the acousto-optic modulator 4 to the first pupil transfer magnifying lens system 5 is inclined by θ so that the diffracted light passes through the optical axis L of the first pupil transfer magnifying lens system 5. Further, as the first pupil transfer magnifying lens system 5 is composed of two groups of lenses, the focal length of the incident side lens group is fin and the focal length of the exit side lens group is fout.

  When the exit surface of the acousto-optic modulator 4 is placed at the focal position of the entrance-side lens group of the first pupil transfer magnifying lens system 5 and the distance between the two lens groups of the first pupil transfer magnifying lens system 5 is fin + fout, The afocal optical system in which the position of the exit surface of the optical modulation element 4 is conjugate to the position of the exit side fout is the deflection center of the first one-dimensional scanning device 11. Further, the pinhole 6 is provided in the vicinity of the intermediate focal plane of the first pupil transmission magnifying lens system 5 so as to transmit only the light beam in the vicinity of the optical axis L, and the pinhole 6 does not need to pass outside the optical axis L. Cut non-diffracted light and higher-order diffracted light.

  Here, when fin <fout, the magnification optical system becomes fout / fin = m1 times, and the separation angle Δθ formed by the acoustooptic modulator 4 can be reduced to 1 / m1. On the other hand, since the beam diameter is enlarged by m1 times, the limiting aperture 7 is provided after the exit side lens of the first pupil transmission magnifying lens system 5 in consideration of the vignetting phenomenon that is caused by the decrease in the amount of peripheral light caused by the subsequent optical system. Inserted to optimize beam diameter.

  The second pupil transmission magnifying lens system 12 has the same configuration as that of the first pupil transmission magnifying lens system 5 described above, and this second pupil transmission magnifying lens system 12 emits from the acoustooptic modulator 4. The surface position and the deflection center of the first one-dimensional scanning device 11 and the deflection center of the second one-dimensional scanning device 13 are kept in a conjugate positional relationship, and an enlargement magnification of m2 which is a magnification larger than 1 is given.

  The light beam emitted from the second pupil transmission magnifying lens system 12 is incident on a second one-dimensional scanning device 13 that deflects the light beam in a direction orthogonal to the deflection direction of the first one-dimensional scanning device 11, thereby The light beam is scanned two-dimensionally. The first one-dimensional scanning device 11 and the second one-dimensional scanning device 13 are connected to the control board 14 and controlled so as to operate in synchronization with the laser light source 1 and the acousto-optic modulation element 4. It is like that.

  The light beam emitted from the second one-dimensional scanning device 13 enters the third pupil transmission magnifying lens system 15 having the same configuration as that of the first pupil transmission magnifying lens system 5 described above. The third pupil transfer magnifying lens system 15 uses the position of the exit surface of the acoustooptic modulator 4, the deflection center of the first one-dimensional scanning device 11, and the deflection center of the second one-dimensional scanning device 13 as the objective lens 16. While maintaining a positional relationship conjugate with the entrance pupil plane, an enlargement magnification of m3 which is a magnification larger than 1 is given.

  As described above, the acoustooptic modulation element is obtained by the optical magnification of m1 × m2 × m3 = m4 times by the first pupil transmission magnifying lens system 5, the second pupil transmission magnifying lens system 12, and the third pupil transmission magnifying lens system 15. 4 is reduced to Δθ / m 4, and the two beams separated by the acoustooptic modulator 4 are incident on the objective lens 16. Thereby, even if the modulation frequency fm of the acousto-optic modulation element 4 is increased, the minute spot formed by the two beams on the surface of the object S can be very close to illuminate the object S. In this way, two beams that are very close to each other and have the same diameter can be obtained, such as a beam LA indicated by a solid line and a beam LB indicated by a dotted line in FIG.

  Further, the frequency of these two beams LA and LB is “light frequency + carrier frequency fc ± modulation frequency fm”. Here, it is assumed that the center distance Δx of the minute spot formed on the surface of the object S by the two close beams LA and LB from the objective lens 16 is set to be equal to or less than the diffraction limit spot diameter K. In this case, each spot does not fall below the diffraction limit of Abbe's theory, but because it is light of a different frequency that is slightly shifted, by performing heterodyne detection, the sum signal or difference signal is obtained. A beat signal can be generated and differential information can be acquired.

  The light receiving element 18 shown in FIG. 1 is a plurality of divided light receiving elements divided into two or more parts along the beam LA and LB separation direction shown in FIG. For example, when the boundary line C shown in FIGS. 2 and 3 extending in a direction perpendicular to the separation direction of the beams LA and LB is formed on the optical axis L, a dark line is formed in parallel to the boundary line C. A plurality of these light receiving elements are arranged, and a beat signal is obtained from the sum signal or difference signal. In this case, scanning the beam using the sum signal shows the average phase difference in the beam, which is substantially equivalent to the differential interference microscope, and scanning the beam using the difference signal corresponds to the beam separation degree. Therefore, it is possible to acquire phase difference information and position information with high lateral resolution. The improvement of the lateral resolution will be described later in detail.

First, speeding up of information acquisition will be described. As shown in FIG. 2, the two beams LA and LB focused by the objective lens 16 become two adjacent spots A and B (shown in FIG. 3). The complex amplitude Ea of the spot A and the complex amplitude Eb of the spot B are expressed by the following equations.
Ea = Aexpj (2π (fo + fc + fm) t)
Eb = Bexpj (2π (fo + fc-fm) t + δ)
Δ in the expression of this complex amplitude Eb represents the phase difference in the height direction of the spot B of the beam LB with respect to the spot A of the beam LA, and fo represents the frequency of light. As described above, the interval between the two spots is determined by the modulation frequency fm applied to the acousto-optic modulation element 4 and the magnification m4 of the magnifying optical system, and is thus independent of the scanning speed.

  On the other hand, the two beams LA and LB reflected by the object S return on the optical path opposite to the illumination light beam described above. That is, by returning the objective lens 16, the third pupil transfer lens system 15, the second one-scanning device 13, the second pupil transfer lens system 12, and the first one-scanning device 11 in order, the deflection component Is canceled and becomes axial light, reflected by the beam splitter 8 and guided to the light receiving element 18. The light receiving element 18 is divided into at least two along the direction in which the beams LA and LB are separated by the acousto-optic modulation element 4. These two or more light receiving elements are each constituted by a photodiode, a photomultiplier tube (PMT), or the like.

  When each of the divided light receiving elements constituting the light receiving element 18 is a photodiode, the cutoff frequency is determined in inverse proportion to the inter-terminal capacitance of the element. Since the capacitance between terminals is generally proportional to the light receiving area, it is desirable to use a photodiode having a small light receiving area in order to increase the speed of the light receiving system. Along with this, a plurality of small photodiodes with a small inter-terminal capacitance are arranged on the array to increase the speed, and the photoelectric conversion unit 19 performs electrical amplification corresponding to each photodiode on a one-to-one basis, The signal comparator 20 can generate a sum signal or a difference signal with a combination of elements as required.

  In the first data processing unit 21, the signal from the signal comparator 20 is heterodyne detected using the modulation signal of the acousto-optic modulation element 4 from the control board 14, and the intensity information and the phase information are obtained by substantial orthogonal transformation. obtain. Further, a three-dimensional image within the focal depth of the objective lens 16 is constructed based on the intensity information and phase information described above in accordance with each control signal from the control board 14 to the one-dimensional scanning devices 11 and 13.

Hereinafter, the relationship between the shape of the object S and the light returning to the divided light receiving elements 18A and 18B of the light receiving element 18 will be described in detail with reference to FIGS. In FIG. 4, the solid line indicates the beam LA, and the dotted line indicates the beam LB. In FIG. 4, the spots A and B by the two beams LA and LB do not appear originally, but in order to facilitate understanding, the spot A is indicated by a solid circle and the spot B is indicated by a dotted circle.
FIG. 4 schematically shows the “sample shape” that is the shape of the object S and the light that returns to the divided light receiving elements 18A and 18B. That is, the light receiving element 18 is constituted by the divided light receiving element 18A and the divided light receiving element 18B, and the signal value of the divided light receiving element 18A is a, and the signal value of the divided light receiving element 18B is b. . Then, the light incident on the plane portion of the object S enters the divided light receiving elements 18A and 18B symmetrically, and if the difference between them is taken, the value of b−a becomes zero.
On the other hand, in FIG. 4A, spots A and B are located on the gentle upward slope of the object S, and the return light from the gentle upward slope of the object S is divided light receiving elements. Since it is biased toward the 18B side, the value of ba becomes positive.

On the other hand, in FIG. 4B, spots A and B are located across a gentle downward slope and a sharp downward slope. In this case, the return light from the gentle downward sloping slope is biased toward the divided light receiving element 18A, and the value of b−a becomes negative. The return light from the steeply downward slope is further biased toward the divided light receiving element 18A, and the value of ba becomes a larger negative value.
At this time, since the phase information obtained from the signal obtained by the light receiving element 18 is differential information, the sign is maintained, and the intensity information is the absolute value thereof, so the sign is positive.

  FIG. 5 is a diagram illustrating a case where the “sample shape” described in FIG. 4 is represented by phase information and intensity information. As described above, since the phase information is a differential value and represents the inclination of the slope of the object S, the shape of the object S can be obtained from the phase information and the intensity information.

  In the above description, the case where two divided light receiving elements are applied has been described. However, the same discussion can be made when a plurality of light receiving elements are separated from the object S in the beam separation direction with the optical axis L as a boundary. In order to obtain a difference output, it is only necessary to perform it between corresponding light receiving elements with the optical axis L as a boundary. If only the sum output of a plurality of light receiving elements is used, the same can be realized by using one light receiving element.

  On the other hand, the optical system is two orthogonal one-dimensional scanning devices coupled by the pupil transmission optical system, but may be a two-dimensional scanning device. Further, the pupil transmission optical system emits these scanning devices and the objective lens 16. It is configured to connect with the pupil plane. Since such a configuration is the same as a typical configuration of a confocal laser scanning microscope, a confocal optical system that is a light receiving optical system of the confocal laser scanning microscope can be incorporated.

The light receiving element 18 detects the phase difference δ between the two beams LA and LB as a beat signal. That is, the intensity I of the two beams on the light receiving element 18 is detected by the photoelectric conversion unit 19 of the light receiving element 18 with a value based on the following expression, and is sent to the signal comparator 20.
I = (Ea + Eb) (Ea + Eb) ^* = A ² + B ² + 2ABcos (2π * 2fmt + δ)
Therefore, the phase difference δ can be measured by performing the phase comparison of the heterodyne detection at the frequency 2fm using the signal comparator 20. In this way, since the modulation frequency fm can be increased and the beam can be made very close, the lateral resolution can be increased and at the same time data can be acquired at high speed.

  That is, the time for performing the phase comparison is inversely proportional to the modulation frequency fm. For example, if data of 1000 points or more is to be acquired at a video rate (horizontal scanning frequency of about 16 KHz), the frequency of information acquisition at one point Is 16 MHz. If the modulation frequency fm is set to 8 MHz, the beat frequency becomes 16 MHz, so that information can be sufficiently acquired at the video rate.

Hereinafter, the depth of focus of the objective lens will be described with reference to FIG.
When the height of the object S, which is a cross-sectional object having a height exceeding the “objective depth of focus” as shown in FIG. 6A, is measured, the result is as follows.
In a conventional laser scanning microscope, a three-dimensional shape can be acquired as a continuous value with a high lateral resolution as in the “conventional lateral resolution” shown in FIG. Therefore, it is necessary to shift the range of the “focus depth of objective lens” to acquire two images, and accordingly, it is difficult to quantify the shape in the depth direction.

  On the other hand, as shown in FIG. 6C, the confocal optical system can acquire numerical information in accordance with the “confocal system height resolution” along the height direction. Height information can be acquired as coordinates. Therefore, by synthesizing the three-dimensional shape of FIG. 6B and the height information of FIG. 6C, as shown in FIG. A resolution image can be acquired.

  As described above, according to this embodiment, a laser capable of acquiring three-dimensional measurement data whose lateral resolution exceeds the Abbe diffraction limit and whose depth direction is quantified with the same resolution as that of a confocal laser scanning microscope. A scanning microscope can be obtained. Therefore, it is possible to observe and measure an object that has been difficult to observe with various conventional optical microscopes and could only be observed with an electron microscope or a scanning probe microscope.

On the other hand, by using a commercially available autostereoscopic display or a three-dimensional display using polarized glasses, it is possible to display a three-dimensional image at a video rate, which is useful in education, research, and medicine. It can be a device. Further, since the degree of overlap of the two beams is made smaller than the beam diameter, there is almost no difference in path between the two beams. Therefore, the influences of disturbance and vibration are also generated simultaneously by the two beams, and these influences are canceled out.

  On the other hand, in the present embodiment, an example in which the beam separation degree is much smaller than the individual beam diameters has been shown. However, by increasing the modulation frequency, the beam separation degree is increased and the beam diameter is approximately equal to the beam diameter. The optical system of the present invention is also useful when a degree of separation is required.

  In the above-described embodiment, the description has been given of the configuration in which two-dimensional scanning is performed by arranging two one-dimensional scanning devices orthogonally. However, if the application requires simple data in only one direction, 1 A similar effect can be obtained even in a system using only one stage of the dimension scanning device. As the one-dimensional scanning device, a micro mirror device, a resonant mirror, a galvano mirror, a rotating polygon mirror, or the like using a micromachine technique can be used. Furthermore, a high-speed scanner using a nonlinear optical crystal or a photonic crystal, which has been developed in recent years, can be used.

  In addition, in the micromirror device using the micromachine technology, there is a device capable of two-dimensional scanning with one device, but if this can be used, the second pupil transmission magnification optical system 12 is required. Disappear. Accordingly, the magnification becomes smaller by m2, but if the magnifications m1 and m3 of the first pupil transmission magnification optical system and the third pupil transmission magnification optical system are increased, the magnification magnification m4 can be maintained. Become.

  In the above, the means for acquiring data at high speed has been described. Next, means for significantly increasing the lateral resolution will be described.

Think in one dimension for simplicity. First, the phase distribution of the surface profile d (x) is set to Ae ^{jθ (x)} . Here, θ (x) = 2πd (x) / λ. In the case of reflection as in this embodiment, the optical path difference is doubled, so half of the observed θ (x) may be used as height information.
Now, when the acousto-optic modulation element is given a product signal (DSB modulation) of the carrier signal fc and the modulation signal fm as described above, the diffracted light is effectively light having two slightly separated frequencies of fc ± fm. It becomes. When converged by the objective lens, it becomes two beams separated by Δx, and each beam profile is u (x). In this case, the Fourier transform of the product of the surface profile and the beam profile is performed at a location away from the objective lens.

In this laser scanning microscope, the beam received by one light receiving element is modulated by ej ^{(ωc-ωm) t} , and is received by the other light receiving element separated by the center distance Δx. This beam is modulated by ej ^{(ωc + ωm) t} . Therefore, the complex amplitude distribution on the light receiving element is as follows.
E = ∫ (Ae ^{jθ (x)} u (x) e ^jkx dx · e ^{j (ωc−ωm) t} + Ae ^{jθ (x + Δx)} u (x) e ^jkx dx · e ^{j (ωc + ωm) t} .

When the intensity I is detected by these light receiving elements, I = EE ^* and further 2 ωm heterodyne detection is performed, so the following equation (1) is obtained.
I (k) = A ² ∫e ^{j (θ (x) −θ (x ′ + Δx ′)} u (x) u (x ′) e ^{jk (x−x ′)} dxdx′e ^−j2ωmt
+ A ² ∫e ^{−j (θ (x) −θ (x ′ + Δx ′)} u (x) u (x ′) e ^{jk (x−x ′)} dxdx′e ^j2ωmt (1)

Then, the approximate center of the irradiation areas A and B where the two beams LA and LB overlap each other is defined as a boundary line C in FIG. 3, and is a position sandwiching the boundary line C and is a separation direction of the beams LA and LB. Two light receiving elements are arranged away from the object S in correspondence with the positions along the separation direction of the respective irradiation areas A and B.
First, consider what the sum signal of the two light receiving elements will be. Since it is considered to be a Fourier transform plane at a position away from the object S, if the maximum spatial frequency that can be received by the light receiving element is Kmax, the intensity I is obtained from the following equation for the sum signal.
I = ∫I (k) dk (integration range is -Kmax to Kmax)
= A ² ∫cos (θ (x) −θ (x ′ + Δx ′) − 2ωmt) u (x) u (x ′) sin (Kmax (x−x ′)) / (x−x ′) dxdx ′

If the light receiving element is placed close to receive light up to a wider spatial frequency,
Since sin (Kmax (x−x ′)) / (x−x ′) = Kδ (x−x ′), the following equation (2) is obtained.
I = A ² ∫cos (θ (x) −θ (x + Δx) −2ωmt) u (x) ² dx (2)

That is, the phase difference between the separation positions of the two beams is integrated by the weight of the beam profile.
When the equation (2) is modified, the following equation is obtained.
Iq = A ² ∫cos (θ (x) −θ (x + Δx) u (x) ² dx · cos (2ωmt)
Ii = A ² ∫sin (θ (x) −θ (x + Δx) u (x) ² dx · sin (2ωmt)

Accordingly, the observed phase difference Θ is represented by the following equation (3) by orthogonal transformation.
Θ = tan ⁻¹ (∫sin (θ (x) −θ (x + Δx)) u (x) ² dx / ∫cos (θ (x) −θ (x + Δx)) u (x) ² dx) ... (3)

On the other hand, considering the difference signal between the two light receiving elements, the following equation is obtained in the same manner as in the case of the sum signal.
I = ∫I (k) dk (integration range is 0 to Kmax) −∫I (k) dk (integration range is −Kmax to 0)
= A ² ∫sin (θ (x) −θ (x ′ + Δx ′) − 2ωmt) u (x) u (x ′) (cos (Kmax (x−x ′) − 1) / (x−x ′ dxdx '

If it is arranged to receive light up to a wider spatial frequency with the light receiving elements close to each other,
Since (cos (Kmax (x−x ′) − 1) / (x−x ′) = δ ′ (x−x ′) + 1 / x (δ (x) −1), the following equation (4) become that way.
I = A ² ∫d / dx (sin (θ (x) −θ (x + Δx) −2ωmt)) u (x) ² dx (4) Equation (4) Then, it becomes as follows.
Iq = A ² d / dx (sin (θ (x) −θ (x + Δx)) u (x) ² dx · cos (2ωmt)
Ii = −A ² ∫d / dx (cos (θ (x) −θ (x + Δx)) u (x) ² dx · sin (2ωmt)

Therefore, the phase difference Θ observed by the orthogonal transformation is expressed by the following equation (5).
Θ = tan ⁻¹ (−∫d / dx (cos (θ (x) −θ (x + Δx)) u (x) ² dx / ∫d / dx (sin (θ (x) −θ (x + Δx )) u (x) ² dx) (5)

Here, the expressions (3) and (5) are compared. The following points are qualitatively understood.
First, equation (3) shows the phase difference obtained as a result of smoothing the phase difference between two points separated by the center distance Δx of the beam with the weight function of u (x). The phase difference is shown. This is a process equivalent to a differential interference microscope.

  On the other hand, in equation (5), the differential of the phase difference between two points separated by the beam center distance Δx is smoothed by the weight function of u (x), so the original function is roughly restored. Will be. Therefore, when the beam is scanned, the phase difference and the position information can be acquired with a lateral resolution corresponding to the degree of beam separation.

  Here, the case where a two-part light receiving element is applied has been described, but a plurality of light receiving elements are separated from the object S along the separation direction of the two beams in the vicinity of the center of the overlapping area of the irradiation areas A and B. The same applies to the case where they are arranged. In particular, when a difference output is obtained, a difference calculation may be performed between a plurality of corresponding light receiving elements among a plurality of light receiving elements arranged corresponding to the vicinity of the center of the optical axis L. If only the sum output of a plurality of light receiving elements is used, the same can be realized by using one light receiving element.

  As described above, by processing the spatial frequency information on the Fourier transform plane, it is possible to bring about a very high lateral resolution improvement, particularly in the difference calculation. In addition, if the profile has an inclination in the beam, the direction in which light is reflected or transmitted is qualitatively different. Therefore, it is easily considered that a difference output as intensity is given to the two light receiving elements. More specifically, for changes in the profile smaller than the beam diameter, interference fringes formed by interference between the zeroth-order diffracted wave and the first-order diffracted wave of the Fourier transform in the region irradiated with light. Since the pattern in the far field is different between the two light receiving elements, the difference signal of the light receiving elements appears as an intensity difference reflected in the inclination of the profile.

In this embodiment, the insertion position of the confocal optical system described in the first embodiment is changed.
FIG. 7 is a block diagram showing the configuration of the embodiment of the present invention. In this embodiment, the beam return beam 10, the focusing lens 22, the pinhole 23, the light receiving element 24, and the photoelectric conversion unit 25 that existed in the first embodiment are removed, and the reflected return light reflected and separated by the polarization beam splitter 8 is used. Is sent to the light receiving element 18.

  In other words, the polarization beam splitter 8 is arranged to transmit the illumination light, which is a laser beam, and the reflected return light transmitted through the quarter-wave plate 9 is reflected at a right angle by the polarization beam splitter 8 and is reflected by the acoustooptic modulator 4. Is incident on the divided light receiving element of the light receiving element 18 that is divided into two symmetrically along the optical axis. In the photoelectric conversion unit 19, electrical amplification corresponding to each photodiode which is each divided light receiving element constituting the light receiving element 18 is performed on a one-to-one basis, and the signal comparator 20 uses a combination of elements as necessary to obtain a sum. A signal or difference signal is to be created.

  Along with this, in the first data processing unit 21, the signal from the signal comparator 20 is heterodyne detected using the modulation signal of the acousto-optic modulation element 4 from the control board 14, and the intensity is obtained by substantial orthogonal transformation. Get information and phase information. In addition, a three-dimensional image within the focal depth of the objective lens 16 is constructed based on the intensity information and the phase information in accordance with the control signals of the one-dimensional scanning devices 11 and 13 from the control board 14.

  On the other hand, in this embodiment, a beam splitter 33 is disposed between the second pupil transmission magnifying lens system 12 near the second one-dimensional scanning device 13 and the second one-dimensional scanning device 13. ing. The reflected return light from the object S returns on the optical path on the optical axis L, the scanning component by the second one-dimensional scanning device 13 is canceled, enters the beam splitter 33 again, and is reflected at a right angle, thereby being illuminated. Separated from the optical path. However, since the deflection component by the first one-dimensional scanning device 11 is not canceled, a linear light beam that is long in the deflection direction by the first one-dimensional scanning device 11 is obtained.

A one-dimensional imaging device 35 in which the imaging lens 34 is disposed in the separated return optical path and the light receiving elements having the diffraction-limited light receiving element size of the imaging lens 34 are arranged in the line-shaped light flux direction is connected to this connection. Arranged in the focal plane of the image lens 34, the photoelectric conversion unit 36 performs an appropriate amplification process.
However, the one-dimensional imaging element 35 may be a light receiving element array in which light receiving elements having the diffraction limited light receiving element size of the imaging lens 34 are arranged in the line direction.

  Since the focal plane of the imaging lens 34 is a conjugate position with the focal plane of the objective lens 16, the amount of light in the one-dimensional image sensor 35 is maximized only when the observation sample is focused, and the focal position is shifted. The light from the spot becomes larger than the size of the light receiving element on the surface of the one-dimensional image sensor 35, and the amount of received light is remarkably reduced.

As a result, the depth of focus becomes remarkably shallow, and the resolution in the height direction is improved.
The objective lens 16 is moved with high accuracy by the moving mechanism 26 similar to that of the first embodiment, and the position information of the one-dimensional scanning devices 11 and 13 from the control circuit 14 and the position information of the moving mechanism 26 are used to determine the above 1 for each pixel. The second data processing unit 27 performs processing for obtaining a focused position where the light intensity received by the two-dimensional image sensor 35 is highest, thereby constructing three-dimensional position information. Note that the effect is the same as a mechanism for moving the object S instead of moving the objective lens 16.

  A three-dimensional image obtained in the first data processing unit 21 within the focal depth of the objective lens 16 based on intensity information and phase information, and a three-dimensional position obtained in the second data processing unit 27 Information is processed in the third data processing unit 31 and quantified in the depth direction with the same resolution as that of the confocal laser scanning microscope, so that surface profile information can be easily derived. indicate.

  From the above, it is possible to measure even the object S having an elevation difference deeper than the focal depth of the objective lens 16. The data processing unit 31 has a function of storing the obtained three-dimensional image data as electronic data. Therefore, according to the present embodiment, laser scanning capable of acquiring three-dimensional measurement data whose lateral resolution exceeds Abbe's diffraction limit and whose depth direction is quantified with the same resolution as a confocal laser scanning microscope. A type microscope can be obtained.

In this embodiment, a confocal optical system is configured by the light receiving element 18.
FIG. 8 is a block diagram showing the configuration of the embodiment of the present invention. Also in the present embodiment, the beam splitter 10, the focusing lens 22, the pinhole 23, the light receiving element 24, and the photoelectric conversion unit 25 existing in the first embodiment are removed, and the reflection return reflected and separated by the polarization beam splitter 8. The light is sent to the light receiving element 18. Further, in the present embodiment, the focusing lens 22 on the optical axis of the reflected return light between the polarizing beam splitter 8 and the light receiving element 18, and the diffraction limited spot diameter of the focusing lens 22 on the focal plane of the focusing lens 22. The pinholes 23 having the diameters are arranged respectively.

  The polarization beam splitter 8 is arranged to transmit the illumination light, and the reflected return light transmitted through the quarter wavelength plate 9 is reflected by the polarization beam splitter 8 at a right angle. The reflected return light passes through the converging lens 22 and the pinhole 23 arranged on the optical axis, and immediately after the pinhole 23, the acoustooptic modulation element 4 divides the beam into a plurality around the optical axis along the direction in which the beam is separated. The divided light receiving element 18 receives the light beam that has passed through the pinhole 23.

  Since the focal plane of the converging lens 22 is a conjugate position with the focal plane of the objective lens 16, the light beam is transmitted through the pinhole 23 only when the object S, which is the observation sample, is focused, and the light receiving element. 18 is received. Further, the photoelectric conversion unit 19 performs electrical amplification corresponding to each photodiode which is each divided light receiving element constituting the light receiving element 18 on a one-to-one basis, and the signal comparator 20 performs summation by combining the elements as necessary. A signal or difference signal can be created.

Along with this, in the first data processing unit 21, the signal from the signal comparator 20 is heterodyne detected using the modulation signal of the acousto-optic modulation element 4 from the control board 14, and the intensity is obtained by substantial orthogonal transformation. Get information and phase information.
Further, in accordance with the control signals from the control board 14 to the one-dimensional scanning devices 11 and 13, the three-dimensional within the depth of focus of the objective lens 16 narrowed by the confocal optical system based on the intensity information and the phase information described above. Build an image.

For light from a location where the focal position is shifted, the light flux on the surface of the pinhole 23 becomes larger than the pinhole diameter, and the amount of light received by the light receiving element 18 is significantly reduced. As a result, the depth of focus is significantly shallower than that of a normal microscope, and the vertical resolution is greatly improved.
Further, the objective lens 16 is moved with high accuracy by the moving mechanism 26 similar to that of the first embodiment, and the focused height for each pixel is stored. Note that the effect is the same as a mechanism for moving the object S instead of moving the objective lens 16.

  The objective lens 16 is moved with high accuracy by the moving mechanism 26, and is received by the light receiving element 18 for each pixel from the positional information of the one-dimensional scanning devices 11 and 13 from the control circuit 14 and the positional information of the moving mechanism 26, The second data processing unit 27 performs processing for obtaining a focused position where the light intensity of the sum signal generated in the signal comparator 20 is the highest, thereby constructing three-dimensional position information.

  As described above, also in this embodiment, the three-dimensional image within the focal depth of the objective lens 16 narrowed by the confocal optical system based on the intensity information and the phase information obtained in the first data processing unit 21; The three-dimensional position information obtained in the second data processing unit 27 is processed in the third data processing unit 31 and quantified in the depth direction with a resolution equivalent to that of a confocal laser scanning microscope. Surface profile information can be easily derived and displayed on the display device 32.

  From the above, it is possible to measure even the object S having an elevation difference deeper than the focal depth of the objective lens 16. The data processing unit 31 has a function of storing the obtained three-dimensional image data as electronic data. Therefore, according to the present embodiment, laser scanning capable of acquiring three-dimensional measurement data whose lateral resolution exceeds Abbe's diffraction limit and whose depth direction is quantified with the same resolution as a confocal laser scanning microscope. A type microscope can be obtained.

In this embodiment, the reflection optical system described in the first embodiment is replaced with a transmission optical system.
FIG. 9 is a block diagram of a transmissive optical system showing the configuration of the embodiment of the present invention.
In the present embodiment, the main optical system deletes the beam splitter 10, the light receiving element 18, the photoelectric conversion unit 19, and the signal comparator 20 in the first embodiment, and instead of the light collected by the objective lens 16. Therefore, the light receiving element 28 is disposed on the opposite side of the objective lens 16 across the object S.

  That is, on the extension line of the optical axis of the objective lens 16, a divided light receiving element of the light receiving element 28 that is divided into a plurality of parts around the optical axis is arranged along the direction in which the beam is separated by the acousto-optic modulation element 4. In the photoelectric conversion unit 29, electrical amplification corresponding to each photodiode which is each divided light receiving element constituting the light receiving element 28 is performed on a one-to-one basis, and in the signal comparator 30, depending on the combination of the divided light receiving elements as necessary. Sum or difference signals can be created. In the first data processing unit 21, the signal from the signal comparator 30 is heterodyne detected using the modulation signal of the acousto-optic modulation element 4 from the control board 14, and the intensity information and the phase information are obtained by substantial orthogonal transformation. obtain.

Further, a three-dimensional image within the depth of focus of the objective lens 16 is constructed based on intensity information and phase information in accordance with the control signals of the one-dimensional scanning devices 11 and 13 from the control board 14.
In the case of the present embodiment, since the light receiving element 28 can be disposed closer to the object S than in the reflective type, the obtainable spatial frequency can be set very high. As a result, since the reproducibility of the spatial frequency of the object S is improved, the lateral resolution can be further improved. In particular, observation and measurement of living organisms and cells can be performed with very high resolution in the state of being alive. This is a feature that is greatly different from a measuring instrument such as an electron microscope that cannot be observed unless the living body is killed even at a high magnification.

  As described above, also in the present embodiment, the three-dimensional image within the focal depth of the objective lens 16 based on the intensity information and the phase information obtained in the first data processing unit 21 and the second data processing unit 27. The three-dimensional position information obtained in step 3 is processed in the third data processing unit 31 and is quantified in the depth direction with the same resolution as that of the confocal laser scanning microscope, and the surface profile information is easily derived. Can be displayed on the display device 32.

  From the above, it is possible to measure even the object S having an elevation difference deeper than the focal depth of the objective lens 16. The data processing unit 31 has a function of storing the obtained three-dimensional image data as electronic data. Therefore, according to the present embodiment, laser scanning capable of acquiring three-dimensional measurement data whose lateral resolution exceeds Abbe's diffraction limit and whose depth direction is quantified with the same resolution as a confocal laser scanning microscope. A type microscope can be obtained.

In this embodiment, the reflection optical system described in the second embodiment is replaced with a transmission optical system.
FIG. 10 is a block diagram of a transmissive optical system showing the configuration of the embodiment of the present invention. The main optical system in this embodiment is the same as that described in Embodiment 4 except that the polarizing beam splitter 8, the quarter wavelength plate 9, the light receiving element 18, the photoelectric conversion unit 19, and the signal comparator 20 in Embodiment 2 are omitted. Further, since the light receiving element 28 which is a light receiving system disposed on the opposite side of the objective lens 16 with the object S interposed therebetween is added, a detailed description is omitted.

This embodiment is an embodiment in the case of using both a reflection optical system and a transmission optical system.
FIG. 11 is a block diagram of an optical system showing the configuration of the embodiment of the present invention. Since the light receiving element 28 which is a light receiving system disposed on the opposite side of the objective lens 16 with the object S interposed therebetween is added to the reflecting optical system described in the first to third embodiments, a detailed description will be given. Omit. A typical example of the present embodiment is a configuration in which a transmission system is added to the configuration of the first embodiment shown in FIG. 11, and according to such a configuration, a reflected image is obtained with one laser scanning microscope. Each transmission image can be picked up and can be picked up at the same time.

  The embodiments according to the present invention have been described above, but the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention.

  The present invention is suitable for various types of microscopes as well as laser scanning microscopes that perform high-speed observation and measurement of the surface shape of opaque objects by scanning with laser light, and observation and measurement of the surface or internal structure of transparent objects. Is.

DESCRIPTION OF SYMBOLS 1 Laser light source 2 Collimator lens 3 Beam shaping optical system 4 Acousto-optic modulation element 5 1st pupil transmission expansion lens system 6 Pinhole 7 Restriction aperture 8 Polarization beam splitter 9 1/4 wavelength plate 10 Beam splitter 11 1st 1 Dimensional scanning device 12 Second pupil transmission magnifying lens system 13 Second one-dimensional scanning device 14 Control board 15 Third pupil transmission magnifying lens system 16 Objective lens 18 First light receiving element 19 Photoelectric conversion unit 20 Signal comparator 21 First data processing unit 22 Converging lens 23 Pinhole 24 Second light receiving element 25 Photoelectric conversion unit 26 Objective lens moving mechanism 27 Second data processing unit 28 Third light receiving element 29 Photoelectric conversion unit 30 Signal comparator 31 3 data processing section
32 Display device 33 Beam splitter 34 Imaging lens 35 One-dimensional imaging device 36 Photoelectric conversion unit S Object

Claims (9)

A laser light source that emits laser light;
An optical modulator that emits light in different directions while modulating the laser light into two lights having mutually different frequencies;
A scanning optical element having a scanning element surface for one-dimensional scanning or two-dimensional scanning of the two lights, and scanning the two lights based on a control signal;
An objective lens that has a pupil position and emits two lights to the object being placed;
The diffracted light exit surface of the optical modulator and the pupil position of the objective lens are positioned so as to be conjugate with each other and positioned between the optical modulator and the objective lens and emitted from the optical modulator. A pupil transfer magnifying lens system that magnifies two lights;
Moving means for moving either the objective lens or the object along an optical axis that is an optical path of two lights, and relatively changing a distance between them;
First light reception comprising two or more divided light-receiving elements that receive reflected light or transmitted light from the object and perform photoelectric conversion, and are arranged along a separation direction of two lights generated by the light modulator. Elements,
Signal comparison means for summing or subtracting each signal photoelectrically converted by the first light receiving element and obtaining phase information and intensity information of the two lights based on the sum or difference;
A first data processing unit for constructing a three-dimensional image within the depth of focus of the objective lens from the phase information and intensity information of the signal comparison unit and the control signal of the scanning optical element;
A second data processing unit that constructs three-dimensional position information from the control signal of the scanning optical element and the amount of movement by the moving unit, based on the light intensity data of the reflected return light from the object;
A third data processing unit for constructing a three-dimensional image quantified by the three-dimensional image within the focal depth of the objective lens and the three-dimensional position information based on the light intensity data;
A laser scanning microscope characterized by comprising: The laser light, which is linearly polarized light emitted from the laser light source, is converted into circularly polarized light, and the reflected return light, which is circularly polarized light from the object, is transmitted again to obtain linearly polarized light whose polarization plane is orthogonal to the laser light. A quarter wave plate,
A polarized beam splitter that separates the reflected return light whose polarization plane is orthogonal to the laser light from the optical path;
A lens for collecting the return light separated by the polarization beam splitter;
A spatial filter disposed in the focal plane of the lens;
A second light receiving element that receives the reflected return light transmitted through the spatial filter and photoelectrically converts the reflected return light; and
The laser scanning microscope according to claim 1, comprising: The scanning optical element
A first scanning optical element that scans the two lights along a diffraction direction of the light modulator;
A second scanning optical element that scans the two lights along a direction orthogonal to the first scanning optical element;
Including
A beam splitter disposed between the first scanning optical element and the second scanning optical element to separate reflected return light from the object;
A lens for focusing the return light separated by the beam splitter to form an image;
A one-dimensional imaging device comprising a plurality of divided light receiving elements positioned as a second light receiving element in the focal plane of the lens and arranged along a separation direction of two lights generated by the light modulator;
Have
Based on the light intensity data of the reflected return light received and photoelectrically converted by the second data processing unit received by the one-dimensional image sensor, a three-dimensional position is determined from the control signal of the scanning optical element and the amount of movement by the moving means. 2. The laser scanning microscope according to claim 1, wherein information is constructed. The laser light that is linearly polarized light emitted from the laser light source is converted into circularly polarized light, and the reflected return light that is circularly polarized light from the object is transmitted again to obtain linearly polarized light whose polarization plane is orthogonal to the laser light 1 / 4 wavelength plate,
A polarized beam splitter that separates the reflected return light whose polarization plane is orthogonal to the laser light from the optical path;
A lens for collecting the return light separated by the polarization beam splitter;
A pinhole located in the focal plane of the lens;
Have
The first light receiving element can also serve as the second light receiving element by receiving the light transmitted through the pinhole,
The first data processing unit constructs a three-dimensional image within the depth of focus of the objective lens narrowed by the confocal optical system in response to light reception by the first light receiving element. The laser scanning microscope described.   The laser scanning microscope according to any one of claims 1 to 4, wherein the first light receiving element receives light that has passed through the object directly under the object. The light modulator is
An acousto-optic modulation element on which laser light emitted from the laser light source is incident;
A signal generator for applying a carrier AC signal and a sine wave signal to the acoustooptic modulator;
The laser scanning microscope according to claim 1, comprising: The optical element includes a one-dimensional scanning element using a galvanometer mirror or a resonant mirror, an optical scanning device using a nonlinear optical crystal or a photonic crystal, a two-dimensional scanning optical system comprising two one-dimensional scanning devices and a pupil transmission magnifying lens system, The laser scanning microscope according to claim 1 , wherein the laser scanning microscope is a one-dimensional or two-dimensional micromirror device. A photoelectric conversion unit that photoelectrically converts light received by the first light receiving element creates a beat signal, and the generated beat signal is generated by all of the plurality of divided light receiving elements that constitute the first light receiving element. The laser according to any one of claims 1 to 7 , wherein the laser is obtained from a sum signal of the divided light receiving elements or a difference signal between the divided light receiving elements at corresponding positions of the plurality of divided light receiving elements. Scanning microscope. 9. The laser scanning microscope according to claim 1, wherein the movement by the moving means is a stage movement driven by a stepping motor, a direct movement by a piezo element, or a stage movement.

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