JP2013235100A - Laser scanning microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser scanning microscope device which obtains adjacent beams and simultaneously increases a beat frequency, and thereby significantly improves resolution and scanning speed.SOLUTION: A laser light source 1, a collimator lens 2, a beam shaping optical system 3 and an acousto-optic modulation element 4 are positioned in a line. Light from the acousto-optic modulation element 4 passes through a first pupil transmission expansion lens system 6, a beam splitter 9 and the like. Photoelectric conversion sections 20 and 25 are connected to light receiving elements 19 and 24 arranged on both sides of the beam splitter 9, and a signal from the photoelectric conversion sections is transmitted to a data processing section 22 through a signal comparator 21. A first one-dimensional scanning device 11 is arranged in the state of being connected to the beam splitter 9, and a second pupil transmission expansion lens system 12, a second one-dimensional scanning device 13 and a third pupil transmission expansion lens system 15 are arranged below the first one-dimensional scanning device 11 in this order. An objective lens 16 facing an object S is arranged on the lower adjacent side of the third pupil transmission expansion lens system 15.

Description

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

  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 with a DC voltage applied to the acoustooptic device, and a sine wave signal is applied to the acoustooptic device to create two spatially separated beams having different frequencies. It was. 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, in a normal acoustooptic device, the frequency required for scanning is within a few tens of MHz to a hundred MHz. If the frequency range is set to about 70 MHz and the phase change between the two beams is captured, the two beams must be close to each other. For example, if the scanning range is set to about 1 mm and it is desired to decompose this scanning range into 1000 points, the frequency is 70 KHz per point. The distance between the two beams at this time is about 1 μm and can be considered to be close to each other.

  However, when it is desired to acquire data at a speed higher than the video rate, for example, it is not possible to meet a demand for real-time observation of cell state changes or a demand for measuring the surface shape accurately and at high speed. That is, when observing at a video rate, the horizontal scanning frequency is about 15 KHz. Therefore, if 1000 points are decomposed, the frequency required per point is about 15 MHz. Therefore, it is virtually impossible to make the two beams close and to increase the frequency.

  In addition, the resolution of a normal microscope is limited by the limits of so-called Abbe's theory. This limit was a result of the diffraction phenomenon of waves, and was regarded as a theoretical limit that could not be exceeded.

JP 59-214706 A

From the above, in the optical heterodyne interferometry described above, the inter-beam distance and the beat frequency for heterodyne detection are in inverse proportion to each other, so that obtaining a close beam and simultaneously increasing the frequency of the detected beat signal is It was virtually impossible.
The present invention has been made in view of the above background, and an object of the present invention is to provide a laser scanning microscope apparatus that obtains a close beam and at the same time increases the beat frequency to remarkably improve the resolution and scanning speed.
Furthermore, another object of the present invention is to provide a laser scanning microscope apparatus that realizes a microscope apparatus having a resolution substantially equal to or higher than the diffraction limit and an optical system that does not cause a decrease in beam utilization efficiency.

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 performing one-dimensional scanning or two-dimensional scanning of the two lights;
The light modulator and the scanning optical element are arranged so as to enlarge two lights emitted from the light modulator and to conjugate the diffracted light emitting surface of the light modulator and the scanning element surface. A pupil transfer magnifying lens system located between
An objective lens that has a pupil position and emits two lights to the object being placed;
A pupil transmission lens system positioned between the scanning optical element and the objective lens so as to conjugately arrange the scanning element surface of the scanning optical element and the pupil position of the objective lens;
A light receiving element that receives reflected light or transmitted light from the object;
A photoelectric conversion unit that photoelectrically converts light received by the light receiving element to create each beat signal; and
A signal comparator for detecting a phase difference or an intensity difference of signals obtained based on the beat signal of the photoelectric conversion unit;
A data processing unit that performs processing based on data obtained by acquiring phase information or intensity information of the signal comparator;
It is set as the laser scanning microscope apparatus characterized by including.

In the present invention, a beam splitter is disposed between the pupil transmission magnifying lens system and the scanning optical element, and the light receiving element includes the first light receiving element and the second light receiving element,
The first light receiving element is disposed at a position conjugate with the scanning optical element, and receives the light transmitted from the pupil transmission magnifying lens system and reflected and separated by the beam splitter,
A second light receiving element is disposed at a position conjugate with the light modulator and receives light reflected from the object and reflected by the beam splitter through the objective lens and the pupil transfer lens. Those are preferred.
Further, in the present invention, the signal comparator includes a phase or intensity of a beat signal corresponding to a frequency difference between the two lights in the photoelectrically converted signal and the object is not present or the object is present. It is preferable to detect the phase difference or the intensity difference from the signal when the objective lens is defocused so that there is no influence.

  On the other hand, an optical modulator according to the present invention includes an acousto-optic device on which laser light emitted from the laser light source is incident, and a signal generator that applies a carrier AC signal and a sine wave signal to the acousto-optic device. In addition, a spatial light modulator to which the laser light emitted from the laser light source is incident, a sinusoidal lattice pattern as amplitude or phase information is written in the spatial light modulator, and a carrier AC signal and a sine wave signal are applied. And a signal generator for moving the checkered pattern in a certain direction.

The scanning optical element according to the present invention is a one-dimensional scanning element using a galvano mirror or a resonant mirror, a two-dimensional scanning optical system comprising two one-dimensional scanning devices and a pupil transfer lens system, or one-dimensional scanning or two-dimensional scanning. A micromirror device is preferably used.
Furthermore, it is preferable that the light receiving element according to the present invention is a plurality of light receiving elements that are divided into at least two parts in the separation direction of two lights emitted in different directions by the light modulator.
In the present invention, the beat signal generated by the photoelectric conversion unit is a sum signal of all the light receiving elements of the plurality of light receiving elements divided into two or more of the light receiving elements, or a divided element divided into two or more. It is preferable to obtain from the difference signal between the light receiving elements at the corresponding positions.

  In the present invention, it is preferable that the pupil transmission lens system in which the scanning element surface of the scanning optical element and the pupil position of the objective lens are arranged in a conjugate manner is an expansion optical system. Further, it is preferable that the scanning optical element is a two-dimensional scanning optical element by combining two one-dimensional scanning optical elements.

  Furthermore, in the present invention, a pupil transmission magnifying lens system in which the diffracted light exit surface of the optical modulator and the scanning element surface of the scanning optical element are conjugated to each other has a curvature in the direction diffracted by the optical modulator. A lens system including a cylindrical lens is preferable. When the one-dimensional scanning optical element closer to the optical modulator scans in the same direction as the direction diffracted by the optical modulator, a pupil transmission lens system in which the pupil position of the scanning optical element is conjugated is provided. A lens system including a cylindrical lens having a curvature in the direction diffracted by the optical modulator is preferable.

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. That is, when an acousto-optic device, which is an optical modulator, is driven by an electrical signal having a frequency fc and a frequency fm, two beams having a frequency fc + fm and a frequency fc-fm are generated by AM modulation using the frequency fc as a carrier. . 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 acoustooptic device becomes considerably large.

  The light emitted from the acousto-optic 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 device 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.

  Furthermore, the photoelectric conversion unit creates each photoelectrically converted beat signal of the light receiving element, and the signal comparator detects the phase difference or intensity difference of the signal obtained based on this beat signal, and the phase of the signal comparator Based on the data obtained by acquiring information or intensity information, the data processing unit processes the surface profile if the target object is a reflective object, and substantially if the target object is a transmissive object. The refractive index difference or thickness is measured.

  Therefore, even if the scanning speed is increased, it is possible to improve the resolution by acquiring data at a sufficiently high rate. The reference phase is, for example, a phase obtained from a part of the outgoing beam separated by the beam splitter and obtained from the beat signal, or an objective lens that does not affect even if there is no object or there is an object The phase obtained by storing the signal in the defocused state together with the scanning signal can be used in the data processing unit.

  As described above, according to the laser scanning microscope apparatus 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.

  Also, since the two beams share almost the same 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 light receiving element that is divided into two or more in a direction perpendicular to the beam separation direction is used as the light receiving element, the sum signal of the outputs of all the light receiving elements is effective. In addition, since the integral value of the region corresponding to the beam diameter of the phase difference corresponding to the degree of separation between 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 each other in two or more divided light receiving elements is acquired, it effectively depends on the degree of separation between the two beams collected by the objective lens. The integrated value of the region corresponding to the differential beam diameter of the 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.

  In addition, the direction orthogonal to the beam separating direction in the magnifying optical system that is the pupil transmission magnifying lens system is substantially irrelevant to the beam separation. Therefore, the optical utilization rate of the beam can be remarkably improved by setting the optical system including the cylindrical lens so that the expansion optical system is only in the direction related to the beam separation and is not expanded in the direction orthogonal thereto. . This is because the amount of light incident on the objective lens is remarkably improved by enlarging in only one direction. As a result, even a very weak signal can be extracted with a high S / N ratio.

  As described above, according to the laser scanning microscope apparatus of 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 apparatus with high light utilization efficiency that obtains three-dimensional information such as height and refractive index distribution at a very high speed with a single two-dimensional scan and an 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 apparatus of the present invention, an acousto-optic modulation element or a spatial modulator that can be modulated 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.

  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 a split type, and at least two light receiving elements having dark lines in the beam separating direction are used, and the sum calculation of all the light receiving elements or the difference calculation is performed between the corresponding light receiving elements. By performing heterodyne detection, extremely high lateral resolution can be obtained particularly in the difference calculation. Furthermore, the use efficiency of light can be improved by using a cylindrical lens in the magnifying optical system, and the S / N ratio of the acquired signal can be improved.

It is a block diagram which shows Example 1 which concerns on the laser scanning microscope apparatus 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 apparatus of this invention. It is a block diagram which shows Example 2 which concerns on the laser scanning microscope apparatus of this invention. It is a block diagram which shows Example 3 which concerns on the laser scanning microscope apparatus of this invention. It is a block diagram which shows Example 4 which concerns on the laser scanning microscope apparatus of this invention. It is a block diagram which shows Example 5 which concerns on the laser scanning microscope apparatus of this invention. It is a block diagram which shows Example 6 which concerns on the laser scanning microscope apparatus of this invention. It is a figure which shows the spatial modulator applied to Example 7 which concerns on the laser scanning microscope apparatus of this invention, Comprising: (A) is a schematic diagram of a spatial modulator, (B) is applied to a spatial modulator. It is a figure which shows the pattern of a voltage and an electric current.

  Embodiments 1 to 8 of the laser scanning microscope apparatus according to the present invention will be described below in detail with reference to the drawings.

Embodiment 1 of a laser scanning microscope apparatus according to 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 apparatus 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 (AOD) 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 built-in signal generator is applied to the control board 14 to the acoustooptic device 4 with a frequency fc as a carrier AC signal and a modulation frequency fm as a sine wave signal.

  Further, the first pupil transmission magnifying lens system 6 composed of two groups of lenses, the limiting aperture 8 for optimizing the beam diameter, and the input laser beam are separated and emitted from the acousto-optic modulator 4. Beam splitters 9 are arranged in order. A pinhole 7 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 transfer magnifying lens system 6. .

  On the other hand, a light receiving element 19 and a light receiving element 24, which are optical sensors, are arranged in positions perpendicular to the direction passing through the optical axis L and adjacent to the beam splitter 9. A cylindrical lens 18 that is a converging lens having a curvature in the direction in which the beam is separated by the acoustooptic modulator 4 is disposed in front of the light receiving element 19.

  Further, the photoelectric conversion unit 20 is connected to the light receiving element 19, the photoelectric conversion unit 25 is connected to the light receiving element 24, and these are respectively connected to a signal comparator 21 that compares signals from the photoelectric conversion units 20 and 25. . The signal comparator 21 is connected to a data processing unit 22 that finally processes data to obtain a profile of the object S to be measured. The data processing unit 22 creates a profile of the object S based on the acousto-optic modulation element 4 controlled by the control board 14, a scanning device control signal to be described later, and a signal from the signal comparator 21.

  Further, the first one-dimensional scanning device 11 for one-dimensionally scanning the input laser beam with respect to the beam splitter 9, and the first one-dimensional scanning device 11 in FIG. A second pupil transmission magnifying lens system 12 and a second one-dimensional scanning device 13 are arranged. 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.

  On the other hand, these laser light sources 1 are gas lasers such as He—Ne, or semiconductor lasers or solid lasers, and each generate 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 as a modulation signal from the control board 14 to these acousto-optic modulation elements 4.

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 transmission magnifying lens system 6 is inclined by θ so that the diffracted light passes through the optical axis L of the first pupil transmission magnifying lens system 6. Further, as the first pupil transfer magnifying lens system 6 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 6 and the distance between the two lens groups of the first pupil transfer magnifying lens system 6 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 7 is provided near the intermediate focal plane of the first pupil transmission enlarging optical system 6 so as to transmit only the light beam in the vicinity of the optical axis L, and the pinhole 7 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 8 is located behind the exit side lens of the first pupil transmission enlarging optical system 6 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 light beam emitted from the limiting aperture 8 passes through the beam splitter 9, but a part of the light is reflected and separated by the beam splitter 9 and is emitted to the light receiving element 24. When the light receiving element 24 receives light, the photoelectric conversion unit 25 performs electrical amplification, and a signal that matches the received light can be formed.

  The second pupil transmission magnifying lens system 12 has the same configuration as that of the first pupil transmission magnifying lens system 6 described above. The second pupil transmission magnifying lens system 12 includes the acoustooptic modulator 4. The exit surface position, 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 6 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 6, 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 the diffraction limit or less. 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 slightly shifted, differential information can be obtained by performing heterodyne detection. it can.

  The light receiving element 19 shown in FIG. 1 is a plurality of 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. At this time, if a sum signal is used, it is substantially equivalent to a differential interference microscope, and if a difference signal is used, a much higher lateral resolution can be obtained. These improvements in 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 9 and guided to the light receiving element 19. The light receiving element 19 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 light receiving element constituting the light receiving element 19 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 having a small inter-terminal capacitance are arranged on the array to increase the speed, and the photoelectric conversion unit 20 performs electrical amplification corresponding to each photodiode on a one-to-one basis, A signal can be formed by a combination of elements as required.

The light receiving element 19 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 19 is detected by the photoelectric conversion unit 20 of the light receiving element 19 with a value based on the following expression, and is sent to the signal comparator 21.
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 21. 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.

  Incidentally, an incident beam of diffracted light generated by the acoustooptic modulator 4 is detected by the light receiving element 24, and a beat signal on this incident beam is created by the photoelectric conversion unit 25. Therefore, the phase difference generated in the optical system or the like up to the acoustooptic modulator 4 is detected by the light receiving element 24 and the photoelectric conversion unit 25, and the light receiving element 24 has a role of providing a phase reference. On the other hand, a beam obtained by adding phase difference information between the two beams A and B is detected by the light receiving element 19, and a beat signal on this beam is generated by the photoelectric conversion unit 20.

  Therefore, the true phase difference δ is detected by comparing the two phases of the photoelectric conversion unit 25 and the photoelectric conversion unit 20 in the signal comparator 21. The true phase difference δ is δh = λδ / 4π, which is the average phase difference between the beam LA and the beam LB, that is, difference information of the average height. Here, λ represents the wavelength of the laser light source. By recording this information together with the scanning information of the plane in the data processing unit 22, the profile information of the surface can be easily derived, and the profile information of the surface is displayed on the display 23 connected to the data processing unit 22. Can be displayed.

  Further, it is possible to acquire data at a higher speed by optimizing the enlargement magnification using an acousto-optic modulator having a Va as large as possible. By using such an optical system, it becomes possible to acquire three-dimensional measurement data at a speed higher than the video rate. 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.

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 apparatus, 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.

The present embodiment is intended to increase the spatial frequency of the reflected return light in the first embodiment.
FIG. 4 is a block diagram of an optical system according to the laser scanning microscope apparatus of the present embodiment. Since reflected return light from the surface of the object S is limited by the opening of the objective lens 16, the component having a high spatial frequency that is vignetted by the opening of the objective lens does not return to the light receiving element 19. For this reason, as shown in FIG. 4, in this embodiment, a light receiving element 26 is incorporated between the objective lens 16 and the object S so that the light beam entering and exiting the objective lens 16 is not vignetted.

  The light receiving element 26 is composed of at least two or more light receiving elements respectively installed along the direction in which the beam is separated by the acousto-optic modulation element 4 around the optical axis L of the objective lens 16. When the light receiving element 26 receives the beam in this manner, the intensity of the beam is detected by the photoelectric conversion unit 27 to which the light receiving element 26 is connected, and a signal is sent to the signal comparator 21.

On the other hand, the light beam reflected by the surface of the object S and entering the aperture of the objective lens 16 returns to the optical path of the illumination light beam and is reflected by the beam splitter 9 as in the first embodiment. The return light is incident on a cylindrical lens 18 having a curvature in the direction in which the beam is separated by the acousto-optic modulation element 4, is temporarily condensed, and is guided to a light receiving element 19 disposed behind the focal plane.
As described above, the light receiving element 19 is divided into at least two in the direction of separating the beam by the acousto-optic modulation element 4. With this configuration, the light receiving element 19 is equivalent to disposing the light receiving element on the object S side of the objective lens 16. Therefore, the light receiving element 19 and the light receiving element 26 can capture the reflected light from the surface of the object S up to a component having a high spatial frequency.

  When the light is received by the light receiving element 19 as described above, the intensity of the beam is detected by the photoelectric conversion unit 20 and sent to the signal comparator 21 as in the first embodiment. Accordingly, the signal from the photoelectric conversion unit 27 and the signal from the photoelectric conversion unit 20 are combined by the signal comparator 21, and the phase comparison between the photoelectric conversion unit 25 and the photoelectric conversion units 20 and 27 is performed in the signal comparator 21. By performing the same as in the first embodiment, the true phase difference δ is detected in the same manner. However, in the present embodiment, since the true phase difference δ is detected with a larger amount of light than in the first embodiment, a more accurate value can be obtained.

In the present embodiment, the main purpose is to easily obtain the reference phase obtained by the light receiving element 24 in the first embodiment by another method.
FIG. 5 is a block diagram of an optical system according to the laser scanning microscope apparatus of the present embodiment. The light receiving element 24 and the photoelectric conversion unit 25 according to the first embodiment are removed as shown in FIG. 5, and the laser is scanned in a state where the object S is not arranged or is considerably defocused even if the object S is present. . Thus, the phase difference is acquired by the light receiving element 19 and stored in the memory in the data processing unit 22 together with the position information in the screen.

  Since this phase information is a phase shift of the optical system and the electrical system, true phase information can be acquired by correcting the phase information in the presence of the object S using this as a reference value. In this case, not only the light receiving element 24 and the photoelectric conversion unit 25 are unnecessary, but if the correction value is obtained before the object S is observed, measurement with higher accuracy is possible.

In this embodiment, an embodiment in which the reflection optical system described in Embodiment 1 is replaced with a transmission optical system will be described.
FIG. 6 shows a block diagram of an optical system according to the transmission type laser scanning microscope apparatus. Since the main optical system is the same as that of the first embodiment, a description thereof will be omitted. However, in the present embodiment shown in FIG. 6, the light collected by the objective lens 16 is transmitted, so that the object S is sandwiched between the objectives. It is characterized in that the light receiving element 28 is arranged facing the lens 16. The light receiving element 28 is composed of at least two or more light receiving elements installed along the direction in which the beam is separated by the acousto-optic modulation element 4 around the optical axis L of the objective lens 16.

  In the case of this structure, the optical axis L of the objective lens 16 extends so that the dark line of each light receiving element constituting the light receiving element 28 extends in the vertical direction with respect to the separation direction of the two beams separated by the acoustooptic modulator 4. Each light receiving element is arranged on the extension line. According to the present embodiment, since the light receiving element can be arranged close to the object S as compared with the reflective type, the spatial frequency that can be acquired 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.

In the present embodiment, the reference phase obtained by the light receiving element 24 in the first embodiment is acquired in advance using a memory as in the third embodiment, and information is acquired in the transmission optical system as in the fourth embodiment. Combined with dots.
FIG. 7 shows a block diagram of an optical system according to the transmission type laser scanning microscope apparatus. As in the third embodiment, the light receiving element 24 and the photoelectric conversion unit 25 in the fourth embodiment are removed, and the laser is scanned in a state where the object S is not disposed or is substantially defocused even if the object S is present. To do. Thus, the phase difference is acquired by the light receiving element 28 and stored in the memory in the data processing unit 22 together with the position information in the screen.
Since this phase information is a phase shift of the optical system and the electrical system, true phase information can be acquired by correcting the phase information in the presence of the object S using this as a reference value.

In this example, in Example 1 to Example 5, when cells or the like are flowed in a microchannel, which is an element for flowing cells or the like in one direction, the cells are seeded after performing a monitor or cell shape determination. An optimal configuration for the above will be described.
FIG. 8 shows a block diagram of an optical system according to the laser scanning microscope apparatus. In this figure, the second pupil transmission enlarging optical system 12 and the second one-dimensional scanning device 13 of Example 5 are omitted, and a flow path such as a micro flow path in which the position of the object S is installed. Microchannels and the like are arranged so that the direction is perpendicular to the scanning direction of the first one-dimensional scanning device 11, and in FIG. 8 is perpendicular to the drawing.

  In this way, the object flows and moves in the direction of the microchannel, so that the two-dimensional scanning can be performed only by the first one-dimensional scanning device 11, and the cells and the like flow through the microchannel. It has a tremendous effect on applications such as monitoring the case and determining the cell shape and then sorting the cells. In addition, since the second pupil transmission enlarging optical system 12 and the second one-dimensional scanning device 13 can be omitted, the optical system is simplified, and the reference phase is a phase data for a very small number of points only in the one-dimensional scanning direction. It is only necessary to store in the memory in the processing unit 22.

This embodiment is characterized in that a spatial modulator is used as a member for applying modulation instead of the acousto-optic modulation element for applying modulation.
FIG. 9 is a conceptual diagram showing a spatial modulator applied in this embodiment. An electrode (not shown) is attached to each pixel so that the magnetic garnet film 53A constituting the spatial modulator 53 as shown in FIG. Is disposed at the position of the acousto-optic modulation element 4 in FIG. By applying voltage and current to each pixel of the magnetic garnet film 53A, the polarization plane of each pixel is rotated by the magneto-optic effect. The degree of rotation of this polarization plane depends on the magnitude of the applied voltage and current. Determined by. As a spatial modulator 53 having such a structure, there is a spatial modulator 53 having 128 × 128 pixels and a response speed of 15 ns.

  The spatial modulator 53 is arranged at the position of the acousto-optic modulation element 4 in FIG. 1, and accordingly, the intensity or phase of the light that has passed through the beam splitter 9 in FIG. 1 becomes a strip-like sine grating. Then, a voltage or current is applied to each pixel in the spatial modulator 53 in the direction perpendicular to the scanning direction as shown in FIG. 9B. At this time, the lattice can be moved at a speed v by causing a single vibration of a frequency fm = ± 2πv / d out of phase with respect to each pixel.

That is, when the pitch of the sine wave lattice is d and the moving speed is v, the following equation is obtained.
Acos {2π / d (x−vt)} = A / 2 (expj {2π / d (x−vt)} + expj {−2π / d (x−vt)})
For this reason, ± 1st-order diffracted light has a modulation frequency of fm = ± 2πv / d. In the case of intensity, a zero-order DC component is generated, but since it is a DC component, there is no influence on the beat signal.

  Here, the ± first-order diffracted light is set to a degree that the beam overlaps to a desired extent according to the pitch of the sine grating and the magnification of the pupil transmission magnifying lens system as in the first embodiment. Further, if the speed v is determined so that the modulation frequency fm is about 8 MHz, the same effect as in the first embodiment can be obtained. The response speed of the spatial modulator 53 is 15 ns, but the current spatial modulator is digitally binary.

  However, analog modulation is possible, and the response speed at that time may be deteriorated by about an order of magnitude. By using it in combination with the pupil transfer magnifying lens system, a modulation frequency of 8 MHz or more can be sufficiently obtained. It is possible to get. In this case, the pupil transmission magnifying lens system is simplified as compared with the first embodiment. This is because the modulation frequency is determined by the response speed of the device, but the beam separation can be reduced by increasing the grating pitch as much as possible.

  Therefore, since the minimum degree of separation is determined by the size of the device, the magnification rate of the pupil transmission magnifying lens system can be reduced or made equal when it is selected appropriately. In this way, the overall length of the optical system can be shortened. Note that the drive circuit and the like can be simplified by making the pixels of the spatial modulator described above into a strip shape as shown in FIG.

In this embodiment, a laser scanning microscope apparatus in which the utilization efficiency of a laser beam is improved will be described.
By using the pupil transmission magnifying optical system as in the above-described embodiment, it becomes possible to provide a laser scanning microscope apparatus capable of high-speed scanning. On the other hand, the use of the magnifying optical system reduces the utilization efficiency of the laser beam. Problems occur.

  That is, when the enlargement magnification is increased to reduce the separation angle, the beam diameter is naturally increased. In this case, since it is common to use a spherical lens in combination as a pupil transfer lens, the beam is evenly expanded in a two-dimensional plane, vignetting is caused by the optical element used, and the utilization efficiency of the beam is lowered.

In this embodiment, paying attention to the fact that the angle component generated by the acousto-optic modulation element or the one-dimensional scanning device is one-dimensional, the diffraction of the acousto-optic modulation element 4 and the curvature in the scanning direction of the one-dimensional scanning devices 11 and 13 are considered. A lens system composed of a cylindrical lens having a lens is used as a pupil transmission optical system.
In the direction of curvature of the cylindrical lens, the diffraction of the acousto-optic modulation element 4, and in the scanning direction of the one-dimensional scanning devices 11 and 13, the pupil transmission magnification optical system, but the diffraction of the acousto-optic modulation element 4 without curvature, And, it does not function as an optical system in the scanning direction of the one-dimensional scanning devices 11 and 13. Therefore, although the beam expands in the direction of curvature, the direction perpendicular to this does not contribute to expansion, and the light intensity is inversely proportional to the area, so that the beam utilization efficiency is greatly improved.

  For example, in the case of a pupil transmission magnifying optical system having an enlargement magnification of m times, when a spherical lens is used, the area of the beam is enlarged to m × m times, but when a cylindrical lens is used, the area of the beam is only m times. By using a cylindrical lens for the pupil transmission enlarging optical system without enlarging, the beam utilization efficiency of m times can be improved.

  As described above, a cylindrical lens can be applied to the first pupil transmission enlarging optical system 6 in the optical system according to the first embodiment. Further, when the deflection direction of the first one-dimensional scanning device 11 is the same as the diffraction direction of the acousto-optic modulation element 4, a cylindrical lens can be applied to the second pupil transmission enlarging optical system 12. However, since the third pupil transmission magnification optical system 15 is a two-dimensional scanning system, it cannot be applied.

  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 microscope apparatuses that perform 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 at high speed. Is.

DESCRIPTION OF SYMBOLS 1 Laser light source 2 Collimator lens 3 Beam shaping optical system 4 Acousto-optic modulation element 6 1st pupil transmission expansion lens system 7 Pinhole 8 Restriction aperture 9 Beam splitter 11 1st one-dimensional scanning device 12 2nd pupil transmission expansion Lens system 13 Second one-dimensional scanning device 14 Control board 15 Third pupil transfer magnifying lens system 16 Objective lens 18 Cylindrical lens 19 Light receiving element 20 Photoelectric conversion unit 21 Signal comparator 22 Data processing unit 23 Display 24 Light receiving element 25 Photoelectric Conversion unit 26 Light receiving element 27 Photoelectric conversion unit 28 Light receiving element 29 Photoelectric conversion unit 53 Spatial modulator 53A Magnetic garnet film S Object

Claims (12)

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 performing one-dimensional scanning or two-dimensional scanning of the two lights;
The light modulator and the scanning optical element are arranged so as to enlarge two lights emitted from the light modulator and to conjugate the diffracted light emitting surface of the light modulator and the scanning element surface. A pupil transfer magnifying lens system located between
An objective lens that has a pupil position and emits two lights to the object being placed;
A pupil transmission lens system positioned between the scanning optical element and the objective lens so as to conjugately arrange the scanning element surface of the scanning optical element and the pupil position of the objective lens;
A light receiving element that receives reflected light or transmitted light from the object;
A photoelectric conversion unit that photoelectrically converts light received by the light receiving element to create each beat signal; and
A signal comparator for detecting a phase difference or an intensity difference of signals obtained based on the beat signal of the photoelectric conversion unit;
A data processing unit that performs processing based on data obtained by acquiring phase information or intensity information of the signal comparator;
A laser scanning microscope apparatus comprising: A beam splitter is disposed between the pupil transmission magnifying lens system and the scanning optical element, and the light receiving element includes a first light receiving element and a second light receiving element;
The first light receiving element is disposed at a position conjugate with the scanning optical element, and receives the light transmitted from the pupil transmission magnifying lens system and reflected and separated by the beam splitter,
A second light receiving element is disposed at a position conjugate with the light modulator and receives light reflected from the object and reflected by the beam splitter through the objective lens and the pupil transfer lens. The laser scanning microscope apparatus according to claim 1.   The objective of the signal comparator is such that the phase or intensity of a beat signal corresponding to the frequency difference between the two lights in the photoelectrically converted signal and the object is not present or affected by the object. 3. The laser scanning microscope apparatus according to claim 1, wherein a phase difference or an intensity difference from a signal in a state where the lens is defocused is detected. The light modulator is
An acousto-optic 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 device;
The laser scanning microscope apparatus according to claim 1, comprising: The light modulator is
A spatial light modulator on which laser light emitted from the laser light source is incident;
A signal generator for writing a sinusoidal lattice pattern as amplitude or phase information to the spatial light modulator, applying a carrier AC signal and a sine wave signal, and moving the lattice pattern in a certain direction;
The laser scanning microscope apparatus according to claim 1, comprising:   The scanning optical element is a one-dimensional scanning element using a galvanometer mirror or a resonant mirror, a two-dimensional scanning optical system including two one-dimensional scanning devices and a pupil transfer lens system, or a one-dimensional scanning or two-dimensional scanning micromirror device. The laser scanning microscope apparatus according to any one of claims 1 to 5, wherein:   7. The light receiving element according to claim 1, wherein the light receiving element is a plurality of light receiving elements that are divided into at least two parts in a separation direction of two lights emitted in different directions by the light modulator. The laser scanning microscope apparatus according to any one of the above.   The beat signal generated by the photoelectric conversion unit is a sum signal of all the light receiving elements of the plurality of light receiving elements divided into two or more of the light receiving element, or light reception at a corresponding position of the divided element divided into two or more. The laser scanning microscope apparatus according to claim 7, wherein the laser scanning microscope apparatus is obtained from a difference signal between elements.   9. The pupil transmission lens system in which the scanning element surface of the scanning optical element and the pupil position of the objective lens are arranged in a conjugate manner is an magnifying optical system. Laser scanning microscope equipment.   10. The laser scanning microscope apparatus according to claim 9, wherein the scanning optical element is a two-dimensional scanning optical element by combining two one-dimensional scanning optical elements.   A lens system including a pupil transmission magnifying lens system in which the diffracted light exit surface of the optical modulator and the scanning element surface of the scanning optical element are conjugated to each other include a cylindrical lens having a curvature in a direction diffracted by the optical modulator. The laser scanning microscope apparatus according to any one of claims 1 to 10, wherein   When the one-dimensional scanning optical element closer to the optical modulator scans in the same direction as the direction diffracted by the optical modulator, the pupil transfer lens system in which the pupil position of the scanning optical element is arranged in a conjugate manner, 11. The laser scanning microscope apparatus according to claim 10, wherein the laser system includes a cylindrical lens having a curvature in a direction diffracted by the optical modulator.

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