JP5589374B2 - Microscope equipment - Google Patents

Description

The present invention relates to a microscope apparatus that irradiates a sample with irradiation light and detects light from the sample based on the irradiation light.

  As a method for obtaining a resolution higher than that of a conventional laser microscope, a super-resolution laser scanning microscope using a plurality of laser beams to obtain one image is known. Non-Patent Document 1 discloses the principle of a super-resolution laser scanning microscope. What is shown in Non-Patent Document 1 is called a STED excitation fluorescence microscope, and is substantially irradiated by irradiating a beam having energy that suppresses the generation of fluorescence close to both sides of the excitation laser spot. In other words, the region where the fluorescence is generated is reduced, and the fluorescence is generated only from a small region below the so-called diffraction limit to obtain super-resolution. In addition, US Pat. No. 5,731,588 relates to a STED fluorescence microscope, which includes an excitation laser for generating fluorescence and irradiation / collection of another laser beam for suppressing the fluorescence phenomenon in the vicinity of the focusing position. A device that performs super-resolution by reducing the region that substantially emits fluorescence by making it light is disclosed.

U.S. Pat.No. 5,731,588

"Breaking the diff-raction resolution limit by stimulated emission: stimulated-emission-depletion fluoresceuce microscopy", Stefan W. Hell and Jan Wichmann, Optics Letters, Vol.19 (1994), pp.780-782 "

  However, since the above-mentioned microscope requires a laser with a different wavelength that suppresses the generation of fluorescence in addition to the normal excitation laser, the apparatus configuration becomes large, and these excitation laser and fluorescence generation are also increased. The combination of lasers that suppresses the light has a limited wavelength, does not correspond to various fluorescent dyes, and has a problem that it is not easy to use.

  An object of the present invention is to provide a microscope apparatus that can perform super-resolution observation with a simple configuration.

Microscope apparatus of the present invention irradiates the illumination light to a sample, the microscope apparatus for detecting light from the sample based on the irradiation light, separates the illumination light having coherence in at least two, isolated and modulation means for applying at least one modulated irradiation light of the irradiation light, the separated illumination light guides to the objective lens, respectively, an irradiation optical system to generate a more generated interference light to the illumination light in the vicinity of the focal point And extracting light having a frequency component corresponding to the interference light from light reflected from the vicinity of the focus or light generated from the vicinity of the focus, and detecting means for detecting the extracted light , and the detection means comprising an image forming means for forming an image data based on the detected light, and that the position of each of the focus of separated the irradiation light are different from each other And butterflies.
According to this microscope apparatus, since coherent illumination light is separated, modulation is applied to at least one of the separated illumination lights, and interference light due to illumination light in the vicinity of the focal point is detected. Thus, super-resolution observation can be performed.

The focal point of the separated irradiation light may be shifted in a direction orthogonal to the optical axis while having a region where the irradiation light overlaps each other.

The focal points of the separated irradiation lights may be shifted in the direction of the optical axis while having regions where the irradiation lights overlap each other.

The irradiation light having coherence is separated into two, and the modulation means applies phase modulation or frequency modulation that changes at a constant frequency to one of them, and changes to interference light that occurs near the focal point at the modulation frequency. to cause intensity modulation, the detecting means may extract light before Symbol modulation frequency component.

The modulating means is a phase modulation of at least two or more different frequencies separated the irradiation light intensity modulation, the addition of any of frequency modulation, two even without least interference light occurring at the focal point Intensity modulation that changes at the sum or difference frequency of the above modulation frequencies may be caused, and the detection means may extract light having a frequency component that is the sum or difference of the at least two modulation frequencies.

  The optical paths of the illumination light separated and modulated by the modulation means may be configured not to overlap each other at the incident position on the objective lens.

  The optical path of the illumination light separated and modulated by the modulation means may be separated into radially partitioned areas that do not overlap each other on the entrance surface to the objective lens.

  According to the microscope apparatus of the present invention, it is easy to separate coherent illumination light, modulate at least one of the separated illumination lights, and detect the interference light due to the illumination light near the focal point. With this configuration, super-resolution observation can be performed.

1 is a block diagram illustrating a configuration of a microscope apparatus according to Embodiment 1. FIG. The block diagram which shows the structure of an optical path splitting apparatus. Sectional drawing which shows the structure of a fine movement inclination stage. Sectional drawing which shows the state of the light beam of a focus position vicinity. The figure which shows intensity distribution of a light beam. The figure which shows intensity distribution of light at the time of matching the phase of the main intersection area | region of two light. FIG. 3 is a diagram illustrating a positional relationship between two light beams on a focal plane in the first embodiment. The figure which shows the example which arrange | positions the focus of three lights on a focal plane. The figure which shows the example which arrange | positions the focus of three lights on a focal plane. The block diagram which shows the structure of an optical path splitting apparatus. The figure which shows intensity distribution of two light beams. The figure which shows intensity distribution of the light which combined two lights, and an interference component. FIG. 6 is a block diagram illustrating a configuration of a microscope apparatus according to a fourth embodiment. FIG. 9 is a block diagram illustrating a configuration of an optical path dividing device used in a microscope apparatus according to a fifth embodiment. It is a figure which shows the structure of a light shielding filter, (a) is a figure which shows the structure of a pair of light shielding filter, (b) is a figure which shows the structure of another pair of light shielding filters. FIG. 10 is a block diagram illustrating a configuration of an optical path dividing device used in a microscope apparatus according to a sixth embodiment.

  Next, an embodiment of a microscope apparatus according to the present invention will be described.

  Hereinafter, the microscope apparatus according to the first embodiment will be described with reference to FIGS.

  FIG. 1 is a block diagram illustrating the configuration of the microscope apparatus according to the first embodiment, and FIG. 2 is a block diagram illustrating the configuration of the optical path dividing apparatus.

  As shown in FIG. 1, the microscope apparatus 1 of the present embodiment has a laser light source apparatus 2 that emits collimated light having a visible wavelength, and an optical path dividing apparatus 3 is disposed on the optical axis thereof.

  As shown in FIG. 2, a beam splitter 30 that receives the laser light from the laser light source device 2 is disposed in the light splitting device 3, and a mirror 31 is disposed in the reflection direction by the beam splitter 30. In FIG. 2, the optical path of the light reflected by the beam splitter 30 is indicated by a broken line, and the other optical paths are indicated by a solid line.

  The mirror 31 is attached to the fine movement tilt stage 32, and its installation angle can be adjusted.

  FIG. 3 is a cross-sectional view showing the configuration of the fine movement tilt stage 32.

  The mirror 31 is fixed to a movable portion 32a of a fine movement tilt stage 32 as shown in FIG. As shown in FIG. 3, in the fine movement tilt stage 32, the fixed portion 32b elastically supports the movable portion 32a, and the movable portion 32a is inclined in two directions by about several mrad by the two or three piezoelectric elements 32c inside. It is configured to be changed. Further, an AOM (acousto-optic element) 33 is disposed on the optical path of the light passing through the beam splitter 30, and an AOM 34 is disposed on the optical path of the light reflected by the mirror 31.

  Here, the AOM 33 is driven at a frequency f1 = 10.1 MHz, and the AOM 34 is driven at a frequency f2 = 10 MHz, and intensity modulation (amplitude modulation) is applied to light passing through each AOM. . The beam light modulated by the AOM 33 and the beam light modulated by the AOM 34 and turned back by the mirror 35 are combined by the beam splitter 36, and their traveling directions are aligned.

  As shown in FIG. 1, a dichroic mirror 4 for changing the direction of the laser and a scanning optical unit 5 for scanning the laser light are arranged at the tip of the optical path dividing device 3. The dichroic mirror 4 has a characteristic of reflecting the wavelength of the laser beam and transmitting the fluorescence of the laser beam. The scanning optical unit 5 includes a variable mirror 5a that can be rotated around one axis, and a variable mirror 5b that can be rotated around one axis that is substantially orthogonal to the axis of the variable mirror 5a.

  A pupil relay lens 6 for converging light, a mirror 7 for deflecting a laser beam, an imaging lens 8 and an objective lens 9 are sequentially arranged along the optical path.

  A stage 11 on which the sample 10 is placed is provided at the tip of the objective lens 9.

  On the other hand, in the fluorescence optical path generated from the sample 10 and transmitted through the dichroic mirror 4, a fluorescence filter 13 that selectively transmits fluorescence, a lens 14 that converges the fluorescence beam, a pinhole 15, and a pinhole 15 are provided. A detector 16 for detecting the passed light is provided. A signal from the detector 16 is supplied to the controller 17 through a band pass filter 19.

  Further, a display monitor 18 is connected to the controller 17. The controller 17 executes scanning control of the optical scanning unit 4 and control of the optical path dividing device 3. The display monitor 18 displays an observation image acquired via the detector 16 and various other information.

  Next, the operation of the microscope apparatus 1 will be described.

  The sample 10 is arranged on the stage 11 with a fluorescent indicator excited by light from the laser light source 2 being introduced.

  The collimated laser beam from the laser light source 2 is guided to the optical path dividing device 3. Inside the optical path splitting device 3, the laser light that is traveling straight through the beam splitter 30 is modulated at a frequency f 1 = 10.1 MHz by passing through the AOM 33. The laser beam reflected by the beam splitter 30 is reflected by the mirror 31 and modulated by the AOM 34 at a frequency f2 = 10 MHz. The light that has passed through the AOM 34 is reflected by the mirror 35. Some of these two laser beams are configured so that their traveling directions are substantially the same by the beam splitter 36.

  These laser beams are guided to the optical scanning unit 5 by the dichroic mirror 4 and scanned by the optical scanning unit 5 in an arbitrary direction. The laser light further passes through the pupil relay lens 6, the reflection mirror 7, and the imaging lens 8 and enters the objective lens 9. The laser light is converged in the vicinity of the sample 10 by the objective lens 9.

  Since the pupil of the objective lens 9 is relayed to the position of the optical scanning unit 5 by the pupil relay lens 6, the light scanned by the optical scanning unit 5 scans the light at the pupil position of the objective lens 9. Thus, the focal point by the objective lens 9 is scanned on the focal plane. Further, by driving the fine movement tilt stage 32, one of the directions of the light divided by the light splitting device 3 is moved minutely so that the focal positions of these two lights are slightly shifted.

  For example, if the wavelength of the laser beam is 40 nm, the objective lens is 100 times magnification, the focal length is 2 mm, and the NA is 1.4, the focal spot size is about 0.17 μm.

  Here, if the fine light tilt stage 32 is adjusted so that the directions of the two lights are different by 50 μrad, the respective focal positions are shifted by about 0.1 μm. FIG. 4 is a cross-sectional view showing the state of the light beam near the focal position at this time. As shown in FIG. 4, the two light beams 41 and 42 converge toward the focal plane 43. However, since the focal positions of the two light beams 41 and 42 are deviated from each other, the light beams 41 and 42 in FIG. It is drawn at a position shifted in the direction perpendicular to the optical axis. In FIG. 4, the Z direction indicates the optical axis direction of the objective lens 9, and the X direction indicates a direction orthogonal to the optical axis direction.

  FIG. 5 is a diagram showing the intensity distribution of the light beams 41 and 42. In FIG. 5, the horizontal axis corresponds to the X direction (FIG. 4) on the focal plane 43, and the vertical axis represents the intensity of the light beams 41 and 42 of each beam.

  FIG. 6 is a diagram showing the light intensity distribution when the phases of the main intersecting regions of the two lights are matched. The horizontal axis in FIG. 6 corresponds to the X direction (FIG. 4) on the focal plane 43, as in FIG.

  When the phase of the main intersection region of the two lights is matched by adjusting the optical path length of the two lights, the intensity distribution of the two lights is shown as a curve 44 in FIG. At this time, the interference component of the two lights becomes a curve 45. The interference component of the two lights includes a modulation component at f1-f2, which is a difference frequency between the modulation frequencies of the two lights.

  The fluorescence indicator of the sample 10 is excited by such laser light and emits fluorescence. This fluorescence is captured by the objective lens 9 and travels in the opposite direction to the laser beam, and is guided to the dichroic mirror 4 through the imaging lens 8, the reflection mirror 7, the pupil relay lens 6, and the optical scanning unit 5. This fluorescence is transmitted through the dichroic mirror 4, a specific wavelength component is selectively transmitted by the fluorescent filter 13, is condensed by the lens 14, and the light that has passed through the pinhole 15 is detected by the detector 16.

  The component excited by the interference light in the fluorescence signal has a modulation component of f1-f2 = 100 kHz as in the case of the laser light. By extracting this 100 kHz component, the fluorescence due to the interference light can be extracted. .

  Here, the pinhole 15 is adjusted so that only the signal corresponding to 46 in FIG. 6 passes so that the fluorescence by the interference light is maximized. The light detected by the detector 16 is sent to the band-pass filter 19 where only the frequency near the difference frequency of 100 kHz is extracted, and excited by the interference light having the intensity distribution shown by the curve 45 in FIG. A signal corresponding to the emitted fluorescence is obtained.

  The signal extracted by the bandpass filter 19 is guided to the controller 17 and converted into a digital signal in synchronization with the scanning control, and image data is created in correspondence with the scanning position and displayed on the display monitor 18. The image data is stored in a memory (not shown) inside the controller 17.

  Here, the full width at half maximum in the X direction on the focal plane of the interference light indicated by the curve 45 is about 40% smaller than the full width at half maximum of the respective excitation light (light beams 41 and 42). Since the illumination range becomes smaller, when the fluorescence due to the interference light is detected by a detection optical system using a pinhole, the resolution in the X direction is improved by about 50% compared to a normal optical microscope.

  In addition, the objective lens 9 or the sample 10 can be moved in the optical axis direction of the objective lens 9 by an actuator (not shown) to observe an image of another depth. Furthermore, by linking the optical scanning unit 5 and the actuator, it is possible to observe an arbitrary cross-sectional image or three-dimensional information. Furthermore, the PSF (point spread function) of the interference light can be obtained, and the inverse operation can be performed to obtain a higher resolution image.

  As described above, according to the microscope apparatus of the present embodiment, super-resolution observation can be performed with a single laser light source apparatus, and thus the complexity of the apparatus can be avoided. In addition, since the wavelength of the excitation laser can be freely selected, various fluorescent dyes can be supported.

  In the above embodiment, the f1-f2 component that is the difference in frequency included in the interference light is extracted. However, the present invention is not limited to this, and the same effect can be obtained by extracting the component of f1 + f2 that is the sum of the frequencies. .

  Further, the same effect can be obtained by applying phase modulation or intensity modulation by two EOMs (electro-optic modulation elements) instead of the modulation by the two AOMs 33 and 34 in the optical path dividing device 3. Further, intensity modulation may be added by other methods.

  Further, the AOMs 33 and 34 may be used as frequency shifters. In this case, the same effect can be obtained even when light is incident on AOMs driven at different constant frequencies and diffracted light subjected to frequency modulation is used.

  Further, AOM or EOM may be inserted into only one of the divided optical paths, and frequency modulation or phase modulation may be added to the light in this optical path. In this case, since the interference component has a component due to the modulation frequency, the same effect can be obtained even if this modulation frequency component is extracted.

  In addition, any configuration can be employed as the modulation means.

  Further, the light source is not limited to the laser, and any light source can be used as long as it is light that generates interference light. The same effect can be obtained even when an extremely short pulse laser beam is used as the light source.

  Furthermore, multiphoton fluorescence generated by excitation light modulated in the intersection region of two lights may be detected, and second harmonics and higher harmonics whose wavelengths are halved may be detected. .

  In the above embodiment, the variable mirror is shown as a method for scanning light. However, the scanning method is not limited to this, as long as the focal point and the sample move relative to each other, and the sample can be moved on the stage. good.

  Further, the pinhole opening can be selected to have an arbitrary size and shape. The aperture corresponding to 47 in FIG. 6 and the aperture corresponding to 48 may be switched, and a high resolution signal may be calculated using each signal.

  In the present embodiment, the fluorescence is detected. However, the present invention is not limited to this, and a configuration for detecting reflected light of illumination light may be used.

  According to the above embodiment, since the focal points of the two light beams are slightly shifted and interfered, and the fluorescence due to the interference light is detected, the full width at half maximum of the interference light is reduced, which is higher than that of the conventional confocal microscope. Resolution can be obtained. In addition, since the interference light is generated by the modulation means, the wavelength of the light can be set freely while avoiding the complexity of the apparatus.

  Hereinafter, the microscope apparatus according to the second embodiment will be described with reference to FIGS. The present embodiment shows a configuration in which the number of light beams is increased compared to the first embodiment.

  FIG. 7 is a diagram illustrating a positional relationship between two light beams on the focal plane in the first embodiment. As shown in FIG. 7, when the optical axis of the objective lens is the Z-axis, the high-resolution image is obtained in the X direction by shifting the focal points of the two lights in the X direction on the focal plane in the first embodiment. .

  On the other hand, FIG. 8 shows an example in which three light focal points are arranged on the focal plane. By irradiating light whose focal positions are shifted in the Y direction, the resolution in the Y direction as well as the X direction can be improved.

  In this case, the optical path is split into three by the optical path splitting device, and each light is modulated with a different frequency. The focal point of these lights is configured to have a positional relationship as shown in FIG. 8 on the focal plane, and the photodetection device is configured to improve the resolution in the X direction by the interference light of the light beam 51 and the light beam 52. The resolution in the Y direction is improved by the interference light between the light beam 51 and the light beam 53. At this time, the modulation frequencies of the interference light in the X direction and the Y direction are different.

  With such a configuration, an image with high resolution can be calculated in both the X direction and the Y direction by performing an operation from a signal obtained by scanning light.

  Further, as shown in FIG. 9, the positional relationship between the three focal points may be set. In this case, the interference component between the light beam 51 and the light beam 52, the interference component between the light beam 51 and the light beam 53, and the interference component between the light beam 52 and the light beam 53 are used. The resolution can be improved. Also in this case, if each interference component has a different modulation frequency, the signals can be separated from each other.

  Hereinafter, the microscope apparatus according to the third embodiment will be described with reference to FIGS.

  The present embodiment is a modification of the first embodiment and improves the resolution in the Z direction (the optical axis direction of the objective lens).

  FIG. 10 is a block diagram showing a configuration of an optical path dividing device 3A used in the microscope apparatus of the present embodiment. Other components are the same as those in the first embodiment. 10, the same components as those in FIG. 2 are denoted by the same reference numerals, and the description thereof is omitted.

  As shown in FIG. 10, in the optical path splitting device 3A, the light bent by the beam splitter 30 is reflected by a concave mirror 37 having a weak curvature. In the present embodiment, EOM 38 is inserted only on one side of the light to add phase modulation.

  When the same objective lens as in Example 1 is used, the full width at half maximum in the Z direction is about 0.6 μm. A concave mirror having a focal point whose focal position is shifted by about 0.4 μm in the optical axis direction is used.

  FIG. 11 is a diagram showing the intensity distribution of two light beams, and FIG. 12 is a diagram showing the intensity distribution of the light and interference components combined with the two lights.

  As shown in FIG. 11, at this time, the intensity distributions of the two light beams 61 and 62 are in a positional relationship in which the peaks are shifted from each other in the Z direction (optical axis direction).

  In FIG. 12, a curve 63 indicates the intensity distribution obtained by combining the two lights, and a curve 64 indicates the intensity distribution of the interference component. Since the interference component includes the modulation component of the EOM 38, the fluorescence due to the interference light indicated by the curve 64 also includes the same modulation component. As in the first embodiment, the full width at half maximum of the interference light is smaller than that of the respective light beams 61 and 62. Therefore, an image with improved resolution in the Z direction can be obtained by imaging the fluorescence signal of the interference light. Can do.

  Hereinafter, the microscope apparatus of Example 4 will be described with reference to FIG. The microscope apparatus according to the fourth embodiment detects reflected and scattered light instead of fluorescence.

  FIG. 13 is a block diagram illustrating a configuration of the microscope apparatus according to the fourth embodiment. The same components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  In the microscope apparatus 1A of the present embodiment, a near-infrared light SLD 2A that emits collimated light is used as a light source. The optical path splitting device 3 is the same as that in the first embodiment. Further, a polarizing beam splitter 60 is used instead of the dichroic mirror 4 in the first embodiment, and a polarizing filter 62 is used instead of the fluorescent filter 13. Further, a quarter wave plate 61 is inserted in front of the objective lens 9.

  The light from SLD 2A has linearly polarized light. This light is divided into two beams by the optical path splitting device 3, modulated, and reflected by the polarization beam splitter 60, and then passes through the same optical path as in the first embodiment, and the ¼ wavelength plate 61. Are each converted into circularly polarized light and focused by the objective lens 9.

  Similar to the first embodiment, two focal points shifted in the X direction are formed in the vicinity of the condensing point, and the full width at half maximum of the interference area of the two lights is smaller than that of the respective lights. The interference light includes intensity modulation at the difference frequency (100 kHz). Reflected and scattered light from the focal point is collected by the objective lens 9, and once again passes through the quarter-wave plate 61, it becomes polarized light having a polarization direction different from the incident light by 90 degrees, and the optical path of the illumination light is reversed. Return. Further, after passing through the polarization beam splitter 60, the polarization component is selected by the polarization filter 62, and further passes through the pinhole 15 and is detected by the detector 16.

  The output signal from the photodetector 16 is sent to the band pass filter 19, where only the frequency near the difference frequency of 100 kHz is extracted. The extracted signal is guided to the controller 17, converted into a digital signal in synchronization with the scanning control, image data is created in correspondence with the scanning position, and displayed on the display monitor 18. The image data is stored in a memory (not shown) inside the controller 17.

  According to the microscope apparatus 1A of the present embodiment, coherent light that illuminates the sample is projected with modulation at different frequencies, and a minute interference region having a full width at half maximum narrower than each light beam near the focal point is projected. Since it is configured so as to detect reflection and scattered light from the interference region, observation with higher resolution than usual is possible. This configuration is effective not only for biological use but also for industrial use for observing metal and semiconductor surfaces.

  Hereinafter, the microscope apparatus according to the fifth embodiment will be described with reference to FIGS. The present embodiment shows a configuration in which a pair of light shielding filters are added to the optical path dividing device of the microscope apparatus of the first embodiment. The configuration of the present embodiment is the same as that of the first embodiment with respect to other components other than the optical path splitting device.

  FIG. 14 is a block diagram illustrating a configuration of an optical path splitting device 3B used in the microscope apparatus according to the fifth embodiment, and FIG. 15 illustrates a light shielding pattern of a light shielding filter. In FIG. 14, the same components as those of the microscope apparatus of the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  As shown in FIG. 14, the light path dividing device 3B is provided with a light shielding filter 301 and a light shielding filter 302. As shown in FIG. 15A, the light shielding filter 301 is radially divided into eight regions, and a light shielding portion 301a that blocks light and a transmission portion 301b that transmits light are alternately assigned to each region. . Similarly, the light shielding filter 302 is radially divided into eight regions, and a light shielding unit 302a that blocks light and a transmission unit 302b that transmits light are alternately assigned to each region.

  As can be seen by comparing the light shielding filter 301 and the light shielding filter 302 shown in FIG. 15A, the light shielding portion and the transmission portion in each of the light shielding filters have a complementary positional relationship with each other, and the two light beams that have passed through the beam splitter 36. These optical paths satisfy the condition that they do not overlap with each other until they reach the objective lens 9, and these optical paths are separated into radial regions that do not overlap each other on the incident surface to the objective lens 9. This condition is satisfied regardless of the angle of the mirror 31 driven by the fine movement tilt stage 32 (see FIG. 1).

  As described above, since the pair of light shielding filters 301 and 302 are provided in the optical path splitting device 3B in the microscope apparatus of the present embodiment, the overlap of the optical paths split in the optical path splitting device 3B is eliminated, and the objective lens 9 The two optical paths overlap only in the vicinity of the focal point formed by condensing. Accordingly, interference between the two lights occurs only in the vicinity of the focal point, so that it is possible to extract a signal from the vicinity of the focal point more selectively than in the microscope apparatus of the first embodiment. Furthermore, due to this effect, for example, even when observing a deep part away from the surface of a biological tissue, a signal near the focal point can be efficiently extracted, so that a deeper part can be observed.

  Further, since the regions of the light shielding portion and the transmission portion in the optical filter 301 and the light shielding filter 302 are radially divided, the size of the focal point of each light is almost the same as that of the microscope apparatus of the first embodiment, and the two light beams The size of the intersecting region can be maintained substantially the same.

  FIG. 15B is a diagram illustrating another light shielding pattern of the pair of light shielding filters.

  In the example of FIG. 15B, the light shielding filter 303 is radially divided into four regions, and a light shielding portion 303a that blocks light and a transmission portion 303b that transmits light are alternately assigned to each region. Similarly, the light blocking filter 304 is radially divided into four regions, and a light blocking portion 304a that blocks light and a light transmitting portion 304b that allows light to pass through are alternately assigned to each region. Further, similarly to the pair of light shielding filters 301 and 302 shown in FIG. 15A, the light shielding portions and the transmission portions in the respective light shielding filters are in a complementary positional relationship, and the optical paths of the two lights that have passed through the beam splitter 36. Do not overlap each other until the objective lens 9 is reached. Therefore, the same function as the pair of light shielding filters shown in FIG.

  Hereinafter, the microscope apparatus of Example 6 will be described with reference to FIG. The present embodiment shows a configuration in which a pair of light shielding filters are added to the optical path splitting device (FIG. 10) of the microscope apparatus of the third embodiment. The configuration of the present embodiment is the same as that of the third embodiment with respect to other components other than the optical path splitting device.

  FIG. 16 is a block diagram illustrating a configuration of an optical path dividing device 3C used in the microscope apparatus according to the sixth embodiment. In FIG. 16, the same components as those in the microscope apparatus of the third embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  As shown in FIG. 16, the light path dividing device 3C is provided with a light shielding filter 303 and a light shielding filter 304 having the light shielding pattern shown in FIG. Similar to the microscope apparatus of the fifth embodiment, the light shielding portions and the transmission portions in the respective light shielding filters have a complementary positional relationship, and the optical paths of the two lights passing through the beam splitter 36 do not overlap each other until reaching the objective lens 9. These optical paths are separated into radial regions that do not overlap with each other on the entrance surface to the objective lens 9. This condition is also satisfied when the two light beams are in a condensing state that differs in the depth direction due to the effect of the concave mirror 37 (see FIG. 10).

  Thus, in the microscope apparatus of the present embodiment, since the pair of light shielding filters 303 and 304 are provided in the optical path splitting device 3C, the overlap of the optical paths split in the optical path splitting device 3C is eliminated, and the objective lens 9 The two optical paths overlap only in the vicinity of the focal point formed by condensing. Therefore, interference between the two lights occurs only in the vicinity of the focal point, so that a signal from the vicinity of the focal point can be extracted more selectively than in the microscope apparatus of the third embodiment. Furthermore, due to this effect, for example, even when observing a deep part away from the surface of a biological tissue, a signal near the focal point can be efficiently extracted, so that a deeper part can be observed.

  In addition, since the regions of the light shielding portion and the transmission portion in the optical filter 303 and the light shielding filter 304 are radially divided, the size of the focal point of each light is larger in the direction of the light shielding portion than in the third embodiment. About the magnitude | size of the intersection area | region of two light, it can maintain substantially equal.

  In the microscope apparatus of the present embodiment, the same effect can be obtained by using a pair of light shielding filters having the light shielding pattern shown in FIG. 15A instead of the pair of light shielding filters shown in FIG. Can do.

  As described above, according to the microscope apparatus of the present invention, the coherent illumination light is separated, the at least one illumination light of the separated illumination light is modulated, and the interference light due to the illumination light in the vicinity of the focal point. Therefore, super-resolution observation can be performed with a simple configuration.

The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied to a microscope apparatus that irradiates a sample with irradiation light and detects light from the sample based on the irradiation light.

3 Optical path splitting device (modulation means)
3A Optical path splitting device (modulation means)
9 Objective lens 16 Detector (detection means)

Claims (7)

In a microscope apparatus for irradiating a sample with irradiation light and detecting light from the sample based on the irradiation light,
Modulation means for separating the irradiation light having coherence into at least two or more and modulating the at least one irradiation light of the separated irradiation light;
An irradiation optical system for guiding the separated irradiation light to the objective lens and generating interference light generated by the irradiation light in the vicinity of the focal point;
A detection means for extracting light of a frequency component corresponding to the interference light from light reflected from the vicinity of the focus or light generated from the vicinity of the focus, and detecting the extracted light,
Image creating means for creating image data based on the light detected by the detecting means;
With
The microscope apparatus characterized in that the positions of the individual focal points of the separated irradiation light are different from each other.   2. The microscope apparatus according to claim 1, wherein the separated focal points of the irradiation light are shifted in a direction orthogonal to the optical axis while having regions where the irradiation lights overlap each other.   2. The microscope apparatus according to claim 1, wherein the focal points of the separated irradiation lights are shifted in the direction of the optical axis while having regions where the irradiation lights overlap each other. The irradiation light having coherence is separated into two,
The modulation means applies phase modulation or frequency modulation that changes at a constant frequency to one of them, and causes interference light that occurs near the focal point to undergo intensity modulation that changes at the modulation frequency,
The microscope apparatus according to claim 1, wherein the detection unit extracts light of the modulation frequency component. The modulating means is a phase modulation of at least two or more different frequencies separated the irradiation light intensity modulation, the addition of any of frequency modulation, two even without least interference light occurring at the focal point Cause intensity modulation that changes at the sum or difference frequency of the above modulation frequencies,
The microscope apparatus according to claim 1, wherein the detection unit extracts light having a frequency component that is a sum or difference of the at least two modulation frequencies.   The optical path of the illumination light separated and modulated by the modulation means is configured not to overlap each other at an incident position on the objective lens. Microscope equipment.   7. The optical path of the illumination light separated and modulated by the modulation means is separated into radially partitioned regions that do not overlap each other on the entrance surface to the objective lens. Microscope equipment.

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