JP2014052532A - Nonlinear optical microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nonlinear optical microscope capable of performing focus adjustment of a laser beam rapidly without causing vibration.SOLUTION: A laser beam B emitted from a laser light source 2 passes through beam expanders 3 and 4 so as to be incident on a first KTN module 7, and passes through relay lenses 8 and 9 and a half wavelength plate 10 so as to be incident on a second KTN module 11. At this time, applied voltages to the first KTN module 7 and the second KTN module 11 are controlled, and condensation is performed by condenser lenses 13 and 17, so that focus adjustment in an optical axis direction is performed and a primary condensing point is moved to a position 61a or a position 61b. The laser beam B subjected to primary condensation passes through a tube lens 23, and then a focus position is adjusted to a position 62a or a position 62b by an objective lens 25 with a fixed position.

Description

  The present invention relates to a nonlinear optical microscope that observes a sample by condensing laser light emitted from a laser light source into a sample by an objective lens to induce a nonlinear optical phenomenon.

  Conventionally, in this type of nonlinear optical microscope, an electric motor or a piezoelectric element is used to adjust the focus by moving an objective lens or a stage on which a sample is placed. In the microscope apparatus disclosed in Patent Document 1, an electric motor is driven at a driving direction and a driving speed determined by an arithmetic circuit, and the relative position between the objective lens and the sample surface is moved in the optical axis direction, thereby focusing. Adjustments are being made.

  However, with such a microscope that moves the objective lens and stage mechanically, it is impossible to quickly adjust the focus, and it is impossible to three-dimensionally capture fast biological phenomena that occur in the order of milliseconds in the life science field. there were. In addition, vibration is generated when laser light is scanned at high speed in the optical axis direction using an electric motor or a piezoelectric element. The vibration may affect the microscope apparatus and the image, and should be reduced as much as possible.

  For this reason, conventionally, a three-dimensional confocal microscope using a liquid deformable lens disclosed in Patent Document 2 and an optical head device using a liquid crystal lens disclosed in Patent Document 3 have been proposed.

  The three-dimensional confocal microscope disclosed in Patent Document 2 includes a surface tension type variable focus lens in front of the objective lens, and the variable focus lens performs scanning in the optical axis direction of the objective lens. The variable focus lens is configured by filling a first liquid (for example, oil) and a second liquid (for example, water) that are not mixed with each other into a box surrounded by a plate, an electrode, and a support base. . The contact angle θ between the first liquid and the support base changes in accordance with the applied voltage to the electrode, and the focal length of the variable focus lens is adjusted by controlling this applied voltage.

  In the optical head device disclosed in Patent Document 3, the Fresnel lens surface and the liquid crystal layer are combined, the orientation of the liquid crystal is changed by applying an electric field, and the focal length is changed by switching the presence or absence of the diffraction effect by the Fresnel lens surface. A liquid crystal lens is used.

JP 2010-8630 A JP 2004-317704 A JP 2007-73088 A

  The above three-dimensional confocal microscope using the liquid deformable lens disclosed in Patent Document 2 changes the focal length at a higher speed than the microscope apparatus that mechanically moves the objective lens disclosed in Patent Document 1. However, the speed limit is still about 50 Hz. Therefore, it is difficult to capture fast biological phenomena in the life science field in three dimensions in real time.

  Further, the liquid crystal lens used in the conventional optical head device disclosed in Patent Document 3 is not used for a nonlinear optical microscope, but even when applied to a nonlinear optical microscope, the speed limit is several tens to 100 Hz. It is still difficult to capture fast biological phenomena in the life science field in three dimensions in real time.

The present invention has been made to solve such problems,
In a non-linear optical microscope in which a laser beam emitted from a laser light source is focused on a sample by an objective lens to induce a non-linear optical phenomenon, the light generated by this non-linear optical phenomenon is detected and the sample is observed,
Light that condenses laser light in a sample by allowing laser light to pass through an electro-optic element that exhibits an electro-optic (EO) effect in which the refractive index inside the element changes according to the applied voltage and controlling the applied voltage. The axial position is variable.

  The refractive index change due to the electro-optic effect in the electro-optic element is derived from the electromagnetic field generated in the crystal when a voltage is applied, so the speed at which the liquid in the liquid lens moves and the orientation of the liquid crystal in the liquid crystal lens change. It happens much faster than it makes it happen. For this reason, according to the present configuration in which the laser light emitted from the laser light source passes through the electro-optic element, the refractive index in the electro-optic element is varied by controlling the voltage applied to the electro-optic element. The position in the optical axis direction where the light is collected in the sample can be varied very quickly. Accordingly, there is provided a nonlinear optical microscope that can adjust the focus of laser light at high speed without causing vibration.

  Further, the invention is characterized in that the electro-optic element is a cubic potassium tantalate niobate crystal (hereinafter referred to as a KTN crystal).

  KTN crystals have been known for a long time to have a very large electro-optic effect and can greatly change the refractive index with respect to the applied voltage. However, a single crystal having a practical size and performance can be obtained by recent technological development. It became possible. In addition, a crystal formed into a cubic crystal by appropriate temperature control is transparent in the visible light to infrared light region, and can be used as an optical component. Such a KTN crystal is particularly suitable for a high-speed focusing device in a nonlinear optical microscope, and according to the nonlinear optical microscope of this configuration using the KTN crystal as a focusing device, a fast biological phenomenon in the life science field is tertiary. Originally, it will be possible to capture sufficiently in real time, which will contribute to technological development in the life science field.

  In the present invention, an applied voltage is applied to one electrode of a pair of electrodes provided in a strip shape on one surface of the electro-optic element, and on the other surface of the electro-optic element facing the one surface. It is applied between the other pair of electrodes provided in a band shape.

  According to this configuration, when a voltage is applied between one pair of electrodes and the other pair of electrodes of the electro-optic element, the electro-optic between each of the one pair of electrodes and the other pair of electrodes. An electric field is generated inside the device. This electric field is also distributed in the element part where one and the other electrodes are not facing each other, but the refractive index change due to the electro-optic effect occurs in proportion to the square of the electric field, so in the element part where the electric field close to the electrode is large. In the element portion where the refractive index changes greatly and the central electric field between the pair of electrodes apart from the electrodes is small, the refractive index does not change much. Since light tends to travel toward a higher refractive index, laser light incident on one surface sandwiched between one pair of electrodes is deflected in the optical axis direction inside the device and sandwiched between the other pair of electrodes. The light is emitted from the other surface. Further, the laser light incident on one side surface sandwiched between the one electrode and the other one electrode is deflected in the optical axis direction inside the device, and is emitted from the other side surface sandwiched between the one and other other electrodes. Since the refractive index increases as a higher voltage is applied between the one and the other electrodes, the laser beam is strongly deflected in the optical axis direction inside the element, and the focal point approaches the electro-optic element. In this way, by controlling the magnitude of the voltage applied between the one and the other electrodes, the focal position in the optical axis direction in the sample can be varied.

  In addition, the present invention is characterized in that the nonlinear optical phenomenon is a multiphoton absorption phenomenon, a harmonic generation phenomenon, a sum frequency or difference frequency generation phenomenon, or a coherent anti-Stokes Raman scattering phenomenon.

  According to this configuration, non-linearity such as a multiphoton excitation fluorescence microscope, a harmonic generation microscope, a sum frequency or difference frequency generation microscope, or a coherent anti-Stokes Raman scattering microscope that can perform laser beam focusing at high speed without causing vibration. An optical microscope is provided.

  According to the present invention, as described above, a non-linear optical microscope capable of performing laser beam focusing at high speed without causing vibration is provided, and fast biological phenomena in the life science field are captured in three dimensions in real time. It will be possible to contribute to technological development in the life science field.

1 is an optical path diagram of a two-photon excitation fluorescence microscope according to an embodiment of the present invention. It is a perspective view which shows the structure of the KTN module which comprises the focus adjustment apparatus of the two-photon excitation fluorescence microscope shown in FIG. FIG. 3 is a perspective view showing a situation where two KTN modules shown in FIG. 2 are combined and laser light is condensed in two orthogonal directions. It is a schematic diagram which shows the optical system of a common optical scanning microscope. It is a schematic diagram which shows the optical system of the two-photon excitation fluorescence microscope shown in FIG. FIG. 2 is a control system diagram of the two-photon excitation fluorescence microscope shown in FIG. 1. It is an optical path figure of the two-photon excitation fluorescence microscope by the modification of one embodiment of this invention.

  Next, an embodiment in which the nonlinear optical microscope according to the present invention is applied to a two-photon excitation fluorescence microscope will be described.

  FIG. 1 is an optical path diagram of a two-photon excitation fluorescence microscope 1 according to this embodiment.

  The two-photon excitation fluorescence microscope 1 according to the present embodiment uses a femtosecond mode-locked titanium sapphire laser as the laser light source 2, and the laser light source 2 emits an ultrashort pulse laser beam only at the moment of femtosecond. Is emitted. Accordingly, the laser light emitted from the laser light source 2 is kept at a low average light intensity, while having a very high density. The laser light passes through the beam expanders 3 and 4 and the shutter 5, is reflected by the high reflectivity mirror 6, and enters the first KTN module 7. The laser light that has passed through the first KTN module 7 passes through the half-wave plate 10 through the relay lenses 8 and 9 and enters the second KTN module 11. The first KTN module 7, the half-wave plate 10 and the second KTN module 11 constitute a focus adjusting device, and scan the laser beam in the z-axis direction of the optical axis direction as will be described later.

  The laser light that has passed through the second KTN module 11 is reflected by the high reflectivity mirror 12, passes through the condenser lens 13 and the relay lens 14, and is reflected by the high reflectivity mirror 15. Thereafter, the light passes through the relay lens 16 and the condenser lens 17, is reflected by the high reflectivity mirrors 18 and 19, and is scanned by the scanning mirrors 20 and 21 in the x axis and y axis directions. The scanned laser light further passes through the relay lens 22, the tube lens 23, and the dichroic mirror 24, and is condensed and irradiated onto the object that becomes the sample 26 by the objective lens 25.

  When the sample 26 is irradiated with laser light, fluorescence is generated from a portion of the spot where the laser light is condensed and stained with a fluorescent dye. Alternatively, the fluorescence inherent in the sample 26 is generated. Since the laser light emitted from the laser light source 2 has a high density of photons as described above, a nonlinear optical phenomenon in the two-photon absorption process occurs in the sample 26 when the laser light is condensed and irradiated. , Fluorescence having an intensity proportional to the square of the light intensity of the incident light is emitted. This fluorescence is collected by the objective lens 25, reflected by the dichroic mirror 24, passes through the relay lens 27 and the infrared cut filter 28, the illumination light is removed, and is split into two by the dichroic mirror 29, and is converted into the type of fluorescence. In response, the signals are amplified by photomultipliers (PMT) 30 and 31.

  The first KTN module 7 and the second KTN module 11 constituting the focus adjustment apparatus have the same structure, and FIG. 2 is a perspective view of the structure.

  Each of the KTN modules 7 and 11 is configured by providing two pairs of electrodes 42a and 42b and 43a and 43b on a KTN crystal 41 which is an electro-optic element. One pair of electrodes 42a, 42b is provided in a strip shape with a gap between the laser light incident surface 41a on one surface of the KTN crystal 41 having a rectangular parallelepiped shape. The other pair of electrodes 43a and 43b is formed in a belt-like shape facing the one pair of electrodes 42a and 42b with the laser beam emitting position 41b sandwiched between the other laser beam emitting surface 41b facing the one surface of the KTN crystal 41. Is provided.

The KTN crystal 41 is an oxide composed of potassium (K), tantalum (Ta), and niobium (Nb), and the chemical formula is represented by KTa _1-x Nb _x O ₃ . This KTN crystal 41 is kept at a temperature several degrees higher than the phase transition temperature of tetragonal crystal and cubic crystal, is used in a cubic state, and is made of two pairs of metal made of gold (Au), platinum (Pt), or the like. By applying a voltage between the electrodes 42a, 42b and 43a, 43b, an electro-optic effect is exhibited. In the two-photon excitation fluorescence microscope 1, laser light is passed through each KTN crystal 41 of the first KTN module 7 and the second KTN module 11 as described above, and two pairs of electrodes 42a, 42b and 43a, 43b are passed. By controlling the applied voltage between them, the refractive index inside the KTN crystal 41 element is changed according to the applied voltage, and the position in the optical axis direction where the laser light is condensed in the sample 26 is varied.

  That is, when a voltage is applied between one pair of electrodes 42a, 42b of the KTN crystal 41 and the other pair of electrodes 43a, 43b, one pair of electrodes 42a, 42b and the other pair of electrodes 43a, 43a, An electric field E is generated inside the KTN crystal 41 between the respective terminals 43b and 43b. This electric field E is distributed also in the element portion where one and the other electrodes 42a, 42b and 43a, 43b are not opposed to each other, but the refractive index change due to the electro-optic effect occurs in proportion to the square of the electric field E. In the element portion where the refractive index changes greatly in the element portion where the electric field E close to the electrodes 42a, 42b and 43a, 43b is large, and in the element portion where the central electric field E between the pair of electrodes apart from the electrodes 42a, 42b and 43a, 43b is small, The refractive index does not change much. Since the light tends to travel toward a higher refractive index, the laser light B incident on the laser light incident surface 41a sandwiched between one pair of electrodes 42a and 42b is deflected in the optical axis direction inside the device. Then, the light is emitted from the laser light emitting surface 41b sandwiched between the other pair of electrodes 43a and 43b. As the higher voltage is applied between the one and the other electrodes 42a, 42b and 43a, 43b, the refractive index change becomes larger, so that the laser beam B is strongly deflected in the direction of the optical axis inside the device, whereby the focal point is the KTN crystal 41. Get closer to. Therefore, the focal position in the optical axis direction (z-axis direction) in the sample 26 can be varied by controlling the magnitude of the voltage applied between the one and the other electrodes 42a, 42b and 43a, 43b. .

  Since the EO effect of the KTN crystal 41 depends on the polarization plane of the incident light, it acts as a cylindrical lens, and condenses the laser beam B only in one direction. Further, since the condensing effect of the KTN crystal 41 strongly depends on the polarization plane of the incident laser beam B, in the two-photon excitation fluorescence microscope 1, the laser focused in the x direction by the KTN crystal 41 of the first KTN module 7 The polarization of the light B is rotated 90 degrees by the half-wave plate 10 and then incident on the second KTN module 11, and the laser light is emitted in the y direction perpendicular to the x direction by the KTN crystal 41 of the second KTN module 11. B is condensed and condensed as conceptually shown in FIG. 3 that are the same as or correspond to those in FIG. 1 are assigned the same reference numerals, and descriptions thereof are omitted.

  FIG. 4 is a schematic diagram showing an optical system of a general optical scanning microscope, and FIG. 5 is a schematic diagram showing an optical system of the two-photon excitation fluorescence microscope 1 of the present embodiment. 5 that are the same as or correspond to those in FIG. 1 are assigned the same reference numerals, and descriptions thereof are omitted.

  In an optical system of a general optical scanning microscope, as shown in FIG. 4, the laser beam B is condensed at a primary condensing point 52 whose position is fixed by a condensing lens 51. Then, the sample is irradiated through the tube lens 53 and the objective lens 54. At this time, the focus position is adjusted to the position 55a or the position 55b by moving the objective lens 54 to the position a indicated by a solid line or the position b indicated by a dotted line.

  On the other hand, in the optical system of the two-photon excitation fluorescence microscope 1 of the present embodiment, the optical system by the focus adjusting device is shown in FIG. 5A, and the microscope optical system coupled thereto is shown in FIG. As described above, the laser light B emitted from the laser light source 2 is incident on the first KTN module 7 through the beam expanders 3 and 4 as shown in FIG. , 9 and the half-wave plate 10 are incident on the second KTN module 11. At this time, the voltage applied to the first KTN module 7 and the second KTN module 11 is controlled, and the light is condensed by the condenser lenses 13 and 17, thereby performing the focus adjustment in the optical axis direction, and the position 61a. The primary condensing point is moved to the position 61b.

  The laser beam B primarily condensed at the positions 61a and 61b by the condensing lenses 13 and 17 passes through the tube lens 23 by the objective lens 25 whose position is fixed as shown in FIG. The focus position is adjusted to the position 62a or the position 62b.

  FIG. 6 is a control system diagram of the two-photon excitation fluorescence microscope 1 of the present embodiment. In the figure, the same or corresponding parts as in FIG.

  The first KTN module 7 and the second KTN module 11 are placed in similar thermostats 71a and 71b, respectively. Each thermostat 71a, 71b is provided with a window 72, in which heaters 73a, 73b and temperature sensors 74a, 74b are installed. The heaters 73a and 73b are connected to temperature control circuits 75a and 75b, and the KTN modules 7 and 11 are connected to high voltage drive circuits 76a and 76b. The temperature sensors 74a and 74b, the temperature control circuits 75a and 75b, and the high voltage drive circuits 76a and 76b are connected to the compensation computers 78a and 78b via the interfaces 77a and 77b.

  The compensation computers 78a and 78b operate in accordance with the software 79a and 79b, and control the temperature control circuits 75a and 75b while referring to the temperatures measured by the temperature sensors 74a and 74b to supply the heaters 73a and 73b. Adjust the energization amount. The interiors of the thermostatic chambers 71a and 71b are set to a temperature several degrees higher than the tetragonal and cubic phase transition temperatures (about 47 ° C.) of the KTN crystal 41 by energizing the heaters 73a and 73b, and the first KTN module. The KTN crystals 41 of the seventh and second KTN modules 11 are used in a cubic state.

  The scanning mirrors 20 and 21 for scanning the laser beam in the x-axis and y-axis directions constitute an x-axis galvano mirror 80 and a y-axis galvano mirror 81, respectively. The x-axis galvano mirror 80 and the y-axis galvano mirror 81 are x-axis. The control circuit 82 and the y-axis control circuit 83 are connected to each other. The high voltage drive circuits 76a and 76b are connected to the z-axis control circuit 84 via the interfaces 77a and 77b. The x-axis control circuit 82, the y-axis control circuit 83, and the z-axis control circuit 84 are commonly connected to a synchronization signal generation circuit 85, and synchronization among them is established. A personal computer (PC) 86 controls the x-axis control circuit 82 and the y-axis control circuit 83 and adjusts the angles of the scanning mirrors 20 and 21 so that the laser light irradiated on the sample 26 is irradiated on the xy plane of the sample 26. Scan in the x-axis and y-axis directions. Further, the PC 86 controls the z-axis control circuit 84 and controls the high voltage drive circuits 76a and 76b, so that the electrodes 42a and 40a provided on the KTN crystals 41 of the first KTN module 7 and the second KTN module 11 are controlled. The applied voltage between 42b and 43a, 43b is varied within a range of, for example, 0 to 1000 [V]. As a result, the focal position of the laser beam condensed in the sample 26 is adjusted in the z-axis direction.

  Further, the fluorescence information from the sample 26 amplified by the photomultiplier (PMT) 30 and 31 is further amplified by the amplifiers 87 and 88 and then stored in the image memory 89. The fluorescence information stored in the image memory 89 is displayed on the screen of the PC 86 as a three-dimensional image by scanning the sample 26 with laser light in the x-axis, y-axis, and z-axis directions. Visualized.

  In such a two-photon excitation fluorescence microscope 1 according to the present embodiment, the refractive index change due to the electro-optic effect in each KTN crystal 41 of the first KTN module 7 and the second KTN module 11 is as shown in FIG. Since it is derived from the electric field E generated in the crystal by applying a voltage, the liquid moving speed in the liquid lens disclosed in the conventional patent document 2 or the liquid crystal lens disclosed in the conventional patent document 3 Occurs much faster than the rate at which the orientation of the liquid crystal changes. For this reason, according to the present configuration in which the laser light emitted from the laser light source 2 is passed through the KTN crystal 41, the voltage applied to the KTN crystal 41 is controlled by the high voltage drive circuits 76a and 76b. By changing the refractive index, the position in the optical axis direction (z-axis direction) where the laser light is condensed in the sample 26 can be changed very quickly. Therefore, a two-photon excitation fluorescence microscope 1 that can adjust the focus of laser light at high speed without causing vibration is provided.

Further, the KTN crystal 41 can be obtained as a single crystal having a practical size and function due to recent technological development, and the KTN crystal 41 in a cubic state has a very large electro-optic effect and has a low voltage. Can greatly change the refractive index. For this reason, such a KTN crystal 41 is particularly suitable for a high-speed focusing device in a nonlinear optical microscope, and the two-photon excitation fluorescence microscope 1 according to the present embodiment using the KTN crystal 41 as a focusing device as described above. According to this, the focus adjustment can be performed at a speed 1000 to 100,000 times that of the conventional art. In addition, although the effect is inferior even with a lithium niobate (LiNbO ₃ ) crystal having an electro-optic effect, a similar microscope can be manufactured and high-speed focus adjustment can be performed. As a result, according to the two-photon excitation fluorescence microscope 1 according to the present embodiment using the KTN crystal 41, such as a crystal having an electro-optic effect in which the refractive index changes with application of voltage, such as the KTN crystal 41 or the LiNbO ₃ crystal. For example, fast biological phenomena in the life science field can be captured in three dimensions in real time, which contributes to technological development in the life science field.

  In the present embodiment described above, the laser light emitted from the laser light source 2 is condensed in the sample 26 by the objective lens 25 to induce a two-photon absorption process as a nonlinear optical phenomenon, and this two-photon absorption process The case where the sample 26 is observed by detecting the generated light has been described. However, the nonlinear optical phenomenon is not limited to the two-photon absorption process, but a multi-photon absorption process such as a three-photon of two or more photons, or a second harmonic generation or a third harmonic generation (Third Harmonic Generation). )), Or non-linear optical phenomena such as Sum Frequency Generation, Difference Frequency Generation, or Coherent Anti-Stokes Raman Scattering. May be. According to these configurations, similarly to the two-photon excitation fluorescence microscope 1 according to the above-described embodiment, the multi-photon excitation fluorescence microscope or the harmonic generation microscope can perform the laser beam focusing at high speed without causing vibration. Alternatively, a non-linear optical microscope such as a sum frequency or difference frequency generation microscope or a coherent anti-Stokes Raman scattering microscope is provided.

  In the above-described embodiment, the case where the relay lenses 8 and 9 are provided between the first KTN module 7 and the second KTN module 11 has been described as shown in FIG. However, as shown in FIG. 7, the relay lenses 8 and 9 are not provided between the first KTN module 7 and the second KTN module 11, and the relay lenses 8 and 9 relay between the two KTN modules 7 and 11. It is good also as a structure which does not. In the figure, the same or corresponding parts as in FIG. Even with such a configuration, the same effects as those of the above-described embodiment can be obtained. Further, the first KTN module 7, the half-wave plate 10 and the second KTN module 11 constituting the focus adjustment apparatus can be provided. One module structure can be connected directly.

  Further, in the present embodiment described above, the method of putting the laser beam B into the KTN crystal 41 is incident on the laser beam incident surface 41a sandwiched between one pair of electrodes 42a and 42b as shown in FIG. The case where light is emitted from the laser light emission surface 41b sandwiched between the other pair of electrodes 43a and 43b has been described. However, the laser beam B is incident on one left side of the figure sandwiched between the one and the other one electrodes 42a and 43a, and the right side of the figure sandwiched between the one and the other other electrodes 42b and 43b. You may make it radiate | emit from another side.

  Even with such a configuration, the refractive index greatly changes in the element portion where the electric field E close to the electrodes 42a, 42b and 43a, 43b is large, and the electric field in the center between the pair of electrodes apart from the electrodes 42a, 42b and 43a, 43b. In the element portion where E is small, the refractive index does not change so much. Therefore, the laser beam B incident on one side surface sandwiched between the one and the other electrodes 42a and 43a is deflected in the optical axis direction inside the element, The light is emitted from the other side surface sandwiched between the other electrodes 42b and 43b. Therefore, even when the laser beam B is put into the KTN crystal 41 in this way, the same effects as those of the above-described embodiment can be obtained.

  Each nonlinear optical microscope, including the two-photon excitation fluorescence microscope 1 of the present embodiment, is a state-of-the-art research field in medicine and biology such as genetic research, brain / neurology, and elucidation of cell functions, optical measurement devices and lasers. As a processing machine, it can be used in various fields such as the industrial field. Such use can make a great contribution to science, industry, and medicine.

DESCRIPTION OF SYMBOLS 1 ... Two-photon excitation fluorescence microscope 2 ... Laser light source 3, 4 ... Beam expander 5 ... Shutter 6, 12, 15, 18, 19 ... High reflectivity mirror 7, 11 ... KTN module 8, 9, 14, 16, 22 , 27 ... Relay lens 10 ... 1/2 wavelength plate 13, 17 ... Condensing lens 20, 21 ... Scanning mirror 23 ... Tube lens 24, 29 ... Dichroic mirror 25 ... Objective lens 26 ... Sample 28 ... Infrared cut filter 30, 31 ... Photomultiplier (PMT)
41 ... KTN crystal (electro-optic element)
41a ... Laser light incident surface 41b ... Laser light emission surface 42a, 42b, 43a, 43b ... A pair of electrodes

Claims (4)

In a non-linear optical microscope in which a laser beam emitted from a laser light source is focused on a sample by an objective lens to induce a non-linear optical phenomenon, the light generated by this non-linear optical phenomenon is detected and the sample is observed,
Light that condenses the laser light in the sample by allowing the laser light to pass through an electro-optic element that exhibits an electro-optic effect in which the refractive index inside the element changes according to the applied voltage and controlling the applied voltage. A nonlinear optical microscope characterized by varying an axial position.   The nonlinear optical microscope according to claim 1, wherein the electro-optical element is a cubic potassium tantalate niobate crystal.   The applied voltage is opposed to the one pair of electrodes on one surface of the electro-optic element, the pair of electrodes provided in a band shape with a space therebetween, and the other surface of the electro-optic element facing the one surface. The nonlinear optical microscope according to claim 1, wherein the nonlinear optical microscope is applied between the other pair of electrodes provided in a strip shape.   The non-linear optical phenomenon is a multi-photon absorption phenomenon, a harmonic generation phenomenon, a sum frequency or difference frequency generation phenomenon, or a coherent anti-Stokes Raman scattering phenomenon, according to any one of claims 1 to 3. The nonlinear optical microscope described.

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