JP4994827B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope.

  Conventionally, when observing multiple-stained fluorescent specimens, laser light with different wavelengths corresponding to each fluorescent dye is irradiated one wavelength at a time in a time-sharing manner to prevent crosstalk between the generated fluorescence. An observation method (a so-called sequential scan method) is known. For example, in the laser scanning microscope disclosed in Patent Document 1, laser beams having different wavelengths from a plurality of laser light sources are combined into one and then passed through an acousto-optic element (AOTF). By performing laser wavelength switching using this AOTF while performing laser scanning, the sequential scanning fluorescence observation method can be executed.

In addition, when observing with the scanning means for observation while performing optical stimulation with a relatively high intensity light at an arbitrary position of the specimen with the scanning means for stimulation, if the stimulation light and the observation light are simultaneously irradiated to the same part of the specimen Fluorescence generated by irradiating the stimulating laser beam is mixed into the observation light, and the luminance of the image is saturated. In order to avoid this, a method of performing irradiation of observation light and stimulation light in a time division manner using an acousto-optic element is known (see, for example, Patent Document 2 and Patent Document 3).
Furthermore, an electro-optic element is known as an element that can be switched at a higher speed than an acousto-optic element (see, for example, Patent Document 4). Also disclosed is a multi-stage configuration in which a plurality of electro-optical elements are provided in series in order to increase the light deflection angle in the electro-optical element (see, for example, Patent Document 5).

JP 2006-31018 A JP 2000-275529 A JP 2005-292273 A JP 2001-324679 A Japanese Patent Laid-Open No. 10-288798

  However, when switching the wavelength using an acousto-optic element as described in Patent Document 1, there is a limitation on the speed of sound of ultrasonic waves propagating in the acousto-optic crystal, and switching faster than a few μsec may be performed. There is an inconvenience that it cannot be done. In a general laser scanning microscope that acquires a scanned image by two-dimensionally scanning one light beam using a galvanometer mirror scanner or the like, the laser wavelength is switched for each line scan of laser light (millimeters). This means that it is difficult to switch the laser wavelength for each pixel (on the order of microseconds).

  Even if switching is performed for each pixel, light passes through a transition state in which the frequency of the ultrasonic wave existing in the acousto-optic crystal is switched. Therefore, the acousto-optic crystal is caused by the cylindrical lens effect in the acousto-optic crystal. The laser beam emitted from the laser beam spreads in one axis direction, and there is an inconvenience that the astigmatism difference is generated when the light is condensed by the objective lens and the resolution is lowered.

  Further, as described above, since the switching time by the acoustooptic device is several μsec, it is difficult to switch between stimulation light and observation light in units of one pixel, and light stimulation and observation are performed simultaneously in units of pixels. There is a problem that it cannot respond to the request to be implemented.

As an element capable of switching laser light coupled to an optical path at high speed, an electro-optic deflection element (EOD) is known as disclosed in Patent Document 4, but an electro-optic deflection element has a small deflection angle. In order to increase the refractive index change, it is necessary to set the space (distance between the electrodes) where voltage is applied to 200 μm or less.
Therefore, because the deflection angle is small, it is difficult to arrange spatially with a commercially available gas laser light source, and a lens with a long focal length is required to guide the laser light into a narrow space (distance between electrodes). The system also requires space and cost. In addition, in order to increase the deflection angle, as disclosed in Patent Document 5, a complicated structure such as arranging a plurality of pairs of electrodes in series is required, which increases the cost.

  The present invention has been made in view of the above-described circumstances, and a commercially available laser light source can be efficiently introduced with a simple configuration, and switching of illumination light such as optical path branching and synthesis of laser beams having different wavelengths can be performed on a pixel basis. It aims at providing the laser scanning microscope which can be performed at the following speeds.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a laser light source, observation scanning means for two-dimensionally scanning a laser beam emitted from the laser light source, a detection optical system for detecting light from the sample, the laser light source, and the observation An electro-optic that is disposed between the scanning means and has an electro-optic crystal in which gradation of refractive index is induced in the direction of current injection by current injection, and switches an optical path of the laser light emitted from the laser light source A deflection element; and a control unit that controls a voltage applied to the electro-optic deflection element in synchronization with scanning by the observation scanning unit, and is arranged to face the electro-optic crystal and applies a voltage to the electro-optic crystal. Thus, a laser scanning microscope is provided in which the laser light is deflected in the current injection direction when the laser light is incident between the electrodes for injecting the current.

  According to the present invention, the laser light emitted from the laser light source is deflected by the operation of the electro-optic deflection element and directed to the observation scanning unit. Then, when the laser beam is scanned two-dimensionally on the sample by the observation scanning means, light such as fluorescence or reflected light emitted from the sample is detected by the detection optical system, and a two-dimensional image of the sample is detected. Can be obtained.

  In this case, since the electro-optic deflection element includes an electro-optic crystal in which a gradation of refractive index is induced by current injection, a large deflection angle can be obtained with a small voltage. That is, in this electro-optic deflection element, the laser light incident on the electro-optic crystal is accumulated as the change in the traveling direction due to refraction proceeds in the crystal due to the refractive index gradient caused by the application of an electric field. In this way, a large deflection angle can be obtained in the direction of the electric field applied to the electro-optic crystal.

  When no electric field is applied, the laser beam travels straight through the crystal, and the deflection angle can be freely controlled according to the strength of the applied electric field. Moreover, since the refractive index gradient is generated by applying an electric field instead of using a sound wave (elastic wave) as in the acoustooptic element, the response is very high compared to the acoustooptic element. Furthermore, the traveling direction of light can be controlled at higher speed.

  Therefore, when the wavelength of the laser beam is switched, the wavelength of the laser beam directed to the observation scanning unit can be switched at a high speed by switching the voltage applied to the electro-optic deflection element. In addition, when laser light from a plurality of light sources is incident on the electro-optic deflection element, the laser light from different light sources directed to the observation scanning means is switched at high speed by switching the voltage applied to the electro-optic deflection element. Can do.

In this case, since a large deflection angle can be obtained with a small voltage, a practical deflection angle can be obtained with a practical voltage even if the distance between the electrodes of the crystal is large. Therefore, a sufficient beam diameter of the laser beam is ensured, and it is not necessary to employ a structure such as a complicated optical system, and a large deflection angle can be obtained with a small voltage. Miniaturization and cost reduction can be achieved.
In this case, it is preferable that the electro-optic crystal is KTa _1-x Nb _x O ₃ .

Wavelength In the above invention, the laser light source, which comprises a plurality of light sources for oscillating laser beams having different wavelengths, the previous SL control means controls the voltage applied to the electro-optical deflecting elements, selected among the different wavelengths of may be the laser beam may switch the emission direction of the laser beam from the electro-optical deflecting element is guided in the observation scanning unit.
In the above invention, the electro-optic deflection element may be disposed at a position where the laser beams from the light sources intersect.

  In this way, by switching the voltage applied to the electro-optic deflecting element in a state where laser beams of different wavelengths from a plurality of light sources are incident on the electro-optic deflecting element from different directions, each laser light is directed in the same direction. Can be emitted. As a result, laser beams of different wavelengths can be guided to the observation scanning unit, and the sample can be irradiated in a time division manner in units of scanning pixels.

  Further, in the above invention, a stimulation scanning unit that adjusts an irradiation position of a laser beam that gives a light stimulus to the sample is provided, and the electro-optic deflection element is connected to the optical path to the observation scanning unit and the stimulation scanning unit. The control means controls the voltage applied to the electro-optic deflection element, and the emission direction of the laser light from the electro-optic deflection element is the optical path to the observation scanning means. The optical path to the stimulation scanning unit may be switched.

  In this way, the laser light from the laser light source can be controlled by controlling the voltage applied to the electro-optic deflection element disposed at the branch point between the optical path to the observation scanning means and the optical path to the stimulation scanning means. The optical path to the observation scanning means or the optical path to the stimulation scanning means can be switched and entered. When the laser beam is incident on the optical path to the observation scanning unit, the laser beam can be scanned two-dimensionally on the sample by the operation of the observation scanning unit to observe the light from the sample. it can. Further, when the laser light is incident on the optical path to the stimulation scanning unit, the laser beam can be irradiated to an arbitrary position on the sample by operating the stimulation scanning unit to give the sample optical stimulation. . In this case, by switching the optical path with the electro-optic deflection element, the observation light and the stimulation light can be switched at a very high speed, and the observation and the light stimulation can be performed almost simultaneously for each scanning pixel.

In the above invention, the laser light source includes a plurality of light sources that oscillate laser beams of different wavelengths, and the electro-optic deflection element is disposed at a position where the laser beams from the light sources intersect. Also good.
By doing so, it is possible to switch the laser beams of different wavelengths as observation light and stimulation light at high speed, and to perform specimen observation and light stimulation almost simultaneously in units of scanning pixels.

In the above invention, the laser light source includes a plurality of light sources that oscillate laser beams of different wavelengths, and the electro-optic deflection elements are emitted from these light sources and are arranged in parallel to each other. It is good also as providing the said two electro-optic crystals which are arrange | positioned adjacent to an optical axis direction of this, and a voltage is applied to a mutually reverse direction.
By doing so, the laser beam deflected in one direction by one electro-optic crystal is deflected in the opposite direction by the other electro-optic crystal, and the laser beam is selectively transmitted to two optical paths that are substantially parallel to each other. Can be emitted.

In the above invention, an optical fiber may be disposed at least one of the electro-optic deflection element and the observation scanning unit or between the electro-optic deflection element and the stimulation scanning unit.
By doing so, it can be accurately incident on the optical fiber while passing through two adjacent electro-optic crystals while maintaining a substantially parallel optical path, and can be guided to the observation scanning means or the stimulation scanning means, respectively. .

Further, in the above invention, the voltage applied to the electro-optic deflection element by the control means to change the incident angle of the laser beam from the electro-optic deflection element to the optical fiber and adjust the amount of incident light to the optical fiber. It is good also as controlling.
By doing in this way, the intensity | strength of the light irradiated to a sample can be adjusted, without providing a light modulation means separately.

In the above invention, the control means may switch the voltage applied to the electro-optic deflection element within each pixel irradiation time of the laser beam to the specimen by the observation scanning means.
By doing so, it is possible to switch the voltage applied to the electro-optic deflection element and irradiate a plurality of switched laser beams for each scanning pixel.

In the above invention, the control means switches between the optical path to the observation scanning means and the optical path to the stimulation scanning means within each one-pixel irradiation time of the laser beam to the specimen by the observation scanning means. The time ratio may be adjusted.
In this way, the ratio between the observation light irradiation time by the observation scanning means and the stimulation light irradiation time by the stimulation scanning means is adjusted, and observation is performed while adjusting the degree of light stimulation in units of scanning pixels. It can be performed.
In the invention described above, the control means may switch the optical path to the stimulation scanning means during a discharge period of a photodetector provided in the detection optical system.

  According to the present invention, a commercially available laser light source can be efficiently introduced with a simple configuration, and illumination light switching such as optical path branching or synthesis of laser light having different wavelengths can be performed at a speed of one pixel unit or less. Play.

A laser scanning microscope 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, a laser scanning microscope 1 according to this embodiment includes a light source unit 2 that emits laser light, an observation optical system 3, a stimulation optical system 4, and these optical systems 3 and 4. The two optical fibers 5 and 6 for guiding the laser light respectively, the dichroic mirror 7 for joining these optical systems 3 and 4, and the specimen S are irradiated with the laser light, and the fluorescence emitted from the specimen S is emitted. The objective lens 8 which condenses, and the control unit 9 which controls these are provided. In the figure, reference numeral 10 denotes an imaging lens that forms an image of light from the sample S collected by the objective lens 8.

  The light source unit 2 oscillates laser light having different wavelengths λ1 and λ2 and emits laser light along optical axes that intersect each other, and these laser light sources. Electro-optical deflecting element 13 disposed at the intersection of laser beams from 11 and 12 and a coupling lens for allowing the laser light that has passed through electro-optical deflecting element 13 to be incident on the end faces of the two optical fibers 5 and 6 14 and 15, and a mirror 16 and a dichroic mirror 17 that guide laser light emitted from the electro-optic deflection element 13 to a predetermined optical path to one coupling lens 15. In the figure, reference numeral 18 denotes a shutter, and reference numeral 19 denotes a dimming ND filter.

The electro-optic deflection element 13 includes an electro-optic crystal 20 in which a gradation of refractive index is induced by current injection, and a pair of electrodes 21 a and 21 b that are opposed to each other with the electro-optic crystal 20 interposed therebetween. For example, the electro-optic crystal 20 has a Kerr constant of 3 × 10 ⁻¹⁶ m ² / V ² or more, and is composed of KTa _1-x Nb _x O ₃ . As such an electro-optic crystal 20, for example, one disclosed in the following document can be used.
"Nakamura et al., Wide-angle, low-voltage
electro-optic beam deflection based on space-charge-controlled mode of
electrical conduction in KTa _1-x Nb _x O ₃ , Applied Physics
Letters 89,
131115,
2006 ''

FIG. 2 shows the relationship between the voltage applied to the electrodes 21a and 21b at both ends of the electro-optic crystal 20 and the gradation of the refractive index.
In the electro-optic deflection element 13, the laser light incident on the electro-optic crystal 20 is accumulated as the change in the traveling direction due to refraction proceeds in the crystal as shown in FIG. In this way, a large deflection angle can be obtained in the direction of the electric field applied to the electro-optic crystal 20.

  When no electric field is applied, the laser light travels straight through the electro-optic crystal 20 as indicated by a broken line in FIG. Therefore, the deflection angle can be freely controlled according to the strength of the applied electric field.

The observation optical system 3 two-dimensionally scans the collimator lens 22 that makes the laser light guided through the optical fiber 5 substantially parallel light, and the laser light that has been made substantially parallel light by the collimator lens 22. A scanning unit (observation scanning unit) 23 and a pupil projection lens 24 that focuses the laser beam scanned by the scanning unit 23 to form an intermediate image are provided.
The scanning unit 23 is, for example, a so-called proximity galvanometer mirror in which two galvanometer mirrors that can swing in two directions intersecting each other are arranged close to each other.

  In the observation optical system 3, a dichroic mirror that condenses the fluorescent light collected by the objective lens 8 and returned through the imaging lens 10, the dichroic mirror 7, the pupil projection lens 24, and the scanning unit 23 from the optical path of the laser light. 25, a confocal lens 26 for condensing the branched fluorescence, a confocal pinhole 27 disposed in the vicinity of the focal position of the confocal lens 26, and fluorescence passing through the confocal pinhole 27 for each wavelength. The dichroic mirror 28 is separated into two, and two photodetectors 29 and 30 for detecting the fluorescence for each separated wavelength are provided. In the figure, reference numeral 31 denotes a mirror, and reference numeral 32 denotes a barrier filter.

  The stimulating optical system 4 two-dimensionally adjusts the position of the collimator lens 33 that makes the laser light guided through the optical fiber 6 substantially parallel and the laser light that has been made substantially parallel by the collimator lens 33. Scanning means (stimulating scanning means) 34, and a pupil projection lens 35 that focuses the laser light scanned by the scanning means 34 to form an intermediate image. By providing the scanning means 34 for the stimulation optical system 4, it is possible to irradiate the stimulation laser light at an arbitrary position independent of the observation position (the position where the observation laser light is irradiated by the observation optical system 3). it can.

  The control unit 9 receives and stores the fluorescence intensity information detected by the photodetectors 29 and 30 and the scanning position information by the scanning means 23 at that time in association with each other. The control unit 9 switches the voltage applied to the electrodes 21a and 21b of the electro-optic deflection element 13 in synchronization with the scanning of the laser beam on the specimen S by the scanning means 23. In the present embodiment, there are four types of patterns A to D as shown in FIG. 2 as methods of applying voltages to the electrodes 21a and 21b. FIG. 4 shows how the laser light travels in each case. FIG. 4 is an enlarged view of the light source unit 2, and (a) to (d) correspond to the cases where a voltage is applied as indicated by A to D shown in FIG. 2.

  In FIG. 4A, the laser light from the laser light source 11 is introduced into the observation optical system 3, and the laser light from the laser light source 12 is deflected out of the optical path. In FIG. 4B, the laser light from the laser light source 11 is deflected out of the optical path, and the laser light from the laser light source 12 is introduced into the observation optical system 3. In FIG. 4C, laser light from the laser light source 11 is introduced into the stimulation optical system 4, and the laser light from the laser light source 12 is deflected out of the optical path. In FIG. 4D, the laser light from the laser light source 11 is deflected out of the optical path, and the laser light from the laser light source 12 is introduced into the stimulation optical system 4.

  Specifically, when the wavelength of the laser light incident on the observation optical system 3 is switched, the voltage applied to the electrodes 21a and 21b of the electro-optic deflection element 13 is inverted within one scanning pixel as shown in FIG. Switch to let This is equivalent to switching A and B in FIG. 2 alternately for each pixel. As a result, the laser beams from the two laser light sources 11 and 12 are alternately directed to the same optical path toward the coupling lens 14 as shown in FIGS. Through the observation optical system 3. In the figure, symbol B indicates an exit from which laser light is emitted.

When the sample S is irradiated with the laser light having the wavelength λ1, the fluorescence emitted from the sample S is detected by the photodetector 29, and when the sample S is irradiated with the laser light having the wavelength λ2, the fluorescence emitted from the sample S is emitted. The photodetector 30 detects each time-division.
By doing in this way, the time division fluorescence detection per scanning pixel unit can be performed, and the time difference of the data acquisition between each wavelength can be made very small. Further, it is not necessary to provide a dichroic mirror for synthesizing wavelengths in the optical path to the observation optical system 3, and the laser light can be guided to the observation optical system 3 with a small loss.

  In addition, when fluorescence observation is performed with a laser beam with a wavelength λ1 while applying light stimulation to a certain point on the sample S with a laser beam with a wavelength λ1, it is added to the electro-optic crystal 20 as shown in FIG. The voltage value is switched between two levels. This is illustrated in FIG. This corresponds to switching C alternately for each pixel. As a result, the gradient of the refractive index generated in the electro-optic crystal 20 is changed, and as shown in FIGS. 4A and 4C, the optical path of the laser beam having the wavelength λ1 toward the coupling lens 14, and the coupling It can be directed to the optical path toward the lens 15, and can be alternately guided to the observation optical system 3 and the stimulation optical system 4 via the optical fibers 5 and 6, respectively. The light stimulus is a pulsed light stimulus that irradiates one point on the sample S intermittently.

  In this way, light stimulation and observation can be performed in a time-sharing manner, without detecting fluorescence that has been maximized by the laser light for stimulation, and accurately grasping the change in fluorescence brightness with respect to the stimulus Can do. Further, since the stimulation laser light is blinked in a high-speed pulse shape, it can be considered as a substantially continuous stimulation.

Further, when the light stimulation is performed during the analog integration discharge period in the light detector 29, the time required for the light stimulation is shorter than the time required for the observation as shown in FIG. In general, it is desirable to increase the stimulation light, and it is desirable to weaken the observation light. Therefore, in this case, by finely adjusting the voltage applied to the electro-optic crystal 20, the angle of the laser light incident on the optical fiber 5 is changed, and the amount of the observation laser light incident on the optical fiber 5 is adjusted. It is preferable to do. Further, an ND filter (not shown) for adjusting the amount of light may be disposed between the electro-optic crystal 20 and the optical fiber 5.
By doing so, since the light stimulation is performed by effectively using the discharge period of the photodetector 29, there is an advantage that the fluorescence detection efficiency is not lowered at all by performing the light stimulation in time division.

  Further, as shown in FIG. 8, the ratio between the irradiation time of the laser light for light stimulation and the irradiation time of the laser light for observation may be changed. For example, in the example shown in FIG. 8, the irradiation time for one pixel (for example, 2 μsec) is divided into 1: 4 for observation and light stimulation. In this way, the light intensity division ratio for light stimulation and observation can be performed only by adjusting the voltage application time to the electro-optic crystal 20, and a separate means for adjusting the division ratio becomes unnecessary. . Therefore, the laser scanning microscope 1 having a simple configuration and high reliability can be provided.

  Further, in the example shown in FIG. 8, observation and light stimulation are divided into four times within one pixel period (2 μsec), and one observation light irradiation time is 0.1 μsec, and one stimulation light irradiation time is set. Irradiation is repeated 4 times alternately at 0.4 μsec. By doing in this way, the continuity of stimulation light irradiation can be improved. Increasing the number of repetitions (number of divisions) of switching within one pixel can further increase the continuity of stimulation.

  Further, as shown in FIG. 9, by performing time-division switching in each pixel with laser light of wavelengths λ1 and λ2 while performing light stimulation with laser light of wavelength λ1 at a certain point on the sample, 2 It is also possible to perform fluorescence observation of the heavily stained specimen S. This corresponds to switching in the order of A, C, B, and C in FIG. 2 within one pixel. Specifically, in the range on the sample S corresponding to one pixel, first, the laser beam for observation with the wavelength λ1 is irradiated, then the laser beam for light stimulation with the wavelength λ1 is irradiated, and then the wavelength λ2 The laser beam for observation is irradiated, and finally, the laser beam for light stimulation having the wavelength λ1 is irradiated again.

  Then, the fluorescence generated from the sample S by irradiating the laser beam with the wavelength λ1 is detected by one photodetector 29, and the fluorescence generated from the sample S by irradiating the laser beam with the wavelength λ2 is detected by the other light. It is detected by the device 30. In this case, light stimulation is performed during the analog integration discharge period of fluorescence detection by the photodetectors 29 and 30.

  In this way, since the time-division fluorescence observation of the double-stained specimen S and the optical stimulation in the time division are switched at the same time in units of scanning pixels, the laser beams having a plurality of different wavelengths for observation are extremely short. Irradiation can be performed with a time difference, and fluorescence observation can be performed without detecting fluorescence that is maximized by a stimulation laser beam. In addition, since light stimulation is performed during the discharge period of the photodetectors 29 and 30, it is not necessary to cause a decrease in fluorescence detection efficiency due to light stimulation performed in a time-sharing manner.

  In the present embodiment, the case where light stimulation is performed on an arbitrary point on the specimen S has been described as an example, but instead, the laser light for light stimulation is scanned on the specimen S in a spiral manner. You may decide to make it. In this case, the optical stimulation can be continuously applied in a spiral manner by making the moving distance of the spot of the laser beam for optical stimulation irradiated intermittently equal to or less than the spot diameter.

  Further, the laser beam for light stimulation may be scanned on the specimen S in an arbitrary region by a reciprocating raster scan, a line scan, or a free line scan method. By doing so, it is possible to perform light stimulation on an arbitrary region in a wider range than point stimulation without impairing the simultaneity of observation and light stimulation.

  In the laser scanning microscope 1 according to the present embodiment, two laser beams having different wavelengths λ1 and λ2 from the two laser light sources 11 and 12 are switched. 12, three laser beams having different wavelengths λ1 to λ3 from the three laser light sources 36 may be switched. In addition to the above embodiment, the laser light source 36 is disposed at a position where the laser light having the wavelength λ3 is directed to the coupling lens 14 in a state where no voltage is applied to the electro-optic crystal 20.

  Then, as shown in FIGS. 11 and 12, the voltage value applied to the electro-optic deflection element and the direction of the voltage are switched, and the laser beams with wavelengths λ1, λ2, and λ3 from the three laser light sources 11, 12, and 36 are switched. Can be directed toward the two coupling lenses 14 and 15, respectively.

Specifically, it is as follows.
(A) When electrode 21a = 0V and electrode 21b = 200V, observation optical system 3 = λ1 (laser light source 11), stimulation optical system 4 = none.
(B) When electrode 21a = 200V and electrode 21b = 0V, observation optical system 3 = λ2 (laser light source 12), stimulation optical system 4 = none.
(C) When the electrode 21a = 0V and the electrode 21b = 0V, the observation optical system 3 = λ3 (laser light source 36) and the stimulation optical system 4 = none.
(D) When the electrode 21a = 0V and the electrode 21b = 100V, the observation optical system 3 = none and the stimulation optical system 4 = λ1 (laser light source 11).
(E) When the electrode 21a = 100V and the electrode 21b = 0V, the observation optical system 3 = none and the stimulation optical system 4 = λ2 (laser light source 12).
(F) When the electrode 21a = 0V and the electrode 21b = 130V, the observation optical system 3 = none and the stimulation optical system 4 = λ3 (laser light source 36).
By doing so, for example, as shown in FIG. 11, fluorescence observation can be performed in units of scanning pixels with laser light with wavelengths λ2 and λ3 while performing light stimulation with laser light with wavelength λ1.

  With this configuration, laser light from ultraviolet light to two-photon excitation near-infrared laser light (for example, 920 nm) is used using the gradient of refractive index generated in the electro-optic crystal 20. Can do.

Next, a laser scanning microscope according to the second embodiment of the present invention will be described below with reference to FIG.
The laser scanning microscope according to this embodiment includes a light source unit 40 shown in FIG.
In the description of the present embodiment, portions having the same configuration as those of the laser scanning microscope 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  The light source unit 40 includes laser light sources 41 and 42 that respectively emit two laser beams having substantially parallel optical axes and different wavelengths, an electro-optic deflection element 43 that transmits the laser light from each of the laser light sources 41 and 42, and Coupling lens 14 for causing laser beams along two parallel optical axes emitted from electro-optic deflection element 43 to enter optical fibers 5 and 6 connected to observation optical system 3 and stimulation optical system 4, respectively. , 15. In the figure, reference numeral 44 denotes a mirror.

  In the present embodiment, the electro-optic deflection element 43 includes two electro-optic crystals 45 and 46 arranged adjacent to each other in the optical axis direction, and counter electrodes 47a and 47b; 48a and 48b sandwiching the electro-optic crystals 45 and 46, respectively. It has. And the same magnitude | size voltage is applied to these counter electrode 47a, 47b; 48a, 48b to a mutually reverse direction.

  That is, as shown in FIG. 14A, the laser light of wavelength λ1 deflected in one direction by one electro-optic crystal 45 is deflected in the other direction by the other electro-optic crystal 46, and is substantially parallel light. The light is incident on one coupling lens 15 while holding the shaft. Then, as shown in FIG. 14 (b), by switching the voltage value applied to the counter electrodes 47a, 47b; 48a, 48b of the electro-optic crystals 45, 46, the other cup is utilized using the change in the deflection angle. The light can enter the ring lens 14.

  Further, as shown in FIGS. 14A and 14D, by reversing the voltage applied to the electro-optic crystals 45 and 46, the laser light having the wavelength λ2 deflected in one direction by the electro-optic crystal 45 is converted into the other. This is deflected in the other direction by the electro-optic crystal 46 and is incident on the coupling lens 14 while maintaining a substantially parallel optical axis. Then, as shown in FIG. 14C, by switching the voltage value applied to the counter electrodes 47a, 47b; 48a, 48b of the electro-optic crystals 45, 46, the change in the deflection angle is used to change the wavelength λ2. Laser light is incident on the coupling lens 15.

  By doing so, laser beams having different wavelengths λ1 and λ2 can be coaxially emitted from the electro-optic deflection element 43 as observation laser light and light stimulation laser light, respectively. Both of these methods have an advantage that a dichroic mirror for wavelength synthesis can be eliminated.

Next, a laser scanning microscope according to the third embodiment of the present invention will be described below with reference to FIG.
The laser scanning microscope according to this embodiment includes a light source unit 50 shown in FIG.
In the description of the present embodiment, portions having the same configuration as those of the laser scanning microscope 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

The light source unit 50 combines three laser light sources 51, 52, and 53 that emit laser beams having three different wavelengths λ1, λ2, and λ3, and laser beams emitted from these laser light sources 51, 52, and 53. Mirror 54 and dichroic mirrors 55 and 56, a diffraction grating 57 that separates the combined laser beams at different angles for each wavelength, and the three separated laser beams that intersect at a predetermined angle. The condensing lenses 58 and 59 that emit light, the electro-optic deflecting element 60 that is disposed at the intersection of the laser beams by the condensing lenses 58 and 59, and the coupling lenses 14 and 15 that are incident on the two optical fibers 5 and 6 And a mirror 16 and a dichroic mirror 17.
The operation of the electro-optic deflection element 60 in this embodiment is the same as the operation in FIGS. 10 to 12 in the first embodiment.

  By doing in this way, the size of the laser light sources 51, 52, 53 is practically large, and in the method of the first embodiment, if the three laser light sources 51, 52, 53 are spatially arranged, a large space is required. Even if necessary, it can be arranged in a relatively small space.

1 is a schematic diagram illustrating an overall configuration of a laser scanning microscope according to a first embodiment of the present invention. 2 is a graph showing the relationship between the voltage and refractive index of an electro-optic deflection element provided in the light source unit of the laser scanning microscope of FIG. FIG. 3 is a schematic diagram illustrating a state of refraction of laser light in an electro-optic crystal of the electro-optic deflection element of FIG. 2. It is explanatory drawing explaining operation | movement of the light source unit of the laser scanning microscope of FIG. It is a graph which shows the relationship between the voltage applied to an electrode at the time of switching operation | movement of Fig.4 (a), (b), and the emitted laser beam. It is a graph which shows the relationship between the voltage applied to an electrode at the time of switching operation | movement of Fig.4 (a), (c), and the emitted laser beam. FIG. 7 is a graph for explaining a case in which stimulation laser light is emitted within a discharge period of the photodetector in the operation of FIG. 6. FIG. 7 is a graph for explaining a case where the ratio of the irradiation time of the observation laser light and the stimulation laser light is changed in the operation of FIG. 6. It is a graph which shows the relationship between the voltage applied to an electrode at the time of switching operation | movement of Fig.4 (a), (b), (c), and the emitted laser beam. FIG. 6 is a schematic diagram showing a light source unit having three laser light sources, which is a modification of the laser scanning microscope of FIG. 1. It is a graph explaining the operation | movement which performs the observation by two different wavelengths, and the optical stimulation by a different wavelength by a time division by the light source unit of FIG. It is explanatory drawing explaining operation | movement of the light source unit of FIG. It is a schematic diagram which shows the light source unit of the laser scanning microscope which concerns on the 2nd Embodiment of this invention. It is explanatory drawing explaining operation | movement of the light source unit of FIG. It is a schematic diagram which shows the light source unit of the laser scanning microscope which concerns on the 3rd Embodiment of this invention.

Explanation of symbols

A Sample λ1, λ2, λ3 Wavelength 1 Laser scanning microscope 3 Observation optical system (detection optical system)
5, 6 Optical fiber 9 Control unit (control means)
11, 12, 36, 41, 42, 51, 52, 53 Laser light source (light source)
13, 43, 60 Electro-optic deflection element 20, 45, 46 Electro-optic crystal 23 Scanning means (observation scanning means)
34 Scanning means (stimulating scanning means)

Claims (12)

A laser light source;
Scanning means for observation that two-dimensionally scans the specimen with laser light emitted from the laser light source;
A detection optical system for detecting light from the specimen;
The laser light emitted from the laser light source, comprising an electro-optic crystal that is disposed between the laser light source and the observation scanning unit and in which a gradation of refractive index is induced in the current injection direction by current injection. An electro-optic deflection element for switching the optical path of
Control means for controlling the voltage applied to the electro-optic deflection element in synchronization with scanning by the observation scanning means,
The laser beam is deflected in the direction of current injection by applying the voltage to the electro-optic crystal and injecting the current between the electrodes disposed opposite to each other with the electro-optic crystal interposed therebetween. Laser scanning microscope. The laser light source includes a plurality of light sources that oscillate laser beams of different wavelengths,
Before SL control means, said electrical controls the voltage applied to the optical deflecting element, the laser light from the electro-optical deflecting element so that the laser beam of the selected wavelength of the different wavelengths are guided to the observation scanning unit the laser scanning microscope according to the emission direction to switch between El claim 1. Before SL electro-optical deflecting element, the laser scanning microscope according to claim 2, which is located where the laser light from the light source. Comprising a scanning means for stimulation for adjusting the irradiation position of the laser beam for applying a light stimulus to the specimen;
The electro-optic deflection element is disposed at a branch point between an optical path to the observation scanning unit and an optical path to the stimulation scanning unit;
The control means controls the voltage applied to the electro-optic deflection element, and determines the emission direction of the laser light from the electro-optic deflection element to the optical path to the observation scanning means and the optical path to the stimulation scanning means. The laser scanning microscope according to claim 1 to be switched. The laser light source includes a plurality of light sources that oscillate laser beams of different wavelengths,
The laser scanning microscope according to claim 4, wherein the electro-optic deflection element is disposed at a position where laser beams from the light source intersect. The laser light source includes a plurality of light sources that oscillate laser beams of different wavelengths,
The two electro-optical elements in which the electro-optic deflecting element is disposed adjacent to the optical path of a plurality of laser beams emitted from these light sources and arranged in parallel to each other, and a voltage is applied in opposite directions to each other. The laser scanning microscope according to claim 2 or 4, comprising a crystal.   The laser scanning microscope according to claim 6, wherein an optical fiber is disposed at least one of the electro-optic deflection element and the observation scanning unit or between the electro-optic deflection element and the stimulation scanning unit.   The control means controls the voltage applied to the electro-optic deflecting element so as to change the incident angle of the laser light from the electro-optic deflecting element to the optical fiber and adjust the amount of light incident on the optical fiber. The laser scanning microscope described. The laser scanning type according to any one of claims 1 to 4, wherein the control unit switches a voltage applied to the electro-optic deflection element within each one-pixel irradiation time of the laser beam to the specimen by the observation scanning unit. microscope.   The said control means adjusts the time ratio which switches the optical path to the scanning means for observation, and the optical path to the scanning means for stimulation within each 1 pixel irradiation time of the laser beam to the sample by the scanning means for observation. 4. A laser scanning microscope according to 4. The electro-optical _{crystal, KTa 1-x Nb x O} 3 laser scanning microscope according to any one of claims 1 to claim 10.   The laser scanning microscope according to claim 10, wherein the control unit switches to an optical path to the stimulation scanning unit during a discharge period of a photodetector included in the detection optical system.

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