JP5551477B2 - Light source device and laser scanning microscope device - Google Patents

Description

  The present invention relates to a light source device and a laser scanning microscope apparatus.

Conventionally, a CARS (coherent anti-Stokes Raman scattering) system using a femtosecond pulse laser is known (for example, see Non-Patent Document 1 and Patent Document 1).
The system of Non-Patent Document 1 uses light obtained by narrowing a pulse laser beam from a femtosecond laser light source as pump light, and wavelength conversion of the pulse laser light from the femtosecond laser light source as Stokes light using a photonic crystal fiber. The two light beams are combined and incident on the specimen, and the CARS light is generated from the molecules using specific vibrations of the molecules in the specimen, and this is detected and observed. is there.

  The system of Patent Document 1 splits a pulsed laser beam from a femtosecond laser light source, converts the wavelength of only one of the pulsed laser beams by a photonic crystal fiber, and then frequency-disperses each pulsed laser beam for frequency difference. The pump light having St. and Stokes light are generated, and the CARS light from the molecule is detected for observation.

  According to these CARS systems, a CARS light image can be obtained by making a pulse laser beam, which is a combination of pump light and Stokes light, incident on the sample, and a multiphoton fluorescence image of the sample can be obtained using the same pulse laser light. Can be obtained.

Kano, “Applied Physics 75 (6)”, p. 682, 2006

US Patent Application Publication No. 2009/0290150

However, when the multiphoton fluorescence observation of the specimen is simultaneously performed by using the light source device of the CARS system in the systems of Non-Patent Document 1 and Patent Document 1, the obtained multiphoton fluorescence image is dark and clear multiphoton fluorescence. There is an inconvenience that observation cannot be performed.
That is, the system of Non-Patent Document 1 has reduced the power by narrowing the pump light, and the Stokes light has reduced the power density by wavelength conversion, so that a sufficient amount of fluorescence is obtained. I can't. In addition, since the system of Patent Document 1 frequency-disperses both the pump light and the Stokes light, the power density is reduced, and a sufficient amount of fluorescence cannot be obtained in the same manner, so that clear multiphoton fluorescence observation can be performed. I can't do it.

  The present invention has been made in view of the above-described circumstances, and provides a light source device and a laser scanning microscope apparatus that can acquire a high-contrast CARS light image and at the same time acquire a bright multiphoton fluorescence image. It is intended to provide.

In order to achieve the above object, the present invention provides the following means.
The present invention generates two pulsed laser beams having a predetermined frequency difference from a laser light source that generates a pulsed laser beam, and the pulsed laser beam emitted from the laser light source. Are combined and input to the scanning microscope, the second optical path for inputting the pulse laser beam emitted from the laser light source to the scanning microscope as it is, the first optical path and the first optical path. In a time-division manner, the pulse laser beam from the laser light source is switched to the first optical path or the second optical path in synchronization with the multiplexing unit that combines the two optical paths and the scanning period of the scanning microscope. Provided is a light source device including an incident light path switching unit.

  According to the present invention, when the pulse laser beam emitted from the laser light source is incident on the first optical path by the operation of the optical path switching unit, the two pulse laser beams having a predetermined frequency difference from the incident pulse laser beam. Are generated and are incident on the scanning microscope in a state where these pulse laser beams are combined. By using one pulse laser beam as pump light and the other pulse laser beam as Stokes light, CARS light can be generated in the specimen, and a CARS light image with high contrast can be obtained.

  On the other hand, when the pulsed laser light emitted from the laser light source is incident on the second optical path by the operation of the optical path switching unit, the incident pulsed laser light is directly incident on the scanning microscope and is caused by the multiphoton excitation effect in the sample. Multiphoton fluorescence is generated. Since the pulsed laser light is incident on the scanning microscope without being dispersed or narrowed, it is possible to collect a pulsed laser light having a sufficient photon density in the sample and obtain a bright multiphoton fluorescence image.

  And since an optical path switching part switches the optical path which makes a pulse laser beam enter in synchronization with the scanning period of a scanning microscope, it becomes possible to acquire simultaneously a CARS image with high contrast, and a bright multiphoton fluorescence image. That is, the CARS image and the multi-photon fluorescence image can be obtained sequentially by performing the switching of the optical path by the optical path switching unit for each scanning period of one frame by the scanning microscope. Further, by switching the optical path by the optical path switching unit for each scanning period of one scanning line by the scanning microscope, it is possible to obtain a CARS light image and a multiphoton fluorescence image with higher synchronization.

In the said invention, the said optical path switching part may be provided with the galvanometer mirror.
In this way, the optical path can be switched at the speed of light with a simple configuration.

A first reference example of the present invention includes a laser light source that generates pulsed laser light, a demultiplexing unit that branches the pulsed laser light emitted from the laser light source into two optical paths, and the demultiplexing unit. The pulse laser provided in one of the branched optical paths, provided in the other optical path branched by the band narrowing mechanism or frequency dispersion mechanism for frequency dispersion of the pulse laser light and the branching unit. A wavelength conversion unit that converts the wavelength of light, a delay adjustment unit that is provided in any one of the optical paths branched by the demultiplexing unit, and that adjusts the optical delay between the pulse laser beams that pass through these optical paths, A multiplexing unit that multiplexes the pulsed laser light that has passed through the optical path and inputs it to a scanning microscope, and the demultiplexing unit determines a branching ratio of the pulsed laser light to two optical paths. Microscope run To provide a light source device is adjustable in synchronization with the period.

According to the first reference example , the pulsed laser light emitted from the laser light source is branched into two optical paths by the demultiplexing unit. The pulse laser beam branched to one optical path is frequency-dispersed by the narrow band or the dispersion adjusting mechanism by the narrow band mechanism, and the wavelength of the pulse laser light branched to the other optical path is converted by the wavelength converter. Then, the two pulse laser beams are combined by the combining unit with the optical delay between them adjusted by the delay adjusting unit, and are incident on the scanning microscope. By using one pulse laser beam as pump light and the other pulse laser beam as Stokes light, CARS light can be generated in the specimen, and a CARS light image with high contrast can be obtained.

  In this case, the demultiplexing unit adjusts the branching ratio of the pulsed light to the two optical paths. Therefore, by setting the branching ratio of the pulsed laser light to the two optical paths to a predetermined ratio, appropriate pump light and Stokes A high-contrast CARS light image can be obtained by generating light. In addition, by adjusting the branching ratio and increasing the amount of pulsed laser light incident on one optical path, the absolute amount of pulsed laser light focused on the specimen can be increased to obtain a bright multiphoton fluorescence image. . By switching the branching ratio in synchronization with the scanning period of the scanning microscope, it is possible to acquire a CARS light image and a multiphoton fluorescence image with high simultaneity.

In the first reference example , the branching unit may be a combination of an electro-optic modulator and a polarization beam splitter, or an acousto-optic modulator.
In this way, by adjusting the voltage applied to the electro-optic modulator, the polarization direction of the pulsed laser light incident on the polarization beam splitter can be changed, and the branching ratio by the polarization beam splitter can be changed. Further, by changing the acoustic wave incident on the acousto-optic modulator, the ratio of the 0th-order light and the diffracted light can be changed, and the branching ratio can be easily adjusted. It is not necessary to perform mechanical switching, and it is possible to acquire a CARS light image and a multiphoton fluorescence image with higher synchronization by switching the branching ratio at high speed.

A second reference example of the present invention includes a laser light source that generates pulsed laser light, a demultiplexing unit that branches the pulsed laser light emitted from the laser light source into two optical paths, and the demultiplexing unit. An acousto-optic tunable filter provided in one of the branched optical paths, an acousto-optic modulator provided in the other optical path branched by the demultiplexing unit, and a wavelength converter that converts the wavelength of the pulsed laser light; A delay adjusting unit that adjusts an optical delay between the pulse laser beams that are provided in any one of the optical paths branched by the branching unit and that passes through these optical paths; and the pulse laser light that has passed through the two optical paths. A multiplexing unit for multiplexing and inputting to the scanning microscope, a modulation signal for narrowing the band of the acousto-optic tunable filter in synchronization with a scanning period of the scanning microscope, and a modulation for widening the band Switching between items, the acousto-optic tunable filter when in is broadband, to provide a light source device and a control unit that sets the modulation signal to block the acoustic-optic modulator.

According to the second reference example , the pulsed laser light emitted from the laser light source is branched into two optical paths by the branching unit. The pulse laser beam branched to one optical path is guided as it is in a form modulated to an appropriate power by applying a single frequency modulation signal to the acousto-optic tunable filter, and the multi-frequency modulation signal is converted into a modulated signal. By giving, it is accompanied in a narrower band form. The output of the pulsed laser beam branched to the other optical path is modulated by the acousto-optic modulator, and the wavelength is converted by the wavelength converter and guided.

  When CARS light observation is performed, a multi-frequency modulation signal is incident on an acousto-optic tunable filter to narrow the band of the pulsed laser light, and the acousto-optic modulator is set to an ON state so that it is placed in another optical path. The incident pulsed light is incident on the wavelength conversion unit, and a predetermined frequency difference is set in the pulsed laser light passing through the two optical paths. As a result, the pulse laser light that has passed through one optical path is used as pump light, the other pulse laser light is used as Stokes light, and the two pulse laser lights are combined while the optical delay between them is adjusted by the delay adjusting unit. By combining with the wave part and entering the scanning microscope, CARS light can be generated in the specimen. In this case, a CARS light image with high contrast can be obtained by appropriately adjusting the ratio of the pump light and the Stokes light.

  On the other hand, when performing multiphoton fluorescence observation, a modulation signal of a single frequency is made incident on an acoustooptic tunable filter, the pulse laser beam whose output is adjusted is guided as it is, and the acoustooptic modulator is turned off. Set to block the pulse laser beam incident on the other optical path. As a result, the pulsed laser beam guided without being narrowed is incident on the sample as it is, and the frequency-dispersed Stokes light is cut off, improving the photon density of the pulsed laser beam focused in the sample. Thus, a bright multiphoton fluorescence image can be obtained. Even in this case, it is not necessary to perform mechanical switching, and the pulsed laser light can be switched at a high speed to obtain a more synchronized CARS light image and multiphoton fluorescence image.

The third reference example of the present invention includes a laser light source that generates pulsed laser light, a demultiplexing unit that branches the pulsed laser light emitted from the laser light source into two optical paths, and the demultiplexing unit. The pulse laser provided in one of the branched optical paths, provided in the other optical path branched by the band narrowing mechanism or frequency dispersion mechanism for frequency dispersion of the pulse laser light and the branching unit. A wavelength conversion unit that converts the wavelength of light, a delay adjustment unit that is provided in any one of the optical paths branched by the demultiplexing unit, and that adjusts the optical delay between the pulse laser beams that pass through these optical paths, Bypassing the narrowing mechanism or the dispersion adjusting mechanism between the multiplexing unit that combines the pulsed laser light that has passed through the optical path and inputs it to the scanning microscope, and the demultiplexing unit and the multiplexing unit And the narrow band The optical path through the mechanism or the dispersion regulating mechanism to provide a light source device and a bypass optical path optical path lengths are different.

According to the third reference example , the pulse laser beam branched to one optical path by the demultiplexing unit is narrowed by the narrowing mechanism, or is frequency-dispersed by the dispersion adjusting mechanism, whereby the Stokes light. Then, the pulse laser beam branched to the other optical path is converted into the Stokes light by being wavelength-converted by the wavelength conversion unit. Then, the optical delay unit adjusts the optical delay between the optical delay units and combines the optical signals in the combining unit and introduces them into the scanning microscope, thereby obtaining a CARS optical image having a high contrast in the sample.

On the other hand, among the above optical paths, a part of the pulse laser beam branched to the optical path having the narrowing mechanism or the dispersion adjusting mechanism is incident on a bypass optical path that bypasses the narrowing mechanism or the dispersion adjusting mechanism. The data is input to the scanning microscope as it is. Since the optical path length of the detour optical path is different from that of the optical path having the band narrowing mechanism or the dispersion adjusting mechanism, the detour optical path is incident on the scanning microscope as pulse laser light that is shifted in time with respect to the pump light. As a result, CARS light and multiphoton fluorescence are generated in a time-separated manner, and a high-contrast CARS light image and a bright and clear multiphoton fluorescence image are acquired while maintaining higher simultaneity. Can do.
The present invention also provides a laser scanning microscope apparatus comprising any one of the light source devices described above and a scanning microscope that makes the pulse laser light from the light source device enter.

  According to the present invention, it is possible to acquire a bright multiphoton fluorescence image at the same time as acquiring a CARS light image with high contrast.

1 is a block diagram showing a light source device and a laser scanning microscope apparatus according to a first embodiment of the present invention. It is a block diagram which shows the light source device and laser scanning microscope apparatus which concern on the 2nd Embodiment of this invention. It is a block diagram which shows the light source device and laser scanning microscope apparatus which concern on the 3rd Embodiment of this invention. It is a block diagram which shows the light source device and laser scanning microscope apparatus which concern on the 4th Embodiment of this invention. It is a block diagram which shows the light source device and laser scanning microscope apparatus which concern on the 5th Embodiment of this invention.

A light source device and a laser scanning microscope apparatus according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a laser scanning microscope apparatus 1 according to this embodiment is used for observing a specimen A by making a light source apparatus 2 according to this embodiment and a pulse laser beam from the light source apparatus 2 incident thereon. A scanning microscope 3 is provided.

  The light source device 2 according to the present embodiment includes a single laser light source 4 that emits a femtosecond pulsed laser light L, and two lasers having a certain frequency difference from the femtosecond pulsed laser light L emitted from the laser light source 4. The first optical path 5 for generating the pulsed laser beams L1 ′ and L2 ′, the second optical path 6 for passing the femtosecond pulsed laser beam L emitted from the laser light source 4 as it is, and the optical paths 5 and 6 are merged. And a light path switching unit 8 for switching the incidence of the femtosecond pulsed laser light L from the laser light source 4 to the optical paths 5 and 6.

  The first optical path 5 includes a beam splitter (demultiplexing unit) 9 that branches the pulse laser beam L from the laser light source 4 into two, and two pulse laser beams L1 and L2 branched by the beam splitter 9, respectively. Two optical paths 10 and 11 to be passed through and an introduction optical system 12 that multiplexes two pulse laser beams L1 ′ and L2 ′ that have passed through the two optical paths 10 and 11 are provided.

  On one optical path 10, a dispersion adjusting glass 14 that imparts frequency dispersion to the pulsed laser light L1 is disposed to generate pump light L1 '(hereinafter also referred to as pump light L1'). In the other optical path 11, a photonic crystal fiber 15 and a dispersion adjusting glass 16 are arranged to generate Stokes light L2 '(hereinafter also referred to as Stokes L2').

The dispersion adjusting glasses 14 and 16 impart predetermined frequency dispersion to the pulse laser beams L1 and L2 that have passed therethrough.
The photonic crystal fiber 15 can change or expand the frequency band of the pulsed laser light L2 that passes therethrough. Further, the pulsed laser light L2 that has passed through the photonic crystal fiber 15 has a frequency dispersion according to the type of the photonic crystal fiber 15 and the conditions of the pulsed laser light L2 that is to be passed through. Further, for example, as the frequency conversion means, any one of a bulk, a thin film, a film, and a photonic crystal structure having the same function and action may be used instead of the photonic crystal fiber 15.

  The multiplexing unit 7 is a polarization beam splitter disposed at the subsequent stage of the dispersion adjusting glass 14 in one optical path 10 that generates the pump light L1 '. The multiplexing unit 7 has a transmission axis set so as to transmit the pulsed laser light L1 ′ that has passed through the dispersion adjusting glass 14, and reflects the pulsed laser light L that has passed through the second optical path 6 described later. Thus, the pulse laser beam L1 ′ that has passed through the optical path 10 is multiplexed.

  The introduction optical system 12 includes a pulse timing adjusting unit 17 that gives a time delay between the pulse laser beams L1 ′ and L2 ′ that have passed through the two optical paths 10 and 11, and these pulse laser beams L, L1 ′, And a laser combiner 18 for multiplexing L2 ′. The pulse timing adjusting means 17 is constituted by a mirror (reflector), for example (not shown). Using at least two or more sets of reflectors, for example, the optical path length of the pulsed laser beam L2 ′ can be changed by turning back the optical path of the pulsed laser beam L2 ′ and adjusting the interval between the at least two sets of reflectors. . As a result, the pulse timing of the pulse laser beam L2 'can be shifted with respect to the pulse laser beam L1', and the amount of time delay can be adjusted.

  The second optical path 6 guides the pulsed laser light L from the laser light source 4 as it is, and in one optical path 10 that generates the pump light L1 ′ in the first optical path 5, in the latter stage of the dispersion adjusting glass 14. The light is combined by a combining unit 7 composed of a polarization beam splitter. A λ / 2 plate 19 is disposed in the second optical path 6, and the pulse laser beam L guided through the second optical path 6 is reflected by the multiplexing unit 7 so that it is totally reflected by the multiplexing unit 7. The polarization direction at the time of incidence can be set.

  The optical path switching unit 8 includes a galvanometer mirror 20 that is swingable about a predetermined axis, and a control unit 21 that controls the swing of the galvanometer mirror 20. The galvanometer mirror 20 selectively enters the pulsed laser light L from the laser light source 4 into either the first optical path 5 or the second optical path 6 by switching the oscillation angle position to two different angular positions. It can be made to.

The control unit 21 outputs a swing angle switching command signal S to the galvanometer mirror 20 in synchronization with the scanning period of the pulse laser beams L, L1 ′, L2 ′ by the scanner 22 of the scanning microscope 3 to be described later. It has become.
For example, each time the pulse laser beams L, L1 ′, and L2 ′ are scanned for one frame on the specimen A by the scanner 22 of the scanning microscope 3, the control unit 21 commands to switch the swing angle of the galvanometer mirror 20. By outputting S, the CARS light image and the multiphoton fluorescence image can be switched and acquired in a frame sequential manner.

  Further, every time the pulse laser beams L, L1 ′, L2 ′ from the scanner 22 of the scanning microscope 3 are scanned for one line on the specimen A, the control unit 21 commands to switch the swing angle of the galvanometer mirror 20. By outputting S, the CARS light image and the multiphoton fluorescence image can be switched and acquired in a line sequential manner. Compared to switching to frame sequential, switching to line sequential can achieve higher simultaneity between the CARS light image and the multiphoton fluorescence image.

  The scanning microscope 3 includes a scanner 22 and a lens group 23 that two-dimensionally scans the pulse laser beams L, L1 ′, and L2 ′ incident from the light source device 2, and the pulse laser beams L and L scanned by the scanner 22. A condensing lens 24 that condenses L1 ′ and L2 ′ on the specimen A, a first photodetector 25 that detects fluorescence generated in the specimen A and collected by the condensing lens 24, and transmitted through the specimen A A condensing lens 26 that condenses the CARS light generated in the direction in which the light is collected, and a second photodetector 27 that detects the CARS light collected by the condensing lens 26.

  In the figure, reference numeral 28 denotes a dichroic mirror, reference numeral 29 denotes a stage, and reference numeral 30 denotes a mirror. Further, the fluorescence generated in the specimen A may be collected by the condenser lens 26 and detected by the second photodetector 27. Further, the CARS light generated in the specimen A may be collected by the condenser lens 24 and detected by the first photodetector 25.

The operation of the light source device 2 and the laser scanning microscope device 1 according to the present embodiment configured as described above will be described below.
According to the light source device 2 according to the present embodiment, the pulsed laser light L emitted from the laser light source 4 is incident on the first optical path 5 or the second optical path 6 by the operation of the optical path switching unit 8. The pulsed laser light L incident on the first optical path 5 is branched into two optical paths 10 and 11 by the beam splitter 9, one of which is given a predetermined frequency dispersion by the dispersion adjusting glass 14 to become pump light L1 ′, On the other hand, the frequency band is changed by the photonic crystal fiber 15 and frequency dispersion is given by the dispersion adjusting glass 16 to become Stokes light L2 ′.

  Then, the pump light L <b> 1 ′ passes through the multiplexing unit 7 and is incident on the laser combiner 18 of the introduction optical system 12. On the other hand, the Stokes light L 2 ′ is incident on the introduction optical system 12, the timing with respect to the pump light L 1 ′ is adjusted by the pulse timing adjusting means 17 in the light guide optical system 12, and then combined in the same optical path by the laser combiner 18. Then, the light enters the scanning microscope 3.

  On the other hand, the pulsed laser light L incident on the second optical path 6 is reflected by being incident on the multiplexing unit 7 after adjusting the polarization direction by passing through the λ / 2 plate 19 and being pumped light L1. It enters the same optical path as'.

  When the pulse laser beams L1 ′ and L2 ′ that have passed through the first optical path 5 are incident on the scanning microscope 3, the pulse laser beams L1 ′ and L2 ′ are scanned two-dimensionally by the scanner 22, and the lens group 23 and The light is condensed on the specimen A by the condenser lens 24, and CARS light is generated at the light condensing position of the specimen A according to the frequency difference between the two pulse laser lights L1 ′ and L2 ′, and the generated CARS light is condensed. The light is collected by the lens 26 and detected by the second photodetector 27.

  When the pulsed laser light L that has passed through the second optical path 6 is incident on the scanning microscope 3, the pulsed laser light L is scanned two-dimensionally by the scanner 22, and the sample is collected by the lens group 23 and the condenser lens 24. By condensing on A, fluorescence is generated by the multiphoton excitation effect at the condensing position, and the generated fluorescence is condensed by the condensing lens 24 and branched from the optical path of the pulsed laser light L by the dichroic mirror 28. And detected by the first photodetector 25.

  In this case, according to the light source device 2 and the laser scanning microscope device 1 according to the present embodiment, the control unit 21 operates the optical path switching unit 8 in synchronization with the scanning cycle of the scanner 22 of the scanning microscope 3. CARS light images and multiphoton fluorescence images can be acquired in a frame sequential manner or in a line sequential manner. And the CARS light image and multiphoton fluorescence image obtained at this time have the advantage that a high-contrast CARS light image and a bright multiphoton fluorescence image can be obtained simultaneously without affecting each other.

  In the present embodiment, the frequency dispersion amount adjustment CARS using the pump light L1 ′ and the Stokes light L2 ′ that are provided with frequency dispersion by transmitting the pulse laser light L through the dispersion adjustment glasses 14 and 16 is used. Although the case has been described as an example, instead of this, the dispersion adjusting glasses 14 and 16 in the two optical paths 10 and 11 are eliminated, and the narrow band that narrows the pulse laser beam L in the optical path 10 of the pump light L1 ′. The present invention may be applied to a case where a mechanism (not shown) is arranged.

Next, a light source device 40 and a laser scanning microscope device 41 according to a second embodiment of the present invention will be described with reference to FIG.
In the description of the light source device 40 and the laser scanning microscope device 41 according to the present embodiment, the same reference numerals are given to the portions having the same configuration as the light source device 2 and the laser scanning microscope device 1 according to the first embodiment described above. A description thereof will be omitted.

  As shown in FIG. 2, the light source device 40 according to the present embodiment passes through the second optical path 6 in the light source device 2 according to the first embodiment out of the two optical paths 10 and 11 of the first optical path 5. The demultiplexing unit 42 is configured to switch the incident amount of the pulsed laser light L that is incident on the optical path 10 of the pump light L1 ′, in common with the optical path 10 of the pump light L1 ′.

  That is, the light source device 40 according to the present embodiment uses an electro-optic modulator 43 and a polarization beam splitter 44 as the demultiplexing unit 42 instead of the beam splitter 9. The electro-optic modulator 43 can change the branching ratio by the polarization beam splitter 44 by switching the polarization direction of the pulsed laser light L by adjusting the incident voltage signal.

  The electro-optic modulator 43 adjusts the incident voltage signal in accordance with the switching command signal S output from the control unit 21 in synchronization with the scanning cycle of the scanner 22. Further, the electro-optic modulator 43 adjusts the incident voltage signal T according to the wavelength of the pulsed laser light L output from the laser light source 4.

  According to the light source device 40 and the laser scanning microscope device 41 according to the present embodiment configured as described above, the switching command signal S output from the control unit 21 in synchronization with the scanning cycle of the scanner 22 of the scanning microscope 3. As a result, the voltage signal incident on the electro-optic modulator 43 is switched, so that the polarization direction of the pulsed laser light L incident on the polarization beam splitter 44 is switched in synchronization with the scanning period of the scanner 22, and the pump light L1. The branching ratio of the pulsed laser light L to the optical path 11 of the 'optical path 10 and the Stokes light L2' is switched.

  For example, by setting the branching ratio of the pump light L1 ′ to the optical path 10 to 70% and the branching ratio of the Stokes light L2 ′ to the optical path 11 to 30%, the pulse laser beams L1 ′ and L2 ′ having a certain frequency difference are used. Are incident on the scanning microscope 3 and the CARS light image of the specimen A can be acquired. Further, by setting the branching ratio of the pump light L1 ′ to the optical path 10 to 100% and the branching ratio of the Stokes light L2 ′ to the optical path 11 being 0%, the light quantity of the pump light L1 ′ is increased, and the sample A A bright multiphoton fluorescence image can be acquired.

As described above, according to the light source device 40 according to the present embodiment, since the pulse laser beams L1 ′ and L2 ′ to be incident on the scanning microscope 3 are switched by the electro-optic modulator 43, it is necessary to provide a mechanical drive system. There is an advantage that it can be switched at high speed. As a result, even in the scanning microscope 3 having the scanner 22 that is driven at a high speed, the acquisition of the CARS light image and the acquisition of the multiphoton fluorescence image can be switched at a high speed, thereby improving the simultaneity of the two types of observations. There is an advantage.
In the present embodiment, the case of the frequency dispersion amount adjustment CARS has been described as an example, but instead, the dispersion adjustment glasses 14 and 16 in the two optical paths 10 and 11 are eliminated, and the optical path of the pump light L1 ′. The present invention may be applied to a case where a narrowing mechanism (not shown) for narrowing the pulse laser beam L in 10 is disposed.

Next, a light source device 50 and a laser scanning microscope device 51 according to a third embodiment of the present invention will be described below with reference to FIG.
In the description of the light source device 50 according to the present embodiment, portions having the same configuration as those of the light source device 40 according to the second embodiment described above are denoted by the same reference numerals and description thereof is omitted.

As shown in FIG. 3, the light source device 50 according to the present embodiment is replaced with an acousto-optic modulator 52 instead of the combination of the electro-optic modulator 43 and the polarization beam splitter 44 of the light source device 40 according to the second embodiment. Is adopted.
The acoustooptic modulator 52 can switch the ratio of the diffracted light generated by the diffraction in the acoustooptic crystal by switching the input modulation signal. In the present embodiment, the optical path 10 of the pump light L1 ′ is arranged in the emission direction of the zero-order light that passes through the acousto-optic modulator 52, and the optical path 11 of the Stokes light L2 ′ in the emission direction of the diffracted light emitted by diffraction. Is arranged.

  For example, by setting the modulation signal to 0%, the pulse laser beam L1 composed of the 0th-order light is incident only on the optical path 10 of the pump light L1 ′, and the pulse laser beam L2 composed of the diffracted light into the optical path 11 of the Stokes light L2 ′. Can be stopped. Further, by setting the modulation signal to 30%, 70% pulsed laser light L1 is incident on the optical path 10 of the pump light L1 ′, and the pulsed laser light L2 composed of 30% diffracted light is incident on the optical path 11 of the Stokes light L2 ′. Can be incident.

Thereby, generation of CARS light and generation of multiphoton fluorescence in the specimen A can be switched at high speed without providing a mechanical drive system, and two types of observations can be performed simultaneously.
Even in this case, since the Stokes light L2 ′ is not generated in the case of multiphoton fluorescence observation, an advantage that a bright multiphoton fluorescence image can be obtained by allowing the pulse laser light L1 ′ having a sufficient amount of light to enter the specimen A. There is. In the case of CARS light observation, it is possible to generate a high-contrast CARS light image by generating pump light L1 ′ and Stokes light L2 ′ in an appropriate ratio.
Also, in the present embodiment, the case of the frequency dispersion amount adjustment CARS has been described as an example, but instead, the dispersion adjustment glasses 14 and 16 in the two optical paths 10 and 11 are eliminated, and the optical path of the pump light L1 ′. The present invention may be applied to a case where a narrowing mechanism (not shown) for narrowing the pulse laser beam L in 10 is disposed.

Next, a light source device 60 and a laser scanning microscope device 61 according to a fourth embodiment of the present invention will be described below with reference to FIG.
In the description of the light source device 60 according to the present embodiment, portions having the same configuration as those of the light source device 40 according to the second embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 4, the light source device 60 according to the present embodiment is replaced with a combination of the beam splitter 9 instead of the combination of the electro-optic modulator 43 and the polarization beam splitter 44 of the light source device 40 according to the second embodiment. After branching to the two optical paths 10, 11, an acousto-optic tunable filter 62 is disposed in the optical path 10 of the pump light L1 ', an acousto-optic modulator 63 is disposed in the optical path 11 of the Stokes light L2', and both the optical paths 10, 11 is different from the light source device according to the second embodiment in that the dispersion adjusting glasses 14 and 16 are removed.

In the light source device 60 according to the present embodiment, a switching command signal S synchronized with the scanning period of the scanner 22 of the scanning microscope 3 is output from the control unit 21 to the acoustooptic tunable filter 62 and the acoustooptic modulator 63, respectively. Has been.
The acousto-optic tunable filter 62 can perform output modulation or narrowing of the pulsed laser beam L1 ′ to be output by inputting a modulation signal having a different predetermined frequency. Further, by multiplexing and inputting modulation signals of these frequencies at the same time, output modulation and band narrowing can be performed simultaneously.
Further, the acousto-optic modulator 63 can switch on and off the output of the pulsed laser light L2 ′ by inputting a modulation signal having a predetermined frequency.

  According to this embodiment configured as described above, the operation of the control unit 21 turns off the acousto-optic modulator 63 in synchronization with the scanning period of the scanner 22, and the acousto-optic tunable filter 62 is simply connected to the acousto-optic tunable filter 62. By inputting a modulation signal of one frequency, the acousto-optic tunable filter 62 performs only output modulation, so that only the pulse laser beam L1 emitted from the laser light source 4 is subjected to output modulation, thereby scanning type. The light can enter the microscope 3. Thereby, a bright multiphoton fluorescence image can be obtained by the pulse laser beam L1 'having a high photon density without dispersion.

Further, the output modulation can adjust the output of the pulsed laser light L1 ′ to an appropriate value, thereby suppressing the occurrence of damage in the specimen A.
In this case, compared with the light source devices 40 and 50 and the laser scanning microscope devices 41 and 51 according to the second and third embodiments described above, the bandwidth of the pulse laser beam L1 ′ is narrowed during multiphoton fluorescence observation. In addition, since frequency dispersion is not performed, there is an advantage that a bright multiphoton fluorescence image can be obtained with almost no output loss.
Also in the present embodiment, the generation of CARS light and the generation of multiphoton fluorescence in the specimen A can be switched at high speed without providing a mechanical drive system, and two types of observations can be performed simultaneously.

  Further, by the operation of the control unit 21, the acousto-optic modulator 63 is turned on to generate the Stokes light L 2 ′ in synchronization with the scanning cycle of the scanner 22, and the multi-frequency modulation signal is sent to the acousto-optic tunable filter 62. By inputting, the acousto-optic tunable filter 62 performs output modulation and band narrowing to generate pump light L1 ′, thereby generating CARS light in the specimen A and obtaining a CARS light image with high contrast. can do.

Next, a light source device 70 and a laser scanning microscope device 71 according to a fifth embodiment of the present invention will be described below with reference to FIG.
In the description of the light source device 70 according to the present embodiment, portions having the same configuration as those of the light source device 40 according to the second embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  In the light source device 70 according to the present embodiment, a beam splitter 9 is disposed instead of the electro-optic modulator 43 and the polarization beam splitter 44 in the second embodiment, and the pump light L1 ′ branched by the beam splitter 9 is provided. The present embodiment is different from the light source device 40 according to the second embodiment in that a bypass optical path 72 that bypasses the dispersion adjusting glass 14 disposed in the optical path 10 is further provided. Reference numerals 73 and 74 in the figure denote λ / 2 plates that adjust the polarization direction of the pulsed laser light L1.

The detour optical path 72 is an optical path formed by two polarization beam splitters 75 and 76 arranged with the dispersion adjusting glass 14 of the optical path 10 of the pump light L1 ′ sandwiched therebetween, and the optical path is folded back by a plurality of mirrors 30. The optical path length is set longer (or shorter) than the optical path 10 passing through the dispersion adjusting glass 14.
As a result, when the pump beam L1 ′ that has passed through the dispersion adjusting glass 14 and the pulse laser beam L1 that has passed through the detour optical path 72 are combined by the polarization beam splitter 76, the pulse that has passed through the two optical paths 10 and 72 is obtained. Laser light L1. L1 ′ is shifted in time so as not to overlap each other.

According to the light source device 70 according to the present embodiment configured in this way, the polarization direction of the pulsed laser light L1 incident on the optical path 10 of the pump light L1 ′ by the beam splitter 9 is changed by the λ / 2 plate 73. By being adjusted and incident on the polarization beam splitter 75, the light is branched at a predetermined branching ratio, one of which is given frequency dispersion by the dispersion adjusting glass 14 to become the pump light L 1 ′, and the other is allowed to pass through the bypass optical path 72. Is given a time delay. Then, the polarization laser beam L1 in the detour optical path 72 is adjusted in its polarization direction by the λ / 2 plate 74 and is combined with the pump light L1 ′ by the polarization beam splitter 76.
Thereafter, the pump light L1 ′ that has passed through the optical path 10 and the pulsed laser light L1 that has passed through the detour optical path 72 and the Stokes light L2 ′ that has passed through the optical path 11 are combined into the scanning microscope 3 by the laser combiner 18. Incident.

  That is, the CARS light can be generated in the sample A by the pump light L1 ′ and the Stokes light L2 ′ generated by the two optical paths 10 and 11, and the time is delayed with respect to the pump light L1 ′. Multiphoton fluorescence can be generated in the specimen A by the pulse laser beam L1. Since the generation of CARS light and the generation of multiphoton fluorescence are shifted in time by giving a time delay to the pulsed laser light L1, it can be performed independently without causing mutual interference.

Since the pulsed laser light L1 that has passed through the detour optical path 72 does not pass through the dispersion adjusting glass 14, it is not given frequency dispersion, and a bright multiphoton fluorescence image can be obtained by improving the photon density at the condensing point in the sample A. . Thereby, a CARS light image with high contrast and a bright multiphoton fluorescence image can be acquired simultaneously.
Also, in the present embodiment, the case of the frequency dispersion amount adjustment CARS has been described as an example, but instead, the dispersion adjustment glasses 14 and 16 in the two optical paths 10 and 11 are eliminated, and the optical path of the pump light L1 ′. The present invention may be applied to a case where a narrowing mechanism (not shown) for narrowing the pulse laser beam L in 10 is disposed.

  In addition, this invention is not limited to embodiment mentioned above, A various change is possible based on the summary of invention. For example, in the above-described example, an example in which a single wavelength is used as irradiation light for obtaining detection light (such as CARS light or fluorescence) from the specimen to be detected is shown. However, the type and purpose of the specimen to be detected Depending on the wavelength, other types of wavelengths may be used as irradiation light, and various detections may be performed by switching to different wavelengths or using them together. Accordingly, for example, there is an advantage that multimodal detection is possible even when applied to multiplex CARS as disclosed in Non-Patent Document 1 described above.

L, L1, L2 Pulse laser light L1 'Pump light (pulse laser light)
L2 'Stokes light (pulse laser light)
1, 41, 51, 61, 71 Laser microscope apparatus.
2, 40, 50, 60, 70 Light source device 3 Scanning microscope 4 Laser light source 5 First optical path 6 Second optical path 7 Multiplexing unit 8 Optical path switching unit 10, 11 Optical path 14, 16 Dispersion adjusting glass (dispersion adjusting mechanism) )
15 Photonic crystal fiber (wavelength converter)
17 Pulse timing adjustment means (delay adjustment unit)
DESCRIPTION OF SYMBOLS 20 Galvano mirror 42 Splitting part 43 Electro-optic modulator 44 Polarizing beam splitter 52 Acousto-optic modulator 62 Acousto-optic tunable filter 63 Acousto-optic modulator 72 Detour optical path

Claims (3)

A laser light source for generating pulsed laser light;
From the pulse laser beam emitted from the laser light source, two pulse laser beams having a predetermined frequency difference are generated, and the generated two pulse laser beams are combined and input to the scanning microscope. The optical path,
A second optical path for inputting the pulsed laser light emitted from the laser light source as it is to the scanning microscope;
A combining unit that combines the first optical path and the second optical path;
A light source device comprising: an optical path switching unit configured to switch the pulse laser light from the laser light source into the first optical path or the second optical path in a time-sharing manner in synchronization with a scanning cycle of the scanning microscope.   The light source device according to claim 1, wherein the optical path switching unit includes a galvanometer mirror. The light source device according to claim 1 or 2 ,
A laser scanning microscope apparatus comprising: a scanning microscope for inputting pulsed laser light from the light source device.

Images