JP5525882B2 - Light source device and scanning microscope - Google Patents

Description

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

2. Description of the Related Art Conventionally, scanning microscopes that can observe different positions in the sample in the optical axis direction without driving the objective lens in the optical axis direction are known (see, for example, Patent Document 1 and Patent Document 2). .
The scanning microscope of Patent Document 1 includes a plurality of light sources, a plurality of illumination light paths, and a plurality of light detection means, so that light guided from separate illumination light paths is at different positions in the optical axis direction in the sample. Condensate at the same time.
The scanning microscope of Patent Document 2 moves the condensing position in the direction of the optical axis by switching the shape of the shape variable mirror disposed in the middle of the illumination optical path.

JP 2004-157246 A JP 2007-316662 A

  However, when the observation target is a scatterer such as a living body, the spot diameter of the excitation light at each condensing position is widened due to the refractive index distribution and fluctuation of the sample. In the scanning microscope of Patent Document 1 that simultaneously collects light at different positions in the optical axis direction, when the light collection positions are close to each other, the detector for detecting fluorescence from one light collection position is connected to the other. Fluorescence from the condensing position is also mixed, and there is a disadvantage that clear fluorescence observation cannot be performed.

  Further, in the scanning microscope of Patent Document 2, excitation light is incident on each condensing position separated in the optical axis direction at different times by switching the shape of the deformable mirror. Although there is no inconvenience, switching the shape of the deformable mirror takes time, and guarantees the synchronism when observing different positions in the optical axis direction of the observation target that changes with time like a living body or living cells in a short time. There is an inconvenience that it cannot be done.

The present invention has been made in view of the above-described circumstances, and a light source device and a scanning microscope that can clearly observe a plurality of points different in the optical axis direction of an observation target while maintaining simultaneity. The purpose is to provide.

In order to achieve the above object, the reference example of the present invention provides the following means.
A reference example of the present invention includes a laser light source that generates pulsed laser light, a pulse dividing unit that generates a plurality of pulsed laser lights spaced from the pulsed laser light emitted from the laser light source, and the pulse dividing unit There is provided a light source device including a fixed wavefront conversion unit that changes the wavefront of the pulsed laser light so that it is condensed at different positions in the optical axis direction for each of the one or more pulsed laser beams generated by the above.

According to the above reference example, when the pulse laser beam emitted from the laser light source is incident on the pulse dividing unit, the pulse dividing unit generates a plurality of pulse laser beams spaced at time intervals, and fixed wavefront conversion. Depending on the part, the wavefront is made different for each of the one or more pulsed laser beams. As a result, pulsed laser beams having different wavefronts are condensed at different positions in the optical axis direction.

  That is, by irradiating the observation target with a plurality of pulse laser beams generated in this manner, the pulse laser beams are condensed at different positions in the direction of the optical axis at different times, and each is detected, thereby Even an observation target made of a scatterer such as a sample can clearly observe different positions close to each other in the optical axis direction without interfering with each other. The time interval of the pulse laser beam only needs to be slightly longer than the lifetime of the light generated in the observation target, and can be set sufficiently short. Therefore, different positions in the optical axis direction can be observed almost simultaneously and clearly.

In the above reference example , for each of the one or more pulsed laser beams generated by the pulse dividing unit, a polarization adjusting unit that changes the polarization direction of the pulsed laser beam is provided, and the wavefront converting unit includes the pulsed laser beam. The wavefront may be selectively converted according to the polarization direction.
In this way, when a pulse laser beam whose polarization direction is changed by the polarization adjusting unit is incident on the wavefront conversion unit, the wavefront is selectively converted according to the polarization direction, so that a time interval is provided. Pulse laser light can be easily condensed at different positions in the optical axis direction.

Moreover, in the said reference example , the said wavefront conversion part is a light source device of Claim 2 provided with the liquid crystal device which converts the wavefront of the pulsed laser beam which has a polarization direction parallel to the orientation direction of a liquid crystal molecule.
In this way, the presence or absence of wavefront conversion in the pulsed laser beam can be easily and quickly switched by applying different polarization laser beams with a polarization direction parallel to and perpendicular to the alignment direction of the liquid crystal molecules. be able to.

In the above reference example , the pulse division unit may include two optical paths having different optical path lengths for dividing and passing the pulse laser light emitted from the laser light source.
In this way, it is possible to easily generate two pulse laser beams with a time interval simply by dividing the pulse laser beam and passing it through two different optical paths.

In the above reference example , the wavefront conversion unit may be arranged in any one of the optical paths of the pulse division unit.
By doing so, wavefront conversion is performed on the pulsed laser light that has passed through the optical path in which the wavefront conversion unit is disposed, and wavefront conversion is not performed on the optical path in which the wavefront conversion unit is not disposed. Since it is emitted, it is possible to easily emit a plurality of pulsed laser beams that can be condensed at different positions in the optical axis direction.

In the above reference example , the wavefront converter may include a liquid crystal device or a variable shape mirror that converts the wavefront of pulsed laser light having a polarization direction parallel to the alignment direction of liquid crystal molecules.
By doing so, not only the liquid crystal device but also the variable shape mirror can emit a plurality of pulsed laser beams that can be condensed at different positions in the optical axis direction with a simple configuration.

In the above reference example , a plurality of units each including the pulse division unit and the wavefront conversion unit may be provided in series, and the pulse division unit in each unit may impart different time intervals to the pulse laser beam. .
By doing in this way, all the pulsed laser beams can be shifted in time, and the condensing position of each pulsed laser beam in the optical axis direction can be shifted for each wavefront conversion unit.

In the above reference example , a wave plate may be disposed between the units.
In this way, the polarization state of the pulsed laser light emitted from one unit can be adjusted by the wave plate and incident on the next unit, and the relative angle of the wavefront conversion unit before and after the wave plate can be changed. It can be set freely.

Further, in the above reference example , the time interval t given to the pulse laser beam by the pulse dividing unit uses the number n of the pulse dividing unit and the repetition frequency R of the pulse laser beam from the laser light source, You may calculate with the following formula | equation.
t = 1 / (2 ⁿ R)
By doing so, it is possible to generate a train of pulsed laser light that is time-multiplexed at a repetition frequency of 2 ⁿ R. Furthermore, the condensing position when each pulse laser beam is irradiated onto the observation target can be spatially multiplexed.

Further, the present invention condenses the laser light source that generates a plurality of pulsed laser beams at intervals and one or more pulsed laser beams emitted from the laser light source at different positions in the optical axis direction. A fixed wavefront conversion unit that changes the wavefront of the pulse laser beam, and a polarization adjustment unit that changes the polarization direction of the pulse laser beam for each of the one or more pulsed laser beams emitted from the laser light source. The light source device includes a light source device in which the wavefront conversion unit selectively converts a wavefront according to a polarization direction of the pulsed laser light .

According to the present invention, the wavefronts of the plurality of pulsed laser beams emitted from the laser light source with a time interval are made different for each of the one or more pulsed laser beams by the fixed wavefront conversion unit. As a result, pulsed laser beams having different wavefronts are condensed at different positions in the optical axis direction. That is, by irradiating the observation target with a plurality of pulse laser beams generated in this way, the pulse laser beams are condensed at different positions in the optical axis direction at different times, and each is detected.
Even an observation target made of a scatterer such as a biological sample can clearly observe different positions close to each other in the optical axis direction without interfering with each other. The time interval of the pulse laser beam only needs to be slightly longer than the lifetime of the light generated in the observation target, and can be set sufficiently short. Therefore, different positions in the optical axis direction can be observed almost simultaneously and clearly.

In the above invention, before Symbol wavefront converting unit may comprise a liquid crystal device that converts the wavefront of the pulsed laser beam having a polarization direction parallel to the orientation direction of the liquid crystal molecules.

  The present invention also provides any one of the above light source devices, a scanner that scans the pulse laser light emitted from the light source device on the observation target, and the pulse laser light scanned by the scanner on the observation target. A scanning microscope is provided that includes an objective lens that illuminates and collects light from the observation target, and a detector that detects the light from the observation target.

  According to the present invention, when the temporally multiplexed pulsed laser light emitted from the light source device is scanned by the scanner and irradiated to the observation target by the objective lens, the position in the observation target is different in the optical axis direction. And the light from the observation target is collected by the objective lens and detected by the detector. Thereby, a plurality of points different in the optical axis direction can be observed almost simultaneously without interfering with each other.

Further, the reference example of the present invention divides a pulsed laser beam to generate a plurality of pulsed laser beams with a time interval, and each of the one or more pulsed laser beams generated by the pulse dividing step. In addition, a polarization step for changing the polarization direction of the pulse laser light and a pulse laser light whose polarization direction has been changed in the polarization step are incident on a wavefront conversion unit that selectively converts the wavefront according to the polarization direction. Thus, there is provided a pulsed light generation method including a wavefront conversion step of converting a wavefront so as to collect light at different positions in the optical axis direction.

  According to the present invention, there is an effect that it is possible to clearly observe a plurality of points different in the optical axis direction of the observation target while maintaining simultaneity.

It is a schematic diagram which shows the light source device and scanning microscope which concern on the 1st Embodiment of this invention. It is a figure which shows the pulse train of two types of pulsed laser beams produced | generated by the pulse division part of the light source device of FIG. 1, (a) In the case of optical path length difference LS, (b) In the case of optical path length difference LS = c / 2R Respectively. It is a figure which shows the state by which the pulse laser beam is condensed by two places spaced apart by the optical axis direction of the objective lens with the light source device of FIG. It is a figure which shows the flowchart of the pulse light generation method which concerns on the 1st Embodiment of this invention. It is a figure which shows the state by which the pulsed laser beam is scanned on 2 planes spaced apart in the optical axis direction of the objective lens with the scanning microscope of FIG. It is a figure which shows the modification of the pulse laser beam produced | generated by the light source device of FIG. It is a schematic diagram which shows the modification of the light source device of FIG. 1, and a scanning microscope. It is a schematic diagram which shows the light source device which concerns on the 2nd Embodiment of this invention. In the wavefront conversion unit in the light source device of FIG. 8, the pulse laser beam having a wavefront that changes the focal position in the direction orthogonal to the optical axis is scanned on two planes spaced in the optical axis direction of the objective lens. It is a figure which shows the state currently performed. It is a schematic diagram which shows the light source device which concerns on the 3rd Embodiment of this invention. It is a figure which shows the pulse train of two types of pulsed laser beams produced | generated by the light source device of FIG. It is a figure which shows the modification of FIG. It is a schematic diagram which shows the modification of the light source device of FIG. It is a figure which shows the pulse train of four types of pulsed laser beams produced | generated by the light source device of FIG.

A light source device 1, a scanning microscope 2, and a pulsed light generation method according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the scanning microscope 2 according to the present embodiment observes a specimen (observation target) A using the light source device 1 according to the present embodiment and pulsed laser light emitted from the light source device 1. A microscope main body 3 is provided.

Light source device 1 includes a laser light source 4 for generating a pulse laser beam L _1, a pulse dividing unit 5 for time-multiplexed by dividing the pulse laser beam L ₁ emitted from the laser light source 4 into two, the pulse division And a wavefront conversion unit 6 that converts the wavefront of _one of the pulse laser beams L ₁ and L ₂ divided by the unit 5.
The laser light source 4 oscillates linearly polarized pulsed laser light L ₁ at a repetition frequency R.

The pulse splitting unit 5 splits the pulse laser light L ₂ from the pulse laser light L ₁ emitted from the laser light source 4 and makes it incident on _two optical paths C ₁ and C ₂ having different optical path lengths, A polarization beam splitter that combines the λ / 2 plate 8 disposed in one optical path C ₂ branched by the beam splitter 7 and the pulsed laser beams L ₁ and L ₂ that have passed through the _two optical paths C ₁ and C _2. 9 and.
In FIG. 1, reference numeral 10 denotes a mirror.

The λ / 2 plate 8 is disposed so as to be inclined by 45 ° with respect to the polarization direction of the pulsed laser light L ₁ emitted from the laser light source 4.
For example, when the pulse laser beam L ₁ emitted from the laser light source 4 has a polarization direction parallel to the paper surface, the polarization direction of the pulse laser beam L ₂ is determined by the λ / 2 plate 8 in the optical path C ₂ . While being converted into a polarization direction perpendicular to the paper surface, the pulsed laser light L _{1 in} the polarization direction parallel to the paper surface is propagated in the other optical path C ₁ .

As a result, the two pulsed laser beams L ₁ and L _{2 that} have reached the polarization beam splitter 9 are transmitted or reflected without waste by the polarization beam splitter 9 and are combined in the same optical path.
The two optical paths C ₁ and C ₂ of the pulse dividing unit 5 have an optical path difference LS. Therefore, the delay time t = L / c is given to the pulse laser beams L ₁ and L ₂ passing through the _two optical paths C ₁ and C ₂ . Here, c is the speed of light.
That is, as shown in FIG. 2A, pulse laser beams L ₁ and L ₂ having polarization directions orthogonal to each other and temporally multiplexed are emitted from the pulse division unit 5. It will be.

Wavefront converter 6 is reflected with the prism 11 which diverts reflects the pulsed laser light L _1, L ₂ emitted from the pulse division unit 5, the pulse laser light L _1, L ₂ reflected by the prism 11 , in time, to modulate one of the pulse wavefront of the laser light L _1, without performing the modulation of the wavefront to the other of the pulsed laser light L _2, a reflective liquid crystal device (LCOS of returning to the prism 11: liquid-crystal on Silicon) 12. The liquid crystal device 12 is divided into many pixels. For each pixel, liquid crystal molecules are sandwiched between a silicon substrate and a transparent substrate facing the silicon substrate, and a voltage is applied to the liquid crystal on the back surface of the mirror-like silicon substrate. It has a structure in which electrodes are formed.

Liquid crystal molecules of the liquid crystal device 12 are oriented parallel to the polarization plane of the substrate and one of the pulsed laser light L _1, when a voltage is applied by electrodes which are formed on a substrate, the liquid crystal molecules are substrates and the other pulsed laser beam It rotates about an axis parallel to the polarization plane of the L _2. Then, with the rotation of the liquid crystal molecules also changes the refractive index distribution of the liquid crystal device 12 to the pulse laser light L ₁ having a polarization plane in the direction of orientation of the liquid crystal molecules.

  The rotation angle of the liquid crystal molecules can be controlled by an applied voltage. When the applied voltage to the liquid crystal device 12 is changed for each pixel, a refractive index distribution can be provided to the liquid crystal device 12. As a result, a desired phase distribution can be given to the incident light. In addition, when the polarization direction of incident light matches the alignment direction of liquid crystal molecules, the phase distribution can be given to the incident light most strongly. Further, when the polarization direction of the incident light and the alignment direction of the liquid crystal molecules are orthogonal, the incident light is not modulated.

  In other words, the liquid crystal device 12 controls the voltage to the liquid crystal molecules directly for each pixel to change the phase of the incident light, thereby freely adjusting the phase with respect to the incident light having the polarization direction that matches the alignment direction of the liquid crystal molecules. It can be modulated.

That is, of the two types of pulsed laser beams L ₁ and L _{2 having} orthogonal polarization directions alternately emitted from the pulse division unit 5, the alignment direction of the liquid crystal molecules of the liquid crystal device 12 included in the wavefront conversion unit 6 is the same. One pulsed laser beam L ₁ having a polarization direction is modulated in wavefront by the liquid crystal device 12, while the other pulsed laser beam L ₂ is reflected by the liquid crystal device 12 without modulating the wavefront. Therefore, the pulsed laser beam L ₁ ′ having a modulated wavefront and the pulsed laser beam L ₂ having no modulated wavefront can be alternately output to the microscope body 3.

Here, the modulation of the wavefront imparted to the _one pulse laser beam L ₁ ′ by the liquid crystal device 12 is performed by changing the position of the light focused by an objective lens (to be described later) of the microscope body 3 as shown in FIG. performed so as to shift the optical axis direction with respect to the condensing position of the light L _2. For example, if the wavefront of the other pulsed laser beam L ₂ is flat, the one of the pulsed laser light L _1, the modulation of the wave front is made so as to have a spherical wavefront having fine curvature axisymmetrically It is like that.

The microscope main body 3 includes a first relay optical system 13 and a second relay optical system 14 that relay the pulse laser beams L ₁ ′ and L ₂ emitted from the light source device 1, and the first and second relay optics. The scanner 15 that is arranged between the systems 13 and 14 and scans the pulse laser beams L ₁ ′ and L ₂ two-dimensionally, and the pulse laser beams L ₁ ′ and L ₂ relayed by the second relay optical system 14. Objective lens 16 that focuses the light on the observation object A, the dichroic mirror 17 that branches the fluorescence generated in the observation object A collected by the objective lens 16 from the pulse laser beams L ₁ ′ and L ₂ , and the dichroic mirror And a photodetector 18 for detecting the fluorescence branched by 17.

  The liquid crystal device 12 of the wavefront modulation unit 6 and the scanner 15 are arranged in an optically conjugate positional relationship by the first relay optical system 13, and the pupil positions of the scanner 15 and the objective lens 16 are arranged by the second relay optical system 14. Are arranged at optically conjugate positions.

A pulse light generation method using the light source device 1 according to this embodiment configured as described above will be described below.
When the pulse laser beam L ₁ emitted from the laser light source 4 is incident on the pulse splitting unit 5, it is branched into _two optical paths C ₁ and C ₂ by the beam splitter 7 as shown in FIG. 4 (dividing step). S1). Since the two optical paths C ₁ and C ₂ have an optical path length difference LS, a time delay corresponding to the optical path length difference LS is given to the pulsed laser light L ₂ that has passed through the longer optical path C ₂ .

In the optical path C ₂ , the λ / 2 plate 8 is disposed with the high-speed axis inclined by 45 ° with respect to the polarization direction of the incident pulse laser light L ₁ , so the pulse laser that has passed through the λ / 2 plate 8. light L _2, the polarization direction is linearly polarized light perpendicular to the pulsed laser light that propagates through the other optical path C ₁ (polarization step S2). Then, the pulse laser beams L ₁ and L ₂ that have propagated through the _two optical paths C ₁ and C ₂ are combined by the polarization beam splitter 9.

That is, two types of pulsed laser beams L ₁ and L ₂ whose polarization directions are orthogonal to each other are time-multiplexed by being combined by the polarization beam splitter 9. When the two types of time-multiplexed pulsed laser beams L ₁ and L ₂ are incident on the liquid crystal device 12 of the wavefront converter 6, the pulsed laser beam L ₁ having a polarization direction horizontal to the alignment direction of the liquid crystal molecules. Only the wavefront is modulated (wavefront conversion step S3). The pulsed laser light L ₂ having a polarization direction perpendicular to the orientation direction of the liquid crystal molecules is directly reflected without modulation of the wavefront is performed.
Thus, from a single pulse laser light L _1, two different types of pulse laser light L ₁ wavefront 'can be generated in a state where the L ₂ was time-multiplexed.

Next, the operation of the scanning microscope 2 according to this embodiment will be described.
As described above, when the two types of pulsed laser beams L ₁ ′ and L ₂ generated by the light source device 1 and the pulsed light generation method according to the present embodiment are incident on the microscope body 3, the first relay optical system 13. The pulse laser beams L ₁ ′ and L ₂ relayed by are scanned two-dimensionally by the scanner 15, relayed by the second relay lens 14, and then focused on the observation object A by the objective lens 16.

Since one of the two types of pulse laser beams L ₁ ′ and L ₂ is wavefront modulated, the two types of pulse laser beams L ₁ ′ and L ₂ are condensed at different positions in the optical axis direction by the objective lens 16. In addition, since the two types of pulsed laser beams L ₁ ′ and L ₂ are time-multiplexed so as to have a minute time interval, the irradiation time to each condensing position is shifted.

Accordingly, the pulsed laser beams L ₁ ′ and L ₂ are condensed at different positions in the optical axis direction with a minute time interval, whereby fluorescence is generated at each light collection position with a minute time interval. Fluorescence generated corresponding to the pulse laser beams L ₁ ′ and L ₂ is collected by the objective lens 16, branched by the dichroic mirror 17, and detected by the photodetector 18.
Then, by storing the fluorescence intensity detected by the light detector 18 at each time and the information of the scanning position of the scanner 15 at that time in association with each other, as shown in FIG. A two-dimensional P ₁ and P ₂ fluorescent image along two planes extending in position can be acquired.

Thus, the light source apparatus 1 according to this embodiment, according to the scanning microscope 2 and pulse light generation method, 2 types of pulse laser light L ₁ ', by multiplexing the L ₂ times, each pulse laser light L ₁ ', L ₂ it is possible to shift the time to be focused at different positions in the optical axis direction, without mixing the fluorescence generated at the two positions can be separately detected.

Moreover, the liquid crystal device 12 can be fixed by generating two types of pulsed laser beams L ₁ ′ and L ₂ according to the polarization direction. Thereby, compared with the case where the wavefront is switched by switching the variable shape mirror as in the prior art, two types of pulsed laser beams L ₁ ′ and L ₂ can be time-multiplexed at a sufficiently short time interval.
As a result, there is an advantage that fluorescence images at different positions in the optical axis direction can be observed almost simultaneously without causing interference with each other.

In the present embodiment, it is preferable to adopt an optical path length difference LS such that t = 1 / 2R as the delay time t provided in the pulse dividing unit 5. That is, it is preferable that LS = c / 2R. By doing so, as shown in FIG. 2B, the pulse laser light L ₁ ′, L repeated at a repetition frequency 2R that is twice the repetition frequency R of the pulse laser light L ₁ from the laser light source 4. ₂ can be generated.

It is possible to select the shortest repetition period 1 / R while ensuring the shortest delay time 1 / 2R that can be detected without mixing the fluorescence generated from the two light collecting positions with each other. there is an advantage that the pulsed laser light L ₁ emitted from the light source 4 most efficiently can be utilized.

Further, in the present embodiment, a case has been described in which the pulsed laser light L ₂ propagating through the optical path C ₂ is multiplexed one by one between the adjacent pulsed laser light L ₁ emitted from the laser light source 4. However, the present invention is not limited to this. For example, as shown in FIG. 6, the delay time L / c provided by the optical path C _{2 is} set to be larger than the required time n / R for n pulses of the pulsed laser light L ₁ emitted from the laser light source 4. Thus, the pulse splitting unit 5 can emit the pulse laser beams L ₁ and L ₂ having different polarization directions in the first n pulse groups and the next n pulse groups.

By doing so, it is possible to continuously irradiate the same type of pulsed laser beams L ₁ ′ and L _2, and it is possible to acquire a bright fluorescent image even when observing an observation target with weak fluorescence. Become.
Further, by setting the delay time to n / R, it is possible to generate pulsed laser beams L ₁ ′ and L ₂ such that pulsed laser beams having different polarization directions are repeated n times at a repetition frequency R / n. it can.

In the present embodiment, the wavefront conversion unit 6 is disposed after the polarization beam splitter 9 and the presence / absence of wavefront modulation is switched according to the polarization direction of the pulse laser beams L ₁ and L ₂ . Instead, as shown in FIG. 7, the wavefront conversion unit 6 may be installed in one of the optical paths C ₁ and C ₂ of the pulse division unit 5 (FIG. 7 illustrates the optical path C _1. It shows the case where it is installed inside.) In this case, the modulating all of the pulse wave front of the laser beam L ₁ propagating through the optical path C ₁ to wavefront conversion unit 6 is provided.

Thus, as the wavefront converting unit 6, although the presence or absence of the modulation of the wave front by the polarization direction may use a different liquid crystal device 12, instead of this, to modulate all of the pulse wave of the laser light L ₁ incident A variable shape mirror and a segment type MEMS mirror (not shown) may be employed.

Next, a light source device 20 according to a second embodiment of the present invention will be described below with reference to the drawings.
In the description of the present embodiment, portions having the same configuration as those of the light source device 20 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

As illustrated in FIG. 8, the light source device 20 according to the present embodiment includes two units 23 and 24 including pulse division units 5 and 21 and wavefront conversion units 6 and 22 arranged in series. A λ / 2 plate 25 is disposed between them.
The pulse division unit 5 provided in the first unit 23 includes two optical paths C ₁ and C ₂ having an optical path length difference LS1, and a delay time t = in the pulse laser beam L ₂ propagating through one optical path C _2. LS1 / c is assigned. The pulse dividing unit 5 is the same as the pulse dividing unit 5 in the first embodiment.

In addition, the liquid crystal device 12 of the wavefront conversion unit 6 provided in the first unit 23 is different in that the presence or absence of wavefront modulation differs depending on the polarization direction of the incident pulse laser beams L ₁ and L ₂ . This is the same as the liquid crystal device 12 of the light source device 1 according to the first embodiment. The liquid crystal device 12 provides a wavefront that allows the pulsed laser light L ₁ ′ with modulated wavefront and the unmodulated pulsed laser light L ₂ to be condensed at a position shifted by a distance 2d in the optical axis direction. ing.

Further, the pulse splitting unit 21 provided in the second unit 24 forms two optical paths C ₃ and C ₄ having an optical path length difference LS2 by two polarization beam splitters 26 and 27, and one optical path C _4. The delay time t = LS2 / c is given to the pulsed laser beams L ₃ and L ₄ propagating through the laser beam. Since two types of pulsed laser beams L ₁ ′ and L ₂ having polarization directions orthogonal to each other are already incident on the pulse dividing unit 21, unlike the pulse dividing unit 5 of the first unit 23, / 2 plate 8 is not provided.

  Lenses 28 to 31 are arranged before and after and inside the second unit 24 to constitute a relay optical system, and the liquid crystal device 12 of the wavefront conversion unit 6 of the first unit 23 and the wavefront of the second unit 24. The liquid crystal device 32 of the conversion unit 22 is arranged at an optically conjugate position. In the figure, reference numeral 33 denotes a prism, and reference numeral 34 denotes a mirror.

In addition, the liquid crystal device 32 of the wavefront conversion unit 22 provided in the second unit 24 also has wavefront conversion depending on the polarization direction of the incident pulse laser beams L ₁ ′, L ₂ , L ₃ , and L _4. Are different from the liquid crystal device 12 of the light source device 1 according to the first embodiment. In this liquid crystal device 32, the pulse laser beams L ₁ ″ and L ₂ ′ whose wavefronts are modulated and the pulse laser beams L ₃ and L ₄ which are not modulated are condensed at a position shifted by a distance d in the optical axis direction. A wavefront is added.

Further, the λ / 2 plate 25 has a polarization direction of _{one of the} two types of pulsed laser beams L ₁ ′ and L ₂ emitted from the wavefront conversion unit 6 of the first unit 23. On the other hand, the high-speed axis is arranged in a direction forming an angle of 22.5 °. As a result, the polarization directions of the two types of pulsed laser beams L ₁ ′ and L ₂ emitted from the wavefront conversion unit 6 of the first unit 23 are both rotated by 45 ° and are incident on the second unit 24. it can. In place of the λ / 2 plate 25, a λ / 4 plate disposed at an angle of 45 ° with respect to the polarization direction of the pulsed laser beams L ₁ ′ and L ₂ may be used.

Thus, even when the direction of the polarization beam splitter 26 of the second unit 24 is arranged in the same direction around the optical axis with respect to the polarization beam splitter 9 in the pulse division unit 5 of the first unit 23, The two types of pulsed laser beams L ₁ ′ and L ₂ emitted from the unit 23 can be equally divided and branched into two optical paths C ₃ and C ₄ .

The operation of the light source device 20 according to this embodiment configured as described above will be described below.
The pulsed laser light L ₁ emitted from the laser light source 4 is time-multiplexed and wavefront modulated only to one side in the same manner as the light source device 1 according to the first embodiment by passing through the first unit 23. Two types of pulsed laser beams L ₁ ′ and L ₂ subjected to the above are generated. At this time, the time interval between the two types of pulsed laser beams L ₁ ′ and L ₂ is t = LS1 / c. The pulse laser beam L ₁ ′ that has passed through _one optical path C ₁ is given a wavefront that varies the condensing position by a distance 2d in the optical axis direction.

Two types of pulsed laser beams L ₁ ′ and L ₂ having polarization directions orthogonal to each other emitted from the first unit 23 are rotated by 45 ° by the λ / 2 plate 8 and incident on the second unit 24. Is done. In the second unit 24, the two types of pulsed laser beams L ₁ ′ and L ₂ are equally divided into two optical paths C ₃ and C ₄ by the polarization beam splitter 26, and propagated through one optical path C _4. Two types of pulsed laser beams L ₃ and L ₄ to which the delay time LS2 / c is given are generated.

Therefore, two types of pulse laser beams L ₁ ′ and L ₂ with delay times 0 and LS1 / c that have propagated through the optical path C ₃ and delay times LS 2 / c and (LS 1 + LS 2) / c that have propagated through the optical path C ₄ Four types of pulse laser beams L ₁ ′, L ₂ , L ₃ and L ₄ including two types of pulse laser beams L ₃ and L ₄ are combined by the polarization beam splitter 27.
Then, these pulse laser beams L ₁ ′, L ₂ , L ₃ , L ₄ pass through the wavefront conversion unit 22, whereby two types of pulse laser beams L ₁ ′, L ₂ that have passed through one optical path C _3. Is provided with a wavefront that varies the condensing position by a distance d in the optical axis direction, and becomes pulsed laser light L ₁ ″, L ₂ ′.

In this case, if the pulse laser beam L ₄ that has passed through the optical path C ₂ and the optical path C ₄ and has the longest delay time (LS1 + LS2) / c is not subjected to wavefront conversion, the pulse laser beam L ₄ , the pulse laser beam L ₁ ″ having the delay time 0 propagated through the optical path C ₁ and the optical path C ₃ is a distance 2d + d = 3d by the wavefront conversion units 6 and 22 of the first and second units 23 and 24, and the optical path C ₁ and the optical path _C is ₄ the pulse laser light _{L 3} of the delay time LS2 / c having propagated wavefront converter 6 only by a distance 2d of the first unit 23, the optical path _{C 2} and the optical path _{C 3} delay time to propagate LS1 / The condensing position of the pulse laser beam L ₂ ′ of c is varied in the optical axis direction by the distance d only by the wavefront conversion unit 22 of the second unit 24.

That is, according to the light source device 20 according to the present embodiment, the pulse laser beams L ₁ ″, L ₂ ′, L ₃ , L are introduced into the microscope main body 3 and within four planes that are different by the distance d in the optical axis direction. ₄ can be collected, and fluorescence images along four planes with different depths can be acquired.Further, four types of pulsed laser beams L ₁ ″, L ₂ ′, L ₃ , and L ₄ are time-multiplexed. Therefore, it is possible to prevent the fluorescence generated at each condensing position from interfering with each other and to obtain a clear fluorescence image almost simultaneously.

In this case, the optical path length difference between the optical paths C ₁ and C ₂ of the pulse division units 5 and 21 is LS1 = c / 2R, and the optical path length difference between the optical paths C ₃ and C ₄ is LS2 = c / 4R. Thus, a time-multiplexed pulse train having a repetition frequency of 4R can be generated.

Moreover, in this embodiment, you may decide to give the wavefront which shifts the condensing position to the direction orthogonal to an optical axis for the modulation | alteration of the wavefront provided in the wavefront conversion part 6 of one unit 23. FIG.
By doing so, as shown in FIG. 9, the pulse laser beams L ₁ ″ and L ₂ ′ are respectively provided at two positions spaced on the two planes P ₁ and P ₂ spaced in the optical axis direction. , L ₃ , L ₄ can be condensed, and pulse laser beams L ₁ ″, L ₂ ′, L ₃ , L ₄ can be focused at _two different points in the direction orthogonal to the optical axis on each plane P ₁ , P ₂ . by irradiating substantially simultaneously, it is possible to improve the acquisition speed of the two-dimensional fluorescence image along each plane P _1, P _2.

Also in this case, since the pulse laser beams L ₁ ″, L ₂ ′, L ₃ , and L ₄ are time-multiplexed, the fluorescence generated at each condensing position is not mixed with each other and is close to each other. There are advantages that light can be condensed at a position and a clear fluorescent image can be acquired.
Further, the number of units 23 and 24 is not limited to two, and an arbitrary number of three or more may be provided.

Next, a light source device 40 according to a third embodiment of the present invention will be described below with reference to the drawings.
In the description of the present embodiment, portions having the same configuration as those of the light source device 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

As shown in FIG. 10, the light source device 40 according to the present embodiment replaces the pulse division unit 5 of the light source device 1 according to the first embodiment with an electro-optic polarization direction of the pulsed laser light L _1. A polarization switching element 41 such as a Pockels cell for switching is employed. The polarization switching element 41 is configured to switch the polarization direction for each of the one or more pulse laser beams L ₁ in synchronization with the output timing of the pulse laser beam L ₁ from the laser light source 4.

According to the light source device 40 according to the thus constructed present embodiment, selectively grant wavefront by the liquid crystal device 12 according to the polarization switching device 41 is switched to the high-speed polarization direction, the pulse laser light L ₁ ', L ₂ can be injected.
11, when switching the polarization direction for each pulse laser light L _1, FIG. 12 shows a case where switching the polarization direction for each n pulsed laser light L _1, respectively. By switching the polarization direction for each n pulsed laser light L _1, even when observing weak observation target fluorescence, there is the advantage that it is possible to acquire a bright fluorescence image.

In the present embodiment, as shown in FIG. 13, in addition to the first unit 42 including the polarization switching element 41 and the wavefront conversion unit 6, the first unit including the polarization switching element 43 and the wavefront conversion unit 22 is used. Two units 44 may be arranged in series.
In this way, the wavefront conversion units 6 and 22 of the two units 42 and 44 provide wavefront modulation according to the polarization directions of the pulse laser beams L ₁ ′, L ₂ , L ₃ , and L _4. Can do. Thus, by a combination of operating states of the polarization switching element 41 and 43, as shown in FIG. 14, at a time interval repetition period of the pulse laser light L ₁ emitted from the laser light source 4 R, 4 kinds of Pulse laser beams L ₁ ″, L ₂ ′, L ₃ , and L ₄ can be generated.

  In the first to third embodiments, a reflective liquid crystal device is used as the liquid crystal device 12. However, the present invention is not limited to this. The same effect as each embodiment is produced.

A Observation object C ₁ , C ₂ , C ₃ , C ₄ optical path L ₁ , L ₁ ′, L ₁ ″, L ₂ , L ₂ ′, L ₃ , L ₄ pulse laser light S 1 division step S 2 polarization step S 3 wavefront conversion Step 1, 20, 40 Light source device 2 Scanning microscope 4 Laser light source 5, 21 Pulse division unit 6, 22 Wavefront conversion unit 8 λ / 2 plate (polarization adjusting unit)
12 Liquid Crystal Device 15 Scanner 16 Objective Lens 18 Photodetector (Detector)
23, 24 unit 25 λ / 2 plate (wave plate)
41, 43 Polarization switching element (polarization controller)

Claims (3)

A laser light source that generates a plurality of pulsed laser beams at time intervals;
A fixed wavefront converter that changes the wavefront of the pulsed laser light so that it is focused at different positions in the optical axis direction for each of the one or more pulsed laser beams emitted from the laser light source;
A polarization adjusting unit that changes the polarization direction of the pulse laser beam for each of one or more pulse laser beams emitted from the laser light source;
The light source device, wherein the wavefront converter selectively converts a wavefront according to a polarization direction of the pulsed laser light. The light source device according to claim 1 , wherein the wavefront conversion unit includes a liquid crystal device that converts a wavefront of pulsed laser light having a polarization direction parallel to an alignment direction of liquid crystal molecules. The light source device according to claim 1 or 2 ,
A scanner that scans an observation target with pulsed laser light emitted from the light source device;
An objective lens for condensing the pulsed laser light scanned by the scanner on the observation object, and condensing the light from the observation object;
A scanning microscope comprising: a detector that detects light from the observation target.

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