JP2007132794A - Multiphoton excitation type observation device, and light source device for multiphoton excitation type observation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve pointing stability of ultra-short pulse laser light entering an observation device body by a simple constitution; to generate efficiently a multiphoton excitation effect; and to acquire a clear multiphoton fluorescent image. <P>SOLUTION: This multiphoton excitation type observation device 1 is equipped with a pulse laser light source 4 for emitting an ultra-short pulse laser light L; the observation device body 3 for irradiating the sample A with the ultra-short pulse laser light L emitted from the pulse laser light source 4, and observing fluorescence emitted from the sample A; and an acoustic optical modulation filter 5 arranged between the pulse laser light source 4 and the observation device body 3, for modulating the ultra-short pulse laser light L emitted from the pulse laser light source 4. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a multiphoton excitation observation apparatus and a multiphoton excitation observation light source apparatus.

Conventionally, as a device for observing the function of cells, etc. by irradiating a sample such as a living body with excitation light from its surface and detecting fluorescence emitted from a relatively deep position below the surface of the sample, a multiphoton excitation type Is known (for example, refer to Patent Document 1).
In Patent Document 1, an acoustic optical modulator (AOM: Acoust Optical) is used as a means for adjusting the intensity of excitation light applied to a sample or turning it on and off.
It is disclosed to use Modulator).
Japanese National Patent Publication No. 10-512959

The AOM is generally a crystal made of tellurium dioxide or molybdate having a square of about 1 cm, and the effective range in which incident laser light can be modulated is relatively narrow. If the ultrashort pulse laser beam incident on the AOM is out of the effective range of the AOM, there is a problem that the beam shape is deteriorated due to the mixing of stray light. When the beam shape deteriorates, the point spread function (PSF) at the focal position of the sample
Function) is deteriorated, the photon density at the light collection position of the sample is lowered, and there is a problem that the multiphoton excitation effect cannot be generated efficiently.

  In addition, although the ultrashort pulse laser light is generally considered to be substantially parallel light, strictly speaking, the ultrashort pulse laser light includes a beam waist having the smallest light beam diameter at a specific position along the optical axis direction, and the beam. A beam divergence that increases the beam diameter at a predetermined spread angle as it moves away from the waist position is provided. Therefore, in order to efficiently generate the multiphoton excitation effect and acquire a clear multiphoton excitation image, the optical axis of the ultrashort pulse laser beam whose beam diameter changes depending on the beam waist position in this way, It is necessary to accurately match within a relatively narrow effective range of AOM. In other words, a reduced beam expander and an alignment adjustment device are required for making the ultrashort pulse laser beam enter the narrow effective range of the AOM.

  Furthermore, since AOM requires several watts of driving power, there is an inconvenience that excessive heat is locally generated in combination with the extremely narrow effective range in which the ultrashort pulse laser beam is incident. As a result, there is an inconvenience that the pointing stability of the light emitted from the AOM is deteriorated.

  The present invention has been made in view of the above-described circumstances, and with a simpler structure, improves the pointing stability of ultrashort pulse laser light incident on the observation apparatus main body, and efficiently multi-photon excitation effect. An object of the present invention is to provide a multiphoton excitation observation device and a light source device for multiphoton excitation observation that can be generated and can obtain a clear multiphoton fluorescence image.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a pulse laser light source that emits an ultrashort pulse laser beam, an observation apparatus main body that irradiates the sample with an ultrashort pulse laser beam emitted from the pulse laser light source, and observes the fluorescence emitted from the sample; Provided is a multiphoton excitation observation device that is disposed between the pulse laser light source and the observation device main body and includes an acousto-optic modulation filter that modulates an ultrashort pulse laser beam emitted from the pulse laser light source.

  According to the present invention, when an ultrashort pulse laser beam emitted from a pulse laser light source is supplied to an observation apparatus body and irradiated on the sample by the observation apparatus body, an ultrashort pulse laser is generated due to the multiphoton excitation effect in the sample. Multiphoton fluorescence is generated at the position where the light is collected. The generated multiphoton fluorescence is detected and observed by the observation apparatus body. The ultrashort pulse laser beam passes through the acousto-optic modulation filter before being incident on the observation device body. Therefore, the ultra-short pulse laser beam is turned on / off or modulated by the acousto-optic modulation filter, and the ultra-short pulse laser beam having a desired light intensity is obtained. Is incident on.

  In this case, according to the present invention, by using the acoustooptic modulation filter, unlike the AOM, the effective range of incidence on the crystal is wide. There is no need to squeeze or precisely adjust the alignment. Therefore, a reduced beam expander and an alignment adjustment device are not required before the acoustooptic modulation filter, and the configuration of the device can be simplified. In addition, since the acousto-optic modulation filter requires lower driving power than the AOM, there are few problems of heat generated locally, and pointing stability can be improved.

In the above invention, the pulse laser light source can emit a plurality of ultrashort pulse laser beams having different wavelengths, and the acoustooptic modulation is performed according to the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source. It is good also as providing the control apparatus which adjusts the intensity | strength and emission angle of the ultra-short pulse laser beam radiate | emitted from a filter.
In this way, even if the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source is changed, the intensity and emission angle of the ultrashort pulse laser light emitted from the acousto-optic modulation filter by the operation of the control device. Is adjusted, the ultrashort pulse laser beam can be emitted in a desired direction. Therefore, it is possible to stably emit ultrashort pulse laser beams having different wavelengths with an appropriate intensity and the same emission angle.

In the above invention, the control device stores the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source and the frequency and amplitude of the drive signal supplied to the acoustooptic modulation filter in association with each other. It is good also as providing.
In this way, when the control device receives the wavelength information of the ultrashort pulse laser beam emitted from the pulse laser light source, the frequency and amplitude of the drive signal stored in the storage unit are selected, and the acousto-optic modulation filter To be supplied. Thereby, even if the wavelength of the ultrashort pulse laser beam is changed, the ultrashort pulse laser beam can be quickly emitted with a desired intensity and an emission angle.

In the above invention, a control device may be provided that controls the acousto-optic modulation filter so that the intensity of the ultrashort pulse laser beam emitted from the acousto-optic modulation filter becomes a specified intensity.
In this way, when the ultrashort pulse laser light emitted from the pulse laser light source is passed through the acousto-optic modulation filter, it is modulated into the ultrashort pulse laser light of the specified intensity by the operation of the control device. Is output. Therefore, a multiphoton excitation effect using an ultrashort pulse laser beam having a desired intensity can be generated in the sample, and observation can be performed with high accuracy.

In the above invention, the control device may further include a storage unit that stores the intensity of the ultrashort pulse laser beam and the frequency and amplitude of the drive signal supplied to the acoustooptic modulation filter in association with each other.
In this way, when the control device specifies the intensity of the ultrashort pulse laser beam, the frequency and amplitude of the drive signal stored in the storage unit are selected and supplied to the acousto-optic modulation filter. As a result, it is possible to emit an extremely short pulse laser beam having a designated intensity quickly.

Moreover, in the said invention, the said control apparatus is equipped with the detector which detects the intensity | strength of the ultrashort pulse laser beam radiate | emitted from the said acousto-optic modulation filter, and this intensity | strength is based on the intensity | strength detected by this detector. The frequency and amplitude of the drive signal supplied to the acousto-optic modulation filter may be adjusted so that becomes the specified intensity.
By doing so, the intensity of the ultra-short pulse laser beam output from the pulse laser light source is detected by the detector and fed back, so that the sample is irradiated with the ultra-short pulse laser beam having a precisely set intensity. It becomes possible.

Moreover, in the said invention, the visible laser light source which radiates | emits visible laser light, The visible laser light which is arrange | positioned between this visible laser light source and the said observation apparatus main body, and is emitted from a visible laser light source and injects into an observation apparatus main body And an acousto-optic modulation filter that modulates.
By doing so, the visible laser light and the ultrashort pulse laser light incident on the observation apparatus main body can be modulated by the same method.

Moreover, in the said invention, the said acousto-optic modulation filter is good also as providing an internal temperature stabilization means like a temperature control apparatus or a heat sink.
By doing so, it is possible to prevent overheating of the acousto-optic modulation filter and further improve the pointing stability of the ultrashort pulse laser beam.

The present invention also provides a multi-photon excitation observation light source device comprising a pulse laser light source that emits an ultrashort pulse laser beam and an acousto-optic modulation filter that modulates the ultrashort pulse laser beam emitted from the pulse laser light source. I will provide a.
According to the present invention, by using an acousto-optic modulation filter, unlike the case of AOM, the effective range of incidence on the crystal is wide. There is no need for precise alignment. Therefore, a reduced beam expander and an alignment adjustment device are not required before the acoustooptic modulation filter, and the configuration of the device can be simplified. In addition, since the acoustooptic modulation filter requires lower driving power than the AOM, there are few problems of locally generated heat, and it is possible to emit an ultrashort pulse laser beam having high pointing stability.

In the above invention, the pulse laser light source can emit a plurality of ultrashort pulse laser beams having different wavelengths, and the acoustooptic modulation is performed according to the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source. It is good also as providing the control apparatus which adjusts the intensity | strength and emission angle of the ultra-short pulse laser beam radiate | emitted from a filter.
In the above invention, the control device stores the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source and the frequency and amplitude of the drive signal supplied to the acoustooptic modulation filter in association with each other. It is good also as providing.

In the above invention, a control device may be provided that controls the acousto-optic modulation filter so that the intensity of the ultrashort pulse laser beam emitted from the acousto-optic modulation filter becomes a specified intensity.
In the above invention, the control device may include a storage unit that stores the intensity of the ultrashort pulse laser beam and the frequency and amplitude of the drive signal supplied to the acoustooptic modulation filter in association with each other.

  Moreover, in the said invention, the said control apparatus is equipped with the detector which detects the intensity | strength of the ultrashort pulse laser beam radiate | emitted from the said acousto-optic modulation filter, and this intensity | strength is based on the intensity | strength detected by this detector. The frequency and amplitude of the drive signal supplied to the acousto-optic modulation filter may be adjusted so that becomes the specified intensity.

Moreover, in the said invention, it is good also as providing the visible laser light source which radiate | emits visible laser light, and the acousto-optic modulation filter which modulates the visible laser light emitted from this visible laser light source.
Moreover, in the said invention, the said acousto-optic modulation filter is good also as providing an internal temperature stabilization means like a temperature control apparatus or a heat sink.

  According to the present invention, by using an acousto-optic modulation filter, it is possible to improve the pointing stability of the ultrashort pulse laser light incident on the observation apparatus main body with a simpler configuration, efficiently generate a multiphoton excitation effect, There is an effect that a clear multiphoton fluorescence image can be obtained.

Hereinafter, a multiphoton excitation observation apparatus according to an embodiment of the present invention will be described with reference to FIGS.
The multiphoton excitation type observation apparatus 1 according to the present embodiment is a multiphoton excitation type microscope, and as shown in FIG. 1, a light source device (multiphoton excitation type light source device for observation) 2 and the light source device 2. And a fluorescence microscope main body (observation apparatus main body) 3 disposed in the latter stage.

  The light source device 2 includes a pulse laser light source 4 that emits an ultrashort pulse laser light L, and an acousto-optic modulation filter (hereinafter, referred to as “on / off” or intensity modulation of the ultrashort pulse laser light L emitted from the pulse laser light source 4). 5), a dispersion compensation optical system 6 that compensates for group velocity dispersion of the modulated ultrashort pulse laser light L, and an ultrashort pulse laser light L that is dispersion compensated and incident on the fluorescence microscope main body 3. And a collimating optical system 7 for adjustment.

  The dispersion compensation optical system 6 includes, for example, a pair of prisms 8 and 9 and a mirror 10 as shown in FIG. By adjusting the distance between the prisms 8 and 9, the dispersion compensation amount is adjusted to compensate for the group velocity dispersion in the entire optical system from the pulse laser light source 4 to the objective lens 3a of the fluorescence microscope main body 3. Yes.

At this time, the optical axis shifts by adjusting the optical path length, and the angle of the optical axis fluctuates due to the change in wavelength. Therefore, by adjusting the positions and angles of the prisms 8 and 9 and the mirror 10 as indicated by arrows. The position and angle of the optical axis of the emitted ultrashort pulse laser beam L are adjusted.
In the figure, reference numeral 11 denotes an ultrashort pulse laser beam L output from the dispersion compensation optical system 6 while reflecting and directing the ultrashort pulse laser beam L emitted from the pulse laser light source 4 to the dispersion compensation optical system 6. It is the mirror arrange | positioned in the position off from the optical axis.

  Further, for example, as shown in FIG. 3, the collimating optical system 7 includes a Galilean beam expander in which a biconvex lens 12 and a planoconcave lens 13 are combined, and a biconvex lens 12 and a planoconcave lens 13 in the optical axis direction. The lens moving mechanisms 14 and 15 are movably supported. The collimating optical system 7 emits the ultrashort pulse laser beam L so that the ultrashort pulse laser beam L is incident on the pupil position of the objective lens 3a of the fluorescent microscope body 3 with a light beam diameter substantially equal to the pupil diameter. The beam diameter and beam divergence of L can be adjusted.

  As shown in FIGS. 4 to 6, the AOTF 5 includes a crystal 16 made of tellurium dioxide, a case 17 for fixing the crystal 16, a transducer 18 made of a piezoelectric element bonded to the crystal 16, and the transducer 18 includes an acoustic wave generating electric circuit 19 for transmitting an electric signal E input to 18, and a heat sink 20 bonded to the crystal 16. Between the crystal 16 and the case 17 and between the crystal 16 and the heat sink 20, an interface material 21 made of, for example, a thermal compound such as silicone grease or an indium foil is interposed. In the figure, reference numeral 22 denotes a sound absorbing material that absorbs an acoustic wave emitted from the transducer 18 and propagated through the crystal 16.

An opening 23 is provided in each of the pair of side walls of the case 17 that are opposed to each other with the crystal 16 interposed therebetween, and an ultrashort pulse laser beam that has entered the ultrashort pulse laser beam L from one opening portion 23 and has passed through the crystal 16. The light L is emitted from the other opening 23.
The heat sink 20 is made of, for example, a material having high thermal conductivity such as aluminum or aluminum alloy, and includes a plurality of plate-like fins 20a arranged at a parallel interval as shown in FIG.

  Inside the fluorescent microscope main body 3, a galvano mirror 301 (for example, a proximity galvanometer mirror) that scans the ultrashort pulse laser beam L in a two-dimensional manner and the scanned ultrashort pulse laser beam L are condensed. A pupil projection lens 302 for forming an intermediate image, an image forming lens 303 for condensing the ultrashort pulse laser light L formed on the intermediate image into a substantially parallel light, and an ultrashort pulse laser light converted to a substantially parallel light. An objective lens 3a for condensing L and forming an image on the sample A; a dichroic mirror 304 for branching the multiphoton fluorescence F emitted from the sample A and collected by the objective lens 3a from the ultrashort pulse laser beam L; A condensing lens 305 that collects the multiphoton fluorescence F and a photomultiplier tube (PMT) 306 that detects the collected multiphoton fluorescence F are provided. Reference numeral 307 denotes a mirror.

The operation of the thus configured multiphoton excitation observation apparatus 1 according to this embodiment will be described below.
The ultrashort pulsed laser light L emitted from the pulsed laser light source 4 passes through the AOTF 5 and is incident on the dispersion compensation optical system 6 in a state of being turned on / off or modulated to a predetermined intensity. The group velocity dispersion amount of the entire optical system of the apparatus 1 is compensated. Thereafter, the ultrashort pulse laser beam L is allowed to pass through the collimating optical system 7, so that the beam diameter and beam divergence are substantially equal to the pupil diameter at the pupil position of the objective lens 3 a of the fluorescent microscope body 3. Correction is made to have a beam diameter. As a result, the ultrashort pulse laser light L emitted from the pulse laser light source 4 is most efficiently collected on the sample A without waste, and the photon density at the beam waist position formed in the sample A is improved, thereby improving efficiency. In particular, a multiphoton excitation effect can be generated.

  In the AOTF 5, an electrical signal E having a predetermined frequency is input to the transducer 18 by the operation of the acoustic wave generating electrical circuit 19, and the ultrashort pulse laser light L in the crystal 16 is generated by the acoustic wave generated by the transducer 18. Acousto-optic effect occurs. Further, by changing the amplitude of the acoustic wave generated by the transducer 18, the diffraction intensity of the ultrashort pulse laser beam L in the crystal 16 is changed, and the intensity of the ultrashort pulse laser beam L emitted from the AOTF 5 is changed to an acoustic level. Modulate at a predetermined ratio according to the amplitude of the wave.

As a result, the incident ultrashort pulse laser beam L passes through the crystal 16 and is emitted from the opening 23 of the case 17 in a state modulated to a predetermined intensity.
In this case, unlike the conventional AOM, the AOTF 5 has a relatively large opening 23, which is an effective range of incidence, so it is not necessary to adjust the beam diameter in the previous stage or to precisely adjust the alignment. However, the ultrashort pulse laser beam L is incident on the crystal 16 within the effective range without leaking. Therefore. There is no ultrashort pulse laser light L that is not incident on the crystal 16 and no ultrashort pulse laser light L that is irregularly reflected by the side surface of the crystal 16. Is not mixed into the ultrashort pulse laser beam L passing through the beam.

  As a result, the occurrence of wavefront disturbance in the ultrashort pulse laser beam L emitted from the crystal 16 is suppressed, and the deterioration of the PSF at the condensing position of the sample A is suppressed. That is, at the condensing position in the sample A formed in front of the objective lens 3a, a decrease in photon density is prevented, and a multiphoton excitation effect can be efficiently generated.

  And according to the multiphoton excitation observation device 1 according to the present embodiment, since the ultrashort pulse laser beam L is modulated by the AOTF 5, the configuration of the light source device 2 can be simplified. That is, unlike the AOM, since the effective range (opening 23) of the incident is wide, the beam diameter of the ultrashort pulse laser beam L is reduced before the incident on the AOTF 5 or the precise alignment adjustment is performed. A reduced beam expander and alignment adjustment device can be omitted. Therefore, there is an advantage that the configuration of the entire apparatus can be simplified, reduced in size, and reduced in cost.

  In addition, according to the multiphoton excitation observation apparatus 1 according to the present embodiment, since the ultrashort pulse laser light L is modulated by the AOTF 5, the pointing stability of the ultrashort pulse laser light L incident on the fluorescence microscope main body 3 is adjusted. Can be stabilized. That is, unlike the AOM, the local overheating of the crystal 16 is small. Therefore, an alignment adjusting means for compensating unstable pointing stability is not required after the AOTF 5, and in this respect, the apparatus can be simplified, reduced in size, and reduced in cost. is there.

  In addition, by turning on and off the acoustic wave input to the crystal 16 by the transducer 18, it is possible to switch on and off the emission and blocking of the ultrashort pulse laser beam L. For example, by turning on and off the ultrashort pulse laser beam L in synchronization with the two-dimensional scanning of the ultrashort pulse laser beam L by the galvanometer mirror 301 of the fluorescence microscope main body 3, the sample A is only in a predetermined scanning range. It becomes possible to irradiate the ultrashort pulse laser beam L.

  The ultrashort pulse laser beam L is diffracted by the acoustooptic effect when it passes through the crystal 16 of the AOTF 5. At this time, since an acoustic wave is input to the crystal 16 from the transducer 18, the crystal 16 locally generates heat due to the energy. The heat generation in the crystal 16 of AOTF5 is sufficiently smaller than the heat generation in the crystal of AOM.

  According to the multiphoton excitation observation apparatus 1 according to this embodiment, the heat capacity of the crystal 16 itself is small, but since the heat sink 20 having a large heat capacity is bonded to the crystal 16, the heat generated in the crystal 16 is generated by the heat sink 20. Is absorbed by. Moreover, since the heat sink 20 is provided with a plurality of fins 20a, the heat absorbed from the crystal 16 is efficiently dissipated to the outside air through these fins 20a. Thereby, the local temperature rise of the crystal | crystallization 16 is suppressed and the fluctuation | variation of the refractive index in the crystal | crystallization 16 is suppressed significantly.

  When the refractive index of the crystal 16 varies, the diffraction of the ultrashort pulse laser beam L in the crystal 16 changes, and the emission direction from the AOTF 5 changes. Therefore, when no countermeasure is taken, the temperature of the crystal 16 changes with time due to the acoustic wave input from the transducer 18, so that the emission direction from the crystal 16 shifts and is not emitted to an appropriate position. It will end up.

  Further, since the amplitude of the acoustic wave input to the crystal 16 differs depending on the use state such as the intensity modulation degree and the on / off frequency, the emission direction of the ultrashort pulse laser light L changes depending on the use state.

  According to the present embodiment, since the temperature change of the crystal 16 is suppressed by the heat sink 20 having a large heat capacity, even if the driving power input to the transducer 18 to generate an acoustic wave is high, local heating in the crystal 16 is performed. The state is prevented, and the emission direction of the ultrashort pulse laser beam L can be kept constant. As a result, it is not necessary to detect the temperature change of the crystal over time, or to adjust the alignment adjusting device each time based on the detected temperature of the crystal 16, and it is set at the start of observation with a simple configuration. Fluorescence observation can be performed by irradiating the appropriate irradiation range of the sample A with the ultrashort pulse laser beam L in the alignment state.

  In the present embodiment, the heat sink 20 is illustrated as having the flat fins 20a, but any other heat sink can be employed instead. In addition, as shown in FIG. 7, instead of the heat sink 20, a temperature adjusting device 24 including a cooler such as a Peltier element or a heater, a temperature measuring instrument such as a heater, and a thermocouple may be arranged.

As shown in FIG. 8, the beam pointing adjustment optical system 25 may be disposed at the subsequent stage of the AOTF 5. For example, as shown in FIG. 9, the beam pointing adjustment optical system 25 includes two mirrors 26a and 26b and two mirror moving mechanisms 27 and 28 for moving the mirrors 26a and 26b, respectively. The first mirror 26a is ultrashort pulsed laser light L that has been incident as shown by an arrow from the first direction Z _1, is deflected in a second direction Z ₂ that is substantially orthogonal to the direction Z ₁ of the first It is like that. The second mirror 26b, the second ultrashort pulsed laser light L incident on the direction Z _2, the first direction Z ₁ and second third direction Z ₃ substantially orthogonal to the direction Z ₂ The beam is emitted after being deflected in the direction.

The first mirror moving mechanism 27, first the a first translating mechanism 27a first along the direction Z ₁ of the translating movement of the first mirror 26a, the third axis line parallel to the direction Z ₃ And a first swing mechanism 27b that swings one mirror 26a. Second mirror moving mechanism 28 includes a second translation mechanism 28a for translating the second mirror 26b along the third direction, the second to the first axis line parallel to the direction Z ₁ A second swing mechanism 28b for swinging the mirror 26b. The first and second translation mechanisms 27a and 28a are driven by, for example, motors 27c and 28c and ball screws 27d and 28d, and the first and second swing mechanisms 27b and 28b are configured by, for example, motors 27e and 28e. Has been.

Thus, if the angle of the optical axis of the ultrashort pulsed laser light L with respect to the first direction Z _1, are shifted in the plane including the first direction Z ₁ and a second direction Z ₂ is actuates a first oscillating mechanism 27b adjusts the angle, if the offset in the plane with respect to the first direction Z ₁ including a second direction Z ₂ and third directions Z ₃ The second swinging mechanism 28b can be operated to adjust its angle. Further, when the position of the optical axis of the ultrashort pulsed laser light L emitted from the beam pointing adjustment optical system 25 is shifted in the first direction Z ₁ actuates the first translation mechanism 27a, the second if the offset of the direction Z ₂ is adapted to be able to actuate the second translation mechanism 28a, to adjust the positional deviation.
This is effective when it is necessary to compensate for misalignment caused by the pulse laser light source 4 and the dispersion compensation optical system 6.

Next, a multiphoton excitation observation apparatus 30 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are assigned to portions having the same configuration as the multiphoton excitation observation apparatus 1 according to the first embodiment described above, and the description thereof is omitted.

  The multiphoton excitation observation apparatus 30 according to the present embodiment branches a pulse laser light source 31 capable of emitting an ultrashort pulse laser beam L having a plurality of wavelengths and an ultrashort pulse laser beam L emitted from the AOTF 5 A detector 32 that detects the intensity, and a controller 33 that adjusts the frequency and amplitude of the acoustic wave (drive signal) supplied to the crystal 16 of the AOTF 5 so that the intensity of the detected ultrashort pulse laser beam L is maximized. And. The control device 33 may include a storage device (not shown) that stores the intensity of the ultrashort pulse laser beam L and the frequency and amplitude of the acoustic wave in association with each other.

The detector 32 includes a beam sampler 34 that branches the ultrashort pulse laser beam L emitted from the AOTF 5, a beam shaping optical system 35 that adjusts the intensity, beam shape, and alignment of the branched ultrashort pulse laser beam L, And a power monitor 36 for detecting the adjusted ultrashort pulse laser beam L.
The beam shaping optical system 35 is configured by, for example, an ND filter, a slit, or a combination of a condenser lens and a slit. The power monitor 36 is constituted by, for example, a calorimeter, a photodiode, a CMOS, or a CCD.

When the acoustic wave supplied to the crystal 16 of the AOTF 5 is fixed and the wavelength of the ultrashort pulse laser light L emitted from the pulse laser light source 4 fluctuates, the intensity of the ultrashort pulse laser light L emitted from the AOTF 5 The emission direction varies.
According to the multiphoton excitation observation apparatus 30 according to the present embodiment, the ultrashort pulse laser beam L detected by the detector 32 when the wavelength of the ultrashort pulse laser beam L emitted from the pulse laser light source 31 changes. Since the frequency and amplitude of the acoustic wave to the crystal 16 of the AOTF 5 are adjusted so that the intensity of the AOTF 5 is maximized, the emission angle from the AOTF 5 is kept constant and appropriate regardless of the wavelength of the ultrashort pulse laser beam L. The emission intensity can be maintained.

Next, a multiphoton excitation observation apparatus 40 according to a third embodiment of the present invention will be described below with reference to FIG.
Also in the description of the present embodiment, the same reference numerals are given to portions having the same configurations as those of the multiphoton excitation observation apparatuses 1 and 30 according to the first and second embodiments described above, and the description thereof is omitted.

The multiphoton excitation observation device 40 according to the present embodiment is based on an input device 41 such as a keyboard for inputting the wavelength of the ultrashort pulse laser light L emitted from the pulse laser light source 31 and an input signal from the input device 41. And a control device 42 for instructing the wavelength of the ultrashort pulsed laser light L to be emitted to the pulsed laser light source 31.
The control device 42 keeps the emission angle from the AOTF 5 constant when the wavelength command signal output to the pulsed laser light source 31 and the ultrashort pulsed laser light L of the wavelength are incident on the AOTF 5, and A storage device 43 is provided that stores the frequency and amplitude of an acoustic wave in association with each other in order to obtain an appropriate emission intensity.

  According to the multiphoton excitation observation device 40 according to the present embodiment, when wavelength information of the ultrashort pulse laser beam L emitted from the pulse laser light source 31 is input from the input device 41, the input wavelength information is converted to the control device. The control device 42 searches the storage device 43 based on the transmitted wavelength information, and acquires the frequency and amplitude information of the acoustic wave stored in association with the wavelength. The acquired frequency and amplitude signal of the acoustic wave is supplied from the control device 42 to the AOTF 5. Thereby, in the AOTF 5, the emission intensity and the emission angle are adjusted according to the wavelength of the emitted ultrashort pulse laser beam L.

  That is, according to the multiphoton excitation observation apparatus 40 according to the present embodiment, an ultrashort pulse can be easily and accurately set at a constant emission angle and with an appropriate emission intensity regardless of the change in the wavelength of the pulse laser light source 31. Laser light L can be emitted.

  As described above, according to the multiphoton excitation observation device 40 according to the present embodiment, even if the wavelength of the ultrashort pulse laser light L emitted from the pulse laser light source 31 is changed, the control device 42 provides the wavelength information. AOTF5 is controlled based on the above. Therefore, even if the wavelength of the ultrashort pulse laser beam L is changed, the ultrashort pulse laser beam L having a light beam diameter substantially equal to the pupil diameter is incident on the pupil position of the objective lens 3a of the fluorescence microscope main body 3. Thus, the multiphoton excitation effect in the sample A can be efficiently generated, and a clear multiphoton excitation image can be obtained.

  In addition, the control device 42 controls the AOTF 5 by acquiring the frequency and amplitude of the acoustic wave supplied to the crystal 16 of the AOTF 5 stored in the storage device 43 in association with the wavelength from the storage device 43. Therefore, even if the wavelength is changed, it is possible to adjust the ultrashort pulse laser light L so that the multiphoton excitation effect is efficiently generated in the sample A simply and quickly.

Next, a multiphoton excitation observation apparatus 50 according to a fourth embodiment of the present invention will be described below with reference to FIG.
Also in the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the multiphoton excitation observation apparatuses 1, 30, and 40 according to the first to third embodiments described above, and the description thereof is omitted.

As shown in FIG. 12, the multiphoton excitation observation apparatus 50 according to the present embodiment emits a visible laser light source 51 and an ultraviolet laser light source 52, and these light sources 51 and 52 in addition to the pulse laser light source 31. AOTFs 53 and 54 for modulating the laser beams L ₁ and L ₂ respectively, optical fibers 55 and 56 such as single mode fibers for propagating the visible laser beam L ₁ and the ultraviolet laser beam L ₂ , and these laser beams L ₁ , L ₂ and the ultrashort pulse laser beam L are combined into the same optical path and supplied to the fluorescence microscope main body 3.

In response to the wavelength command input from the input device 41, the control device 42 outputs laser light L. to be emitted to each of the laser light sources 31, 51, 52. L ₁ . The wavelength of the L ₂ is adapted to the instructions. Further, the storage device 43 includes laser light L.L emitted from each laser light source 31, 51, 52. L ₁ . And wavelength information of the L _2, and the acoustic wave frequency and amplitude information are stored in association supplied to each crystal 16 in AOTF5,53,54 at that time.

The multiplexing optical system 57 includes collimating lenses 58 and 59 for making the visible laser beam L ₁ and the ultraviolet laser beam L ₂ propagated through the optical fibers 55 and 56 substantially parallel lights, and a dichroic mirror for joining the optical paths. 60, 61. In the figure, reference numeral 62 denotes a mirror.
Each of the laser light sources 31, 51, 52 has a plurality of laser beams L.P. L ₁ . The L ₂ is a continuous or pulsed laser light source capable of emitting. Either a method of switching a plurality of single wavelength laser beams or a variable wavelength method may be used.

According to the multiphoton excitation observation device 50 according to the present embodiment configured as described above, the pulse laser light source 31 and the visible laser light source 51 are the same as the multiphoton excitation observation device 40 according to the third embodiment. When the wavelength information of the laser beams L, L ₁ and L ₂ emitted from the ultraviolet laser light source 52 is input from the input device 41, the input wavelength information is sent to the control device 42 and sent by the control device 42. The storage device 43 is searched based on the received wavelength information, and the frequency and amplitude information of the acoustic wave stored in association with the wavelength information is acquired. The acquired frequency and amplitude information of the acoustic wave is supplied from the control device 42 to the AOTFs 5, 53, 54.

Thereby, in the AOTFs 5, 53, 54, the emission intensity and the emission angle are adjusted in accordance with the wavelengths of the emitted laser beams L, L ₁ , L ₂ .
That is, according to the multiphoton excitation observation apparatus 50 according to the present embodiment, regardless of changes in the wavelengths of the laser light sources 31, 51, 52, a simple and accurate constant emission angle and appropriate emission intensity can be obtained. Laser beams L, L ₁ and L ₂ can be emitted.

  In this case, according to the multiphoton excitation observation apparatus 50 according to the present embodiment, the plurality of laser light sources 31, 51, 52 are modulated by the AOTFs 5, 53, 54, respectively, so that the control signals are set to substantially the same level. There is an advantage that the timing control of the AOTFs 5, 53 and 54 can be easily performed.

1 is an overall configuration diagram showing a multiphoton excitation observation device and a multiphoton excitation observation light source device according to a first embodiment of the present invention. It is a figure which expands and shows the dispersion compensation optical system of the multiphoton excitation observation apparatus of FIG. 1, and the light source apparatus for multiphoton excitation observation. It is a figure which expands and shows the collimation optical system of the multiphoton excitation observation apparatus of FIG. 1, and the light source device for multiphoton excitation observation. It is a longitudinal cross-sectional view which expands and shows AOTF of the multiphoton excitation observation device and the multiphoton excitation observation light source device of FIG. It is a perspective view of AOTF of FIG. It is a longitudinal cross-sectional view which shows the internal structure of AOTF of FIG. It is a longitudinal cross-sectional view which shows the modification of AOTF of FIG. It is a whole block diagram which shows the multiphoton excitation observation apparatus and the multiphoton excitation observation light source apparatus which concern on the 2nd Embodiment of this invention. It is a perspective view which expands and shows the beam pointing adjustment optical system used for the multiphoton excitation observation apparatus of FIG. It is a whole block diagram which shows the multiphoton excitation observation apparatus and the multiphoton excitation observation light source apparatus which concern on the 3rd Embodiment of this invention. It is a whole block diagram which shows the multiphoton excitation type observation apparatus which concerns on the 4th Embodiment of this invention. It is a whole block diagram which shows the multiphoton excitation type observation apparatus which concerns on the 5th Embodiment of this invention.

Explanation of symbols

L Ultrashort pulse laser beam L ₁ Visible laser beam L ₂ Ultraviolet laser beam 1, 30, 40, 50 Multiphoton excitation observation device 2 Multiphoton excitation observation light source device 3 Fluorescence microscope body (observation device body)
4,31 Pulsed laser light source 5,53,54 AOTF (acousto-optic modulation filter)
20 Heat sink (internal temperature stabilization means)
24 Temperature control device (internal temperature stabilization means)
32 Detector 33, 42 Control device 43 Storage device (storage unit)

Claims (16)

A pulse laser light source that emits ultrashort pulse laser light;
An observation apparatus main body for irradiating the sample with an ultrashort pulse laser beam emitted from the pulse laser light source and observing fluorescence emitted from the sample;
A multi-photon excitation observation device that is disposed between the pulse laser light source and the observation device main body and includes an acousto-optic modulation filter that modulates ultrashort pulse laser light emitted from the pulse laser light source. The pulse laser light source can emit a plurality of ultrashort pulse laser beams having different wavelengths,
The control apparatus which adjusts the intensity | strength and the emission angle of the ultrashort pulse laser beam radiate | emitted from the acousto-optic modulation filter according to the wavelength of the ultrashort pulse laser beam radiate | emitted from this pulse laser light source. Multiphoton excitation observation device.   The said control apparatus is provided with the memory | storage part which matches and memorize | stores the wavelength of the ultra-short pulse laser beam radiate | emitted from a pulse laser light source, and the frequency and amplitude of the drive signal supplied to an acousto-optic modulation filter. Multiphoton excitation observation device.   The multiphoton excitation observation apparatus according to claim 1, further comprising a control device that controls the acoustooptic modulation filter so that the intensity of the ultrashort pulse laser beam emitted from the acoustooptic modulation filter becomes a specified intensity. .   The multi-photon excitation observation apparatus according to claim 4, wherein the control device includes a storage unit that stores the intensity of the ultrashort pulse laser beam and the frequency and amplitude of the drive signal supplied to the acousto-optic modulation filter in association with each other. .   The control device includes a detector that detects the intensity of the ultrashort pulse laser beam emitted from the acoustooptic modulation filter, and the intensity becomes a specified intensity based on the intensity detected by the detector. The multiphoton excitation observation apparatus according to claim 4 or 5, wherein the frequency and amplitude of the drive signal supplied to the acoustooptic modulation filter are adjusted as described above. A visible laser light source that emits visible laser light;
The acousto-optic modulation filter which is arrange | positioned between this visible laser light source and the said observation apparatus main body, and modulates the visible laser beam emitted from the visible laser light source and injected into the observation apparatus main body. The multiphoton excitation observation apparatus according to any one of the above.   The multi-photon excitation observation device according to any one of claims 1 to 7, wherein the acousto-optic modulation filter includes an internal temperature stabilization means such as a temperature control device or a heat sink. A pulse laser light source that emits ultrashort pulse laser light;
A multiphoton excitation observation light source device comprising an acousto-optic modulation filter for modulating an ultrashort pulse laser beam emitted from the pulse laser light source. The pulse laser light source can emit a plurality of ultrashort pulse laser beams having different wavelengths,
The control apparatus which adjusts the intensity | strength and emission angle of the ultra-short pulse laser beam radiate | emitted from the acousto-optic modulation filter according to the wavelength of the ultra-short pulse laser beam radiate | emitted from this pulse laser light source. Multiphoton excitation type observation light source device.   The said control apparatus is provided with the memory | storage part which matches and memorize | stores the wavelength of the ultra-short pulse laser beam radiate | emitted from a pulse laser light source, and the frequency and amplitude of the drive signal supplied to an acoustooptic modulation filter. Multiphoton excitation type observation light source device.   The multiphoton excitation type observation device according to claim 9, further comprising a control device that controls the acoustooptic modulation filter so that an intensity of the ultrashort pulse laser beam emitted from the acoustooptic modulation filter becomes a specified intensity. Light source device.   The multi-photon excitation type observation according to claim 12, wherein the control device includes a storage unit that stores the intensity of the ultrashort pulse laser beam and the frequency and amplitude of the drive signal supplied to the acousto-optic modulation filter in association with each other. Light source device.   The control device includes a detector that detects the intensity of the ultrashort pulse laser beam emitted from the acoustooptic modulation filter, and the intensity becomes a specified intensity based on the intensity detected by the detector. The multi-photon excitation observation light source device according to claim 12 or 13, wherein the frequency and amplitude of the drive signal supplied to the acousto-optic modulation filter are adjusted as described above.   The multiphoton excitation observation according to any one of claims 9 to 14, further comprising: a visible laser light source that emits visible laser light; and an acousto-optic modulation filter that modulates the visible laser light emitted from the visible laser light source. Light source device.   The multi-photon excitation observation light source device according to any one of claims 9 to 15, wherein the acousto-optic modulation filter includes internal temperature stabilization means such as a temperature control device or a heat sink.

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