JP2007127524A - Multiphoton excitation observation device and multiphoton excitation observing light source device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To acquire the fine output modulation using an acoustic optical device, various optical stimulation or fluorescent observation due to on-off and the bright and sharp fluorescent image in the deep region of a sample. <P>SOLUTION: The multiphoton excitation observation device 1 is equipped with a pulse laser beam source 2, an observation device body 3 for irradiating the sample A with an extremely short pulse laser beam L to observe the fluorescence F emitted from the sample A and the introducing optical system 4 arranged between the pulse laser beam source 2 and the observation device body 3 to regulate the extremely short pulse laser beam L emitted from the pulse laser beam source 2 to be introduced into the observation device body 3. The introducing optical system 4 is equipped with a first light path 20 including an acoustic optical device 31 for adjusting the on-off or output of the extremely short pulse laser beam L emitted from the pulse laser beam source 2 and introsdued into the observation device body 3. The introducing optical system 4 is equipped with a first light path 20 which includes an acoustic optical device 31 for performing the adjustment of the on-off of the extremely short pulse laser beam L emitted from the pulse laser beam sourse 2 and a second light path 22 which bypasses the acoustic optical device 31 in parallel to each other and also equipped with a light path selecting device 24 for passing the extremely short pulse laser beam L through at least one of the light paths 20 and 22. <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 (See, for example, Patent Document 1 and Patent Document 2).
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 for turning it on and off.
The use of an acousto-optic device such as a Modulator is disclosed. By using the acousto-optic device, the intensity of the excitation light can be controlled, and by using the high-speed shutter function, it is possible to irradiate the excitation light with high precision only to the part that requires light stimulation, and an unnecessary place. The problem of damaging the sample by irradiating the sample with excitation light can be prevented.

Also, in Patent Document 2, an acoustooptic device such as AOM or AOTF that strongly disperses the excitation light is disposed adjacent to the optical fiber in order to prevent the pulse shape of the excitation light from being disturbed by a nonlinear effect in the optical fiber. An arranged microscope is disclosed.
Japanese Patent Laid-Open No. 10-512959 JP 2000-206415 A

  However, when the acoustooptic device is used for modulating excitation light, pulse dispersion in the acoustooptic device, vignetting when entering the aperture of the acoustooptic device, disturbance of PSF (point spread function), and incident beam alignment There is a problem that the amount of laser light loss due to the entire optical system is large. When the amount of laser light decreases, it becomes difficult to supply high-intensity laser light to a deep part of the sample, and there is a problem that a fluorescent image of a deep part of the sample cannot be obtained.

  The present invention has been made in view of the circumstances described above, and performs various optical stimulation and fluorescence observation by fine output modulation and on / off using an acousto-optic device, while deeply moving the sample without moving the sample. An object of the present invention is to provide a multiphoton excitation observation apparatus and a multiphoton excitation observation light source apparatus capable of acquiring a bright and clear fluorescent image at a site.

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; An introduction optical system that is disposed between the pulse laser light source and the observation apparatus main body, adjusts an ultrashort pulse laser beam emitted from the pulse laser light source and introduced into the observation apparatus main body, the introduction optical system, A first optical path including an acoustooptic device that performs on / off or output adjustment of an ultrashort pulsed laser beam emitted from a pulsed laser light source and a second optical path that bypasses the acoustooptic device are provided in parallel. A multi-photon excitation observation apparatus including an optical path selection device that allows an ultrashort pulse laser beam to pass through at least one of them.

  According to the present invention, at least one of the first optical path and the second optical path is selected by the operation of the optical path selection device of the introduction optical system, and the ultrashort pulse laser beam is allowed to pass through the selected optical path. To be introduced. When the first optical path is selected, the ultrashort pulse laser light is allowed to pass through the acousto-optic device. Thereby, the ultrashort pulse laser beam is introduced into the observation apparatus main body in a state of being turned on / off at high speed or output adjusted, and is irradiated onto the sample through the observation apparatus main body. Therefore, it is possible to perform optical stimulation or fluorescence observation of a sample by irradiating only a predetermined necessary region of the sample with an ultrashort pulse laser beam with an output adjusted accurately and appropriately.

  On the other hand, when the second optical path is selected, the ultrashort pulse laser beam is bypassed without passing through the acoustooptic device, introduced into the observation device body, and irradiated on the sample. Since it does not pass through the acousto-optic device, the ultrashort pulse laser light is irradiated to the sample while maintaining a high intensity without being attenuated so much. Therefore, it is possible to supply high-intensity ultrashort pulse laser light to a deep part of the sample, and a fluorescent image of the deep part of the sample can be obtained.

  In this case, the ultra-short pulse laser beam that passes through the acousto-optic device and the ultra-short pulse laser beam that does not pass through the acousto-optic device are alternatively or simultaneously incident on the same observation device body. On the other hand, the sample can be irradiated with the ultrashort pulse laser beam finely turned on / off or output-modulated and the high-intensity ultrashort pulse laser beam reaching the deep part of the sample without moving the sample.

Moreover, in the said invention, the said optical path selection apparatus is good also as providing the shutter arrange | positioned in a said 2nd optical path.
Since the acousto-optic device is disposed in the first optical path, the ultrashort pulse laser beam in the first optical path can be easily turned on and off by utilizing the shutter function of the acousto-optic device. On the other hand, by arranging a shutter in the second optical path, the ultrashort pulse laser beam in the second optical path can be easily turned on and off. Therefore, by selectively operating these acousto-optic devices and the shutter, it is possible to allow the ultrashort pulse laser beam to pass through the two optical paths alternatively or simultaneously. As the shutter, a mechanical shutter that is not attenuated when passing an ultrashort pulse laser beam is preferable.

Moreover, in the said invention, the said optical path selection apparatus is good also as providing the total reflection mirror arrange | positioned so that insertion / removal is possible at the branching point and confluence | merging point of a said 1st optical path and a 2nd optical path.
In this way, when the total reflection mirror is removed from the branch point of the first optical path and the second optical path, the ultrashort pulse laser beam is incident on the first optical path (or the second optical path). Can be made. On the other hand, when the total reflection mirror is inserted at the branch point between the first optical path and the second optical path, the ultrashort pulse laser light is reflected by the total reflection mirror, and thus the optical path through which the ultrashort pulse laser light passes. Can be changed to the second optical path (or the first optical path).

  Further, if the total reflection mirror is removed from the confluence of the first optical path and the second optical path, the ultrashort pulse laser beam that has passed through the first optical path (or the second optical path) is allowed to pass. The light can enter the observation apparatus main body. On the other hand, when the total reflection mirror is inserted at the junction of the first optical path and the second optical path, the ultrashort pulse laser beam is reflected by the total reflection mirror, so that the second optical path (or the first optical path) is obtained. The ultrashort pulse laser beam that has passed through the optical path) can be incident on the observation apparatus body. By using the total reflection mirror, it is possible to select an optical path alternatively without substantially attenuating the ultrashort pulse laser beam.

  The present invention also provides an observation in which a sample is irradiated with two pulse laser light sources that emit ultra-short pulse laser light and the ultra-short pulse laser light emitted from each pulse laser light source, and the fluorescence emitted from the sample is observed. An apparatus main body, and an introduction optical system that is disposed between the pulse laser light source and the observation apparatus main body, adjusts an ultrashort pulse laser beam emitted from the pulse laser light source and introduced into the observation apparatus main body, and the introduction An optical system passes a first optical path including an acousto-optic device that performs on / off or output adjustment of an ultrashort pulse laser beam emitted from one pulse laser light source, and an ultrashort pulse laser beam emitted from the other pulse laser light source. Provided is a multiphoton excitation observation apparatus that includes a second optical path that does not include an acousto-optic device to be paralleled.

  According to the present invention, ultrashort pulse laser beams that are separately transmitted through two optical paths from two pulse laser light sources are introduced into the same observation apparatus body. The ultrashort pulse laser beam emitted from the first pulse laser light source and passing through the first optical path is allowed to pass through the acoustooptic device. Thereby, the ultrashort pulse laser beam is introduced into the observation apparatus main body in a state of being turned on / off at high speed or output adjusted, and is irradiated onto the sample through the observation apparatus main body. Therefore, it is possible to perform optical stimulation or fluorescence observation of a sample by irradiating only a predetermined necessary region of the sample with an ultrashort pulse laser beam with an output adjusted accurately and appropriately.

  On the other hand, the ultrashort pulse laser light emitted from the second pulse laser light source and passing through the second optical path bypasses without passing through the acousto-optic device, is introduced into the observation device body, and is irradiated onto the sample. . Since it does not pass through the acousto-optic device, the ultrashort pulse laser light is irradiated to the sample while maintaining a high intensity without being attenuated so much. Therefore, it is possible to supply high-intensity ultrashort pulse laser light to a deep part of the sample, and a fluorescent image of the deep part of the sample can be obtained.

  In this case, the ultra-short pulse laser beam that passes through the acousto-optic device and the ultra-short pulse laser beam that does not pass through the acousto-optic device can alternatively or both be incident on the same observation device body. Without moving the sample, the sample can be irradiated with the ultrashort pulse laser beam finely turned on / off or output-modulated and the high-intensity ultrashort pulse laser beam reaching the deep part of the sample.

In the above invention, the first optical path and the second optical path of the introduction optical system are provided with an incident adjustment device that adjusts the beam diameter and beam divergence of the ultrashort pulse laser beam emitted from the pulse laser light source. It is good.
By doing so, the beam diameter and beam divergence of the ultrashort pulse laser beam propagated from the pulse laser light source are adjusted by the operation of the incidence adjusting device, and are incident on the observation device main body.

  Therefore, due to individual differences when the pulse laser light source is replaced, or when a plurality of pulse laser light sources with different wavelengths are arranged in parallel, the ultrashort pulse laser light with different wavelengths is sampled. Even if the beam waist position of the ultrashort pulse laser beam fluctuates due to the change of the wavelength when irradiating the light, or the change of the dispersion compensation amount accompanying the change of the wavelength, the observation device main body The beam waist position can be adjusted by adjusting the beam diameter and beam divergence of the ultra-short pulse laser beam when entering the laser beam, and the multiphoton excitation effect can be generated accurately at a fixed depth position of the sample. It becomes. As a result, the same position in the depth direction of the sample can be observed regardless of the variation in wavelength or the like.

In the above invention, the observation apparatus main body includes an objective lens that condenses the ultrashort pulse laser beam on the sample, and the incident adjustment apparatus has a beam diameter substantially equal to the pupil diameter at the entrance pupil position of the objective lens. The beam diameter and beam divergence may be adjusted so that the ultrashort pulse laser beam is incident.
In this way, in addition to always achieving a constant beam waist position in front of the objective lens, it is possible to make the ultrashort pulse laser beam enter the objective lens most efficiently. As a result, it is possible to improve the photon density at the beam waist position, efficiently generate a multiphoton excitation effect, and obtain a clear multiphoton fluorescence image.

In the invention described above, the incident adjustment device may include a plurality of lenses and an adjustment movement mechanism that moves at least one of the lenses in the optical axis direction.
With this configuration, it is possible to easily adjust the beam diameter and / or beam divergence by adjusting the distance between the lenses by operating the adjustment moving mechanism or by moving a plurality of lenses. Therefore, the position of the beam waist of the ultra-short pulse laser beam may vary depending on the individual difference of the pulse laser source, the wavelength of the ultra-short pulse laser beam emitted from the pulse laser source, the dispersion compensation amount due to the pre-chirp optical system, etc. Even if it fluctuates, it is possible to adjust the beam diameter reduction ratio and / or beam divergence by operating the adjustment moving mechanism, and to efficiently generate a multiphoton excitation effect at a certain beam waist position in front of the objective lens. It becomes possible.

In the above invention, the light beam diameter of the ultrashort pulse laser beam is arranged between the acoustooptic device of the first optical path and the pulse laser light source so as to be incident within an effective range of the acoustooptic device. An incident correction device may be provided.
According to the present invention, since the incident correction device is provided between the acousto-optic device and the pulse laser light source, the light flux of the ultrashort pulse laser light propagated from the pulse laser light source by the operation of the incident correction device. The diameter is reduced and incident on the acousto-optic device. As a result, the ultrashort pulse laser light incident on the acousto-optic device is completely incident without departing from the effective range set by the size of the crystal constituting the acousto-optic device. In addition, it is possible to prevent light that has not been able to enter the crystal and is removed from being mixed with ultrashort pulse laser light emitted from the crystal as stray light. As a result, it is prevented that the point spread function at the focusing position of the sample of the ultrashort pulse laser beam that is output from the acousto-optic device is modulated and the multi-photon excitation effect is efficiently generated in the sample. And a clear multiphoton fluorescence image can be obtained.

In the above invention, the incident correction device may include a plurality of lenses and a correction movement mechanism that moves at least one of the lenses in the optical axis direction.
With this configuration, the distance between lenses can be adjusted by operating the correction movement mechanism, or the reduction rate of the light beam diameter and / or the beam divergence can be adjusted by moving a plurality of lenses. Therefore, the position of the beam waist of the ultrashort pulse laser beam is changed by the individual difference of the pulse laser source, the wavelength of the ultrashort pulse laser beam emitted from the pulse laser source, the dispersion compensation amount by the dispersion compensation optical system, etc. Even if it fluctuates in direction, it is possible to adjust the beam diameter reduction ratio and / or beam divergence by operating the correction movement mechanism, and to make sure that it is incident within the effective range of the acousto-optic device and efficiently multi-photon excitation. An effect can be generated.

Moreover, in the said invention, the said incident correction apparatus is good also as being comprised by spatial filters, such as an aperture stop, a pinhole, and a slit.
With this configuration, it is possible to easily reduce the beam diameter of the ultrashort pulse laser beam incident on the incident correction device and make it incident within the effective range of the acoustooptic device.

Moreover, in the said invention, the said observation apparatus main body is good also as providing the two scanning devices which each scan the ultra-short pulse laser beam which passed two optical paths of the said introduction optical system.
By doing in this way, the ultra-short pulse laser beam which has passed through the two optical paths can be individually scanned independently by the two scanning devices. Therefore, for example, optical stimulation that precisely irradiates a desired region with ultrashort pulse laser light that has been finely turned on / off or appropriately adjusted by an acousto-optic device, and fluorescence in which the ultrashort pulse laser light is incident on a deep part of the sample. Observation can be performed simultaneously.

  The present invention also includes a pulse laser light source that emits an ultrashort pulse laser beam, and an introduction optical system that adjusts the ultrashort pulse laser beam emitted from the pulse laser light source, the introduction optical system comprising a pulse laser light source. A first optical path including an acousto-optic device that performs on / off or output adjustment of an ultrashort pulsed laser beam emitted from the light source and a second optical path that bypasses the acousto-optic device, and an optical path that switches between these optical paths Provided is a multiphoton excitation observation light source device including a selection device.

  According to the present invention, at least one of the first optical path and the second optical path is selected by the operation of the optical path selection device of the introduction optical system, and the ultrashort pulse laser beam is transmitted through the selected optical path and emitted. . When the first optical path is selected, the ultrashort pulse laser light is allowed to pass through the acousto-optic device. As a result, the ultrashort pulse laser beam is emitted in a state where the output is adjusted at high speed on / off. Therefore, it is possible to perform light stimulation or fluorescence observation of a sample by irradiating only a predetermined necessary region of the sample with an ultrashort pulse laser beam accurately and appropriately adjusted in output. Pulse laser light can be emitted.

  On the other hand, when the second optical path is selected, the ultrashort pulsed laser light is detoured and emitted without passing through the acoustooptic device. Since it does not pass through the acousto-optic device, the ultrashort pulse laser beam is emitted while being maintained at a high intensity without being attenuated so much. Accordingly, it is possible to emit a high-intensity ultrashort pulse laser beam that reaches a deep part of the sample and makes it possible to obtain a fluorescent image of the deep part of the sample.

  According to the present invention, while performing various optical stimulation and fluorescence observation by fine output modulation and on / off using an acousto-optic device, a bright and clear fluorescent image in a deep part of the sample is acquired without moving the sample. There is an effect that can be.

A multiphoton excitation observation apparatus 1 according to a first embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the multiphoton excitation observation apparatus 1 according to this embodiment includes an infrared pulse laser light source 2 capable of emitting an ultrashort pulse laser beam L and an ultrashort pulse laser beam L on a sample A. A fluorescence microscope main body (observation apparatus main body) 3 for irradiating and observing the obtained fluorescence F, and introduction optics for introducing the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 into the fluorescence microscope main body 3 System 4 is provided.
The infrared pulse laser light source 2 and the introduction optical system 4 constitute a multiphoton excitation observation light source device 5 according to this embodiment.

  The fluorescence microscope main body 3 includes a galvanometer mirror (scanning device) 6 that two-dimensionally scans an extremely short pulse laser beam L incident from the introduction optical system 4, and an ultrashort pulse laser that is scanned by the galvanometer mirror 6. A pupil projection lens 7 for condensing the light L to form an intermediate image, an imaging lens 8 for condensing and converting the ultrashort pulse laser light L that forms the intermediate image into substantially parallel light, The ultrashort pulse laser beam L converted into substantially parallel light by the image lens 8 is condensed to form an image on the sample A, while the fluorescence F emitted when the fluorescent substance is excited in the sample A is condensed. The objective lens 9, the excitation dichroic mirror 10 that branches the fluorescence F collected by the objective lens 9 from the ultrashort pulse laser light L, and the collection lens that collects the fluorescence F branched by the excitation dichroic mirror 10 11 and A barrier filter 12 that removes the ultrashort pulse laser light L component in the fluorescence F collected by the condenser lens 11, a fluorescence branching dichroic mirror 13 that branches the fluorescence F that has passed through the barrier filter 12 for each wavelength, For example, a photodetector 14 such as a photomultiplier tube (PMT) that detects fluorescence F branched for each wavelength is provided. In the figure, reference numeral 15 denotes a mirror, reference numeral 16 denotes a band pass filter, and reference numeral 17 denotes a condenser lens.

  The introduction optical system 4 includes a dispersion compensation optical system 18 that imparts negative group velocity dispersion by passing the ultrashort pulse laser light L incident from the infrared pulse laser light source 2, and the dispersion compensation optical system 18. A first optical system having a first optical path 20 and an alignment adjustment device 19 that allows the optical axis and angle of the ultrashort pulse laser beam L to be changed by making the ultrashort pulse laser beam L dispersion-compensated by. 21, a second optical system 23 having a second optical path 22, and an optical path selection device 24 that can alternatively select these two optical paths 20 and 22.

  The dispersion compensation optical system 18 includes, for example, a pair of prisms 25 and 26 and a mirror 27. As shown in FIG. 2, by adjusting the distance between the prisms 25 and 26, the dispersion compensation amount is adjusted for each wavelength, and the entire distance from the infrared pulse laser light source 2 to the objective lens 9 of the fluorescence microscope main body 3 is adjusted. The group velocity dispersion in the optical system is compensated.

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 25 and 26 and the mirror 27 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 28 denotes an ultrashort pulse laser beam reflected from the dispersion compensation optical system 18 while reflecting and directing the ultrashort pulse laser beam L emitted from the infrared pulse laser light source 2 to the dispersion compensation optical system 18. It is a mirror disposed at a position off the optical axis of the light L.

As shown in FIG. 3, the alignment adjusting device 19 includes, for example, two mirrors 19a and 19b and two mirror moving mechanisms 29 and 30 that move the mirrors 19a and 19b, respectively. The first mirror 19a 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, the ultrashort pulsed laser light L incident on the second direction Z _2, in the first direction Z ₁ and second third direction Z ₃ substantially orthogonal to the direction Z ₂ The light is deflected and emitted.

The first mirror moving mechanism 29, first the a first translating mechanism 29a for translating the first mirror first along the direction Z _1, wherein the third axis line parallel to the direction Z ₃ 1 And a first swing mechanism 29b for swinging the mirror 19a. Second mirror moving mechanism 30, first to the third and second translation mechanism 30a to the second mirror 19b along the direction Z ₃ translating, the first about an axis parallel to the direction Z ₁ And a second swing mechanism 30b that swings the second mirror 19b. The first and second translation mechanisms 29a, 30a are driven by, for example, motors 29c, 30c and ball screws 29d, 30d, and the first and second swing mechanisms 29b, 30b are configured by, for example, motors 29e, 30e. 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 29b 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 swing mechanism 30b 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 alignment adjusting device 19 is shifted in the first direction Z ₁ actuates the first translation mechanism 29a, the second direction If the image is shifted Z ₂ actuates the second translation mechanism 30a, thereby making it possible to adjust the positional deviation.

  As a result, the ultrashort pulse laser beam L emitted from the alignment adjusting device 19 can accurately determine the angle and position of the optical axis regardless of the angle and position of the optical axis of the incident ultrashort pulse laser beam L. It can be adjusted to a certain position well.

  The first optical system 21 includes an acoustooptic device 31 that performs on / off or intensity modulation of the ultrashort pulse laser beam L that has been dispersion-compensated by the dispersion compensation optical system 18, and the acoustooptic device 31 and the infrared pulse laser light source 2. And a beam shaping optical system (incident correction device) 32 that adjusts the ultrashort pulse laser light L incident on the acoustooptic device 31 and emitted from the acoustooptic device 31 and incident on the fluorescence microscope main body 3 And a microscope incident correction device 33 for adjusting the ultrashort pulsed laser beam L.

  4 to 6, the acoustooptic device 31 is, for example, an AOM, which is a crystal 34 made of tellurium dioxide, a case 35 for fixing the crystal 34, and a crystal 34 bonded thereto. A transducer 36 composed of a piezoelectric element, an acoustic wave generating electrical circuit 37 that transmits an electrical signal E input to the transducer 36, and a heat sink (heat radiating member) 38 bonded to the crystal 34 are provided. An interface material 39 made of, for example, a thermal compound such as silicone grease or an indium foil is interposed between the crystal 34 and the case 35 and between the crystal 34 and the heat sink 38. In FIG. 6, reference numeral 40 denotes a sound absorbing material that absorbs an acoustic wave emitted from the transducer 36 and propagated in the crystal 34.

An opening 41 is provided in each of the pair of side walls of the case 35 facing each other with the crystal 34 interposed therebetween, and the ultrashort pulse laser beam that has entered the ultrashort pulse laser beam L from one opening 41 and passed through the crystal 34 is provided. The light L is emitted from the other opening 41.
The heat sink 38 is made of a material having a high thermal conductivity such as aluminum or an aluminum alloy, and includes a plurality of flat fins 38a arranged at parallel intervals as shown in FIG. .

The beam shaping optical system 32 reduces the beam diameter of the ultrashort pulsed laser light L that has passed through the dispersion compensation optical system 18 and is aligned by the alignment adjusting device 19, and the crystal 34 of the acoustooptic device 31 (see FIG. 4). It is configured so that it is incident on the effective range X without leakage.
In this embodiment, the beam shaping optical system 32 includes, for example, a Galilean beam expander that combines a biconvex lens 42 and a plano-concave lens 43, a biconvex lens 42, and a plano-concave lens 43, as shown in FIG. The lens moving mechanisms 44 and 45 are movably supported in the optical axis direction. The beam shaping optical system 32 is preferably disposed between the infrared pulse laser light source 2 and the acoustooptic device 31 and closer to the acoustooptic device 31 than the center.

The microscope incident correction device 33 includes an alignment adjustment device 46 and a first collimation optical system 47.
The alignment adjustment device 46 has, for example, the same structure as the alignment adjustment device 19 shown in FIG. 3 and accurately adjusts the position and deflection of the optical axis of the ultrashort pulse laser beam L incident on the fluorescence microscope main body 3. Can be done.

  The first collimating optical system 47 can move, for example, a Kepler-type beam expander having a plurality of convex lenses 48 and at least one of the convex lenses 48 in the optical axis direction, as shown in FIG. And a lens moving mechanism 49 that supports the lens. As a result, the first collimating optical system 47 emits the ultrashort pulse laser light L so that it enters the pupil position of the objective lens 9 of the fluorescence microscope body 3 with a light beam diameter substantially equal to the pupil diameter. The beam diameter and beam divergence of the ultrashort pulse laser beam L can be adjusted.

The ultrashort pulse laser beam L has a relationship in which the beam diameter W, the wavefront curvature R, and the beam divergence D are W = R · D. These three variables W, R, and D are all functions of the wavelength λ of the ultrashort pulse laser beam L, the beam diameter W _{0 at the} beam waist position, and the optical axis direction distance from the beam waist position. Therefore, by determining any two of these three variables W, R, and D, the ultrashort pulse laser beam L can be uniquely determined. That is, adjusting the beam diameter W and the beam divergence D is equivalent to adjusting the beam diameter W and the wavefront curvature R, and adjusting the wavefront curvature R and the beam divergence D.

  The second optical system 23 includes a mechanical shutter 50 that turns on and off the ultrashort pulsed laser light L incident on the second optical path 22 and a second view having the same structure as the first collimating optical system 47. The quasi-optical system 51 is provided. The second collimating optical system 51 emits an extremely short pulse laser beam L so that the ultrashort pulse laser beam L enters the pupil position of the objective lens 9 of the fluorescence microscope main body 3 with a light beam diameter substantially equal to the pupil diameter. The beam diameter and beam divergence of the short pulse laser beam L can be adjusted. In the figure, reference numeral 52 denotes an optical path setting mirror, and reference numeral 53 denotes an ND filter.

  The optical path selection device 24 includes two total reflection mirrors 54 and 55 that are detachably disposed at a branch point and a junction point between the first optical path 20 and the second optical path 22, and the total reflection mirror 54. , 55 to move the mirror moving device (not shown). With the operation of the optical path selection device 24, as shown by a chain line in FIG. 1, the mirror moving device moves the total reflection mirror 54 to place the total reflection mirror 54 at the branch point between the first optical path 20 and the second optical path 22. When inserted, the second optical path 22 is blocked by the total reflection mirror 54, and the ultrashort pulse laser light L is reflected by the total reflection mirror 54 and directed to the first optical path 20. On the other hand, when the mirror moving device moves the total reflection mirror 54 and arranges the total reflection mirror 54 at a position away from the branch point between the first optical path 20 and the second optical path 22, the ultrashort pulse laser beam L is Further, the light beam is allowed to enter the second optical path 22 without being blocked by the total reflection mirror 54.

  When the mirror moving device moves the total reflection mirror 55 and inserts and arranges the total reflection mirror 55 at the junction of the first optical path 20 and the second optical path 22, the extremely short distance that has passed through the first optical path 20. The pulse laser beam L is reflected by the total reflection mirror 55 and directed to the fluorescence microscope main body 3. On the other hand, when the mirror moving device moves the total reflection mirror 55 and arranges the total reflection mirror 55 at a position deviated from the joining point of the first optical path 20 and the second optical path 22, it passes through the second optical path 22. The ultrashort pulse laser beam L thus made is allowed to enter the fluorescent microscope main body 3 as it is without being blocked by the total reflection mirror 55. Thereby, the second optical path 22 bypasses the first optical path 20 including the acoustooptic device 31 and enters the ultrashort pulse laser light L into the fluorescence microscope main body 3 without passing through the acoustooptic device 31. It can be made to.

  The adjusting devices (dispersion compensating optical system 18, alignment adjusting devices 19, 46, beam shaping optical system 32, first and second collimating optical systems 47, 51, acousto-optic device 31, and optical path selecting device 24). Is connected to a control device (not shown). In the control device, the wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 and the optimum operating state of each adjusting device when the wavelength is selected, for example, the dispersion compensation optical system 18 The positions and angles of the prisms 25 and 26, the movement positions of the mirror moving mechanisms 29 and 30 of the alignment adjusting devices 19 and 46, the lens moving mechanisms 44 and 45 constituting the beam shaping optical system 32 and the collimating optical systems 47 and 51, respectively. 49 is provided with a memory device (not shown) for storing the movement position 49 and the frequency of the acoustic wave input to the crystal 34 by the transducer 36 of the acoustooptic device 31 in association with each other. Since the operating states of each adjusting device may be related to each other, it is preferable that the wavelength and the operating state are set for all the adjusting devices.

  The wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is, for example, the wavelength information output from the infrared pulse laser light source L (or the ultrashort pulse laser light L input from the outside). The control device searches the memory device based on the acquired wavelength information, and automatically adjusts each adjusting device so as to be in an operating state corresponding to the wavelength. Yes.

  This allows the ultrashort pulse laser light L to pass through the effective range X of the crystal 34 of the acoustooptic device 31 without leaking, regardless of changes in the wavelength of the infrared pulse laser light source 2, or the objective lens. The multi-photon excitation effect in the sample A can be efficiently generated by making the ultrashort pulse laser beam L substantially equal to the pupil diameter incident on the pupil position 9.

The operation of the thus configured multiphoton excitation observation apparatus 1 according to this embodiment will be described below.
According to the multiphoton excitation observation apparatus 1 according to the present embodiment, when the type of observation is determined, the wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is determined, and the wavelength information is It is input to the control device from the infrared pulse laser light source 2 or from an external input device.

  The control device operates the optical path selection device 24 in accordance with the type of observation, and the mirror movement mechanism operates to cause the total reflection mirrors 54 and 55 to be the branching and joining points of the first and second optical paths 20 and 22. Insert and remove. Specifically, when the acoustooptic device 31 is used to irradiate the ultrashort pulse laser beam L in a finely adjusted region at a relatively shallow position of the sample A, the total reflection mirror 54 is used. , 55 are inserted to pass the ultrashort pulse laser beam L through the first optical path 20. On the contrary, when the ultrashort pulse laser beam L is irradiated to a relatively deep position of the sample A, the total reflection mirrors 54 and 55 are extracted and the ultrashort pulse laser beam L is passed through the second optical path 22.

Next, in the control device, the inside of the memory device is searched based on the wavelength information of the input ultrashort pulse laser beam L, and the operating state of each adjusting device stored in association with the wavelength information is acquired. .
A control apparatus controls each adjustment apparatus so that it may be in the acquired operation state.

  Specifically, first, when the wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is determined, the multiphotons of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 are determined. Since the group velocity dispersion amount in the entire optical system of the excitation type observation apparatus 1 is determined, the dispersion compensation optical system 18 is controlled to an operation state in which the dispersion compensation amount compensates for the group velocity dispersion. In the dispersion compensation optical system 18, the distance between the two prisms 25 and 26, the angle of the two prisms 25 and 26, and the position of the mirror 27 are controlled so as to be in an operating state instructed by the control device. As a result, the ultrashort pulse laser light L appropriately dispersion compensated is output from the dispersion compensation optical system 18.

  Next, when the wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is determined, the emission direction from the infrared pulse laser light source 2 and the emission direction from the dispersion compensation optical system 18 are determined. . Therefore, the control device controls the alignment adjusting device 19 so that the mirror moving mechanisms 19a and 19b obtained from the memory device are in an operating state, and the light of the ultrashort pulse laser light L incident on the beam shaping optical system 32 is obtained. The position and angle of the axis are adjusted so as to be constant regardless of the wavelength of the ultrashort pulse laser beam L.

Here, a case where the first optical path 20 is selected by the optical path selection device 24 will be described.
When the wavelength of the ultrashort pulse laser beam L emitted from the infrared pulse laser light source 2 is determined, the beam waist position and beam divergence of the ultrashort pulse laser beam L output from the dispersion compensation optical system 18 are determined. Therefore, the control device controls the beam shaping optical system 32 so that the lens moving mechanisms 44 and 45 acquired from the memory device are in operation, and adjusts the beam diameter of the emitted ultrashort pulse laser beam L.

  In this case, the beam shaping optical system 32 is configured to reduce the beam diameter of the ultrashort pulsed laser light L so that it enters the effective range X of the crystal 34 of the acoustooptic device 31. The pulse laser beam L is allowed to pass through the crystal 34 without leakage.

  When the wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is determined, the emission direction of the ultrashort pulse laser light L after modulation from the acoustooptic device 31 becomes a predetermined direction. Since the frequency of such an acoustic wave is determined, the control device controls the acousto-optic device 31 so that the acoustic wave generating electric circuit 37 acquired from the memory device is in an operating state, and the output is adjusted on / off. The ultrashort pulse laser beam L is output in a predetermined direction.

  In the acoustooptic device 31, an electrical signal E having a predetermined frequency is input to the transducer 36 by the operation of the electrical circuit 37 for generating an acoustic wave, and an ultrashort pulse laser in the crystal 34 is generated by the acoustic wave generated by the transducer 36. The acoustooptic effect of the light L occurs. Further, by changing the amplitude of the acoustic wave generated by the transducer 36, the diffraction intensity of the ultrashort pulse laser beam L in the crystal 34 is changed, and the intensity of the ultrashort pulse laser beam L emitted from the acoustooptic device 31 is changed. Is modulated at a predetermined ratio according to the amplitude of the acoustic wave.

As a result, the incident ultrashort pulse laser beam L passes through the crystal 34 and is emitted from the opening 41 of the case 35 in a state modulated to a predetermined intensity.
In this case, since the ultrashort pulse laser beam L is incident on the crystal 34 by the beam shaping optical system 32 without leaking, the ultrashort pulse laser beam L not incident on the crystal 34 and the side surface of the crystal 34 are used. There is no ultrashort pulse laser beam L that is irregularly reflected. Therefore, the ultrashort pulse laser light L does not become stray light and is not mixed into the ultrashort pulse laser light L that normally passes through the crystal 34.

  As a result, the occurrence of wavefront disturbance in the ultrashort pulse laser beam L emitted from the crystal 34 is suppressed, and the deterioration of the PSF (point spread function) 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 9, a decrease in photon density is prevented, and a multiphoton excitation effect can be efficiently generated.

  Further, by turning on and off the acoustic wave input to the crystal 34 by the transducer 36, it is possible to switch on and off the emission and blocking of the ultrashort pulse laser beam L. For example, by turning on / 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 6 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 34 of the acoustooptic device 31. At this time, since an acoustic wave is input from the transducer 36 to the crystal 34, the crystal 34 locally generates heat due to the energy.

  According to the multiphoton excitation observation apparatus 1 according to the present embodiment, the crystal 34 itself has a small heat capacity, but since the heat sink 38 having a large heat capacity is bonded to the crystal 34, the heat generated in the crystal 34 is generated by the heat sink 38. Is absorbed by. Further, since the heat sink 38 is provided with a plurality of fins 38a, the heat absorbed from the crystal 34 is efficiently dissipated to the outside air through these fins 38a. Thereby, the local temperature rise of the crystal | crystallization 34 is suppressed and the fluctuation | variation of the refractive index in the crystal | crystallization 34 is suppressed significantly.

  When the refractive index of the crystal 34 fluctuates, the diffraction of the ultrashort pulse laser beam L in the crystal 34 changes, and the emission direction from the acoustooptic device 31 changes. Therefore, when no countermeasure is taken, even if the position and deflection of the optical axis of the ultrashort pulse laser beam L are strictly adjusted by the alignment adjusting devices 19 and 46, the acoustic wave input from the transducer 36 is used. As a result, the temperature of the crystal 34 changes with time, and the emission direction from the crystal 34 shifts, and the crystal 34 is not emitted at an appropriate position.

Further, since the amplitude of the acoustic wave input to the crystal 34 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.
In particular, when the acoustooptic device 31 is an AOM, the drive power input to the transducer 36 to generate an acoustic wave is high, and the effective area in which the ultrashort pulse laser beam L is incident is small. If no measures are taken, the inside of the crystal 34 is locally heated.

According to this embodiment, since the temperature change of the crystal 34 is suppressed by the heat sink 38 having a large heat capacity, even if the driving power input to the transducer 36 to generate an acoustic wave is high, local heating in the crystal 34 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 a change in temperature of the crystal 34 over time or to adjust the alignment adjusting devices 19 and 46 each time based on the detected temperature of the crystal 34, and start observation with a simple configuration. Fluorescence observation can be performed by irradiating the appropriate irradiation range of the sample A with the ultrashort pulsed laser light L while maintaining the alignment state that is sometimes set.
In the present embodiment, as the form of the heat sink 38, the one provided with the flat fins 38a is exemplified, but instead of this, a heat sink of any other form can be adopted.

  When the wavelength of the ultrashort pulse laser beam L emitted from the infrared pulse laser light source 2 is determined, the emission direction of the ultrashort pulse laser beam L from the acoustooptic device 31 is determined. Therefore, the control device controls the alignment adjusting device 46 so as to be in the operating state of the mirror moving mechanism acquired from the memory device, and the light of the ultrashort pulse laser light L incident on the first collimating optical system 47. The position and angle of the axis are adjusted so as to be constant regardless of the wavelength of the ultrashort pulse laser beam L.

  When the wavelength of the ultrashort pulse laser beam L emitted from the infrared pulse laser light source 2 is determined, the beam waist position and beam divergence of the ultrashort pulse laser beam L output from the acoustooptic device 31 are determined. The Therefore, the control device controls the first collimating optical system 47 so that the lens moving mechanism 49 acquired from the memory device is in the operating state, and the beam diameter and beam divergence of the emitted ultrashort pulse laser beam L. Adjust.

  In the first collimation optical system 47, the light is emitted from the infrared pulse laser light source 2 and passes through the dispersion compensation optical system 18, the alignment adjustment device 19, the beam shaping optical system 32, the acoustooptic device 31, and the alignment adjustment device 46. By correcting the light beam diameter and beam divergence of the ultrashort pulse laser beam L again, the pupil position of the objective lens 9 of the fluorescent microscope body 3 is corrected so as to have a light beam diameter substantially equal to the pupil diameter. As a result, the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 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. The multiphoton excitation effect can be generated efficiently.

  The ultrashort pulse laser beam L incident on the fluorescence microscope main body 3 is two-dimensionally scanned by the galvanometer mirror 6 or positioned at a predetermined irradiation position, and the pupil projection lens 7, the imaging lens 8 and the objective lens 9. The light is condensed at a predetermined irradiation position of the sample A via Thereby, the light stimulation of the sample A can be performed. That is, according to the present embodiment, the sample A is irradiated with the ultrashort pulse laser light L finely turned on / off or output-adjusted by the acousto-optic device 31, so that a desired region of the sample can be accurately and appropriately irradiated. Can be stimulated by the ultrashort pulse laser beam L.

  On the other hand, when the type of observation is fluorescence observation, a multiphoton excitation effect is generated at the beam waist position formed in the sample A, and the generated fluorescence F is collected by the objective lens 9 to be excited by the excitation dichroic mirror 10. Is branched from the ultrashort pulse laser beam L, transmitted through the mirror 15, the condenser lens 11, and the barrier filter 12, then branched for each wavelength by the fluorescence branching dichroic mirror 13 and detected by the photodetector 14. . Thereby, the fluorescence image of the sample A can be acquired.

  In this case, the operation of the acousto-optic device 31 can irradiate only the necessary region with the ultrashort pulse laser beam L with high accuracy and appropriate intensity, and prevent the occurrence of inconvenience such as discoloration of the sample A. can do.

Next, a case where the second optical path 22 is selected by the optical path selection device 24 will be described.
The control device controls the mechanical shutter 50 to turn on and off the ultrashort pulsed laser light L emitted from the alignment adjusting device 46. When the mechanical shutter 50 is turned off and the ultrashort pulse laser light L is allowed to pass through the second optical path 22, the ultrashort pulse laser light L is adjusted in intensity by passing through the ND filter 53. The light enters the second collimating optical system 51. By using the mechanical shutter 50, the ultrashort pulse laser beam L can be transmitted without being attenuated when the mechanical shutter 50 is in the OFF state.

  When the wavelength of the ultrashort pulse laser beam L emitted from the infrared pulse laser light source 2 is determined, the beam waist position and beam divergence of the ultrashort pulse laser beam L emitted from the alignment adjusting device 19 are determined. . Therefore, the control device controls the second collimating optical system 51 so that the lens moving mechanism acquired from the memory device is in an operating state, and the beam diameter and beam divergence of the emitted ultrashort pulse laser beam L are controlled. Adjust.

  In the second collimating optical system 51, the beam diameter and beam divergence of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 and passing through the dispersion compensation optical system 18 and the alignment adjusting device 19 are corrected again. By performing the correction again, the pupil position of the objective lens 9 of the fluorescent microscope body 3 is corrected so as to have a light beam diameter substantially equal to the pupil diameter. As a result, the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 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. The multiphoton excitation effect can be generated efficiently.

  In this case, the ultrashort pulse laser beam L passing through the second optical path 22 does not pass through an optical element having a large attenuation unlike the acoustooptic device 31, and therefore the ultrashort pulse laser beam from the infrared pulse laser light source 2 is used. L can be introduced into the fluorescence microscope main body 3 with almost no attenuation. Therefore, it is possible to supply the high-intensity ultrashort pulse laser beam L to a deep part of the sample A, and a bright and clear fluorescent image of the deep part of the sample A can be obtained.

  Thus, according to the multiphoton excitation observation apparatus 1 and the multiphoton excitation observation light source apparatus 5 according to the present embodiment, the wavelength of the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is changed. Even so, the control device controls each adjusting device based on the wavelength information. 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 9 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, according to the multiphoton excitation observation device 1 and the multiphoton excitation observation light source device 5 according to the present embodiment, the first optical path 20 or the second optical path 22 is selected by the operation of the optical path selection device 24. Selected. Therefore, according to the observation method, the ultra-short pulse laser light L that has been finely turned on and off and adjusted in output by the acousto-optic device 31 according to the type of observation is incident on the sample A to perform high-precision optical stimulation and fluorescence observation. An observation method for performing fluorescence observation of a deep part of the sample A with the high-intensity ultrashort pulse laser beam L without attenuation can be selected.

  In this case, according to the multiphoton excitation observation apparatus 1 and the multiphoton excitation observation light source apparatus 5 according to the present embodiment, the above selection is performed only by inserting and removing the total reflection mirrors 54 and 55. It is not necessary to move the sample A between different types of observations. Therefore, when observing a fine area with high magnification, there is an advantage that observation can be performed without losing sight of the observation range between different types of observations.

Even if the wavelength is changed, the beam waist position of the ultrashort pulse laser beam L can be prevented from being shifted in the optical axis direction, and the same position of the sample A can be focused regardless of the wavelength.
Further, the control device controls each adjusting device according to the operating state of each adjusting device stored in the memory device in association with the wavelength. 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.

In the multi-photon excitation observation light source device 5 according to the present embodiment, the dispersion compensation optical system 18 has a structure including two prisms 25 and 26 and a mirror 27. In addition, it may be constituted by a grating pair. In this case, the amount of dispersion compensation is adjusted for each wavelength by adjusting the interval between the gratings, thereby compensating for the group velocity dispersion in the entire optical system from the infrared pulse laser light source 2 to the objective lens 9 of the fluorescence microscope body 3. can do.
In addition to the combination of the prisms 25 and 26 and the mirror 27, a dispersion compensation optical system including a combination of a plurality of prisms, a combination of a plurality of gratings, and a combination of a prism and a grating may be adopted.

  In this embodiment, the wavelength and the operating state are associated with each other in advance and stored in the memory device. Instead, the alignment state of the ultrashort pulse laser beam L is measured, and the measurement result is obtained. May be fed back to control the alignment adjusting devices 19 and 46. By doing in this way, even when a wavelength changes continuously, it can adjust so that the position and direction of the optical axis of ultrashort pulse laser beam L may become fixed.

  Further, the beam diameter of the ultrashort pulse laser beam L input to the beam shaping optical system 32 or the ultrashort pulse laser beam L output from the beam shaping optical system 32 is measured, and based on the measurement result, a predetermined beam flux is measured. The lens moving mechanism may be automatically adjusted so that the diameter and beam divergence can be achieved. Further, the movement of the beam waist position and the variation of the beam divergence caused by the change of the wavelength may be detected, and the respective adjusting devices may be automatically adjusted so that the desired beam waist position and beam divergence can be achieved, respectively.

In the multiphoton excitation observation apparatus 1 and the multiphoton excitation observation light source apparatus 5 according to the present embodiment, the Galilei type beam shaping optical system 32 and the Kepler type collimation optical systems 47 and 51 are employed. Instead, a Kepler type beam shaping optical system and a Galilean type collimation optical system may be employed.
Further, as shown in FIG. 8, the beam shaping optical system 32 may be configured by disposing a pair of concave mirrors 56 and 57 facing each other. With this configuration, the beam diameter of the ultrashort pulse laser beam L is reduced by the reflection optical system, so that the group velocity dispersion does not occur when passing through the beam shaping optical system 32, and the pulse width is increased. Can be prevented.

  Further, as shown in FIG. 9, a diaphragm 58 (or a spatial filter such as a slit or a pinhole) may be employed as the beam shaping optical system 32. By doing so, there is an advantage that the light beam diameter of the ultrashort pulse laser beam L can be reduced with a simple configuration and the adjustment is easy.

  When the beam shaping optical system 32 and the collimation optical systems 47 and 51 are constituted by two or more lenses 42, 43, and 48, the lens moving mechanisms 44, 45, and 49 have at least one lens 42 and What is necessary is just to be comprised so that 43 and 48 may be moved to an optical axis direction.

  Further, when the wavelengths that can be used by the ultrashort pulse laser beam L are limited to a finite number, as shown in FIG. 10, a lens fixed in a state adjusted in advance to a distance and angle suitable for each wavelength. A plurality of correction optical units 32A to 32C made of a group or a concave mirror and a switching mechanism 59 for switching the correction optical units 32A to 32C may be provided. Also in this case, the wavelength and the correction optical units 32A to 32C are associated with each other and stored in the memory device, and the corresponding correction optical units 32A to 32C are searched by searching the memory device based on the obtained wavelength information. If it is selected and switched by the switching mechanism 59, a highly accurate multiphoton fluorescence image can be easily acquired as described above.

A similar structure can be adopted for the collimating optical systems 47 and 51. In the beam shaping optical system 32 and the collimation optical systems 47 and 51 adopting this structure, the number of correction optical units may be two or more.
Further, the lens configurations of the collimating optical systems 47 and 51 and the beam shaping optical system 32 are not limited to the combinations as shown in the figure, and are configured by a combination of arbitrary lenses having positive or negative power. It may be a thing.

Next, a multiphoton excitation observation apparatus 100 and a multiphoton excitation observation light source apparatus 101 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 given to portions having the same configurations as those of the multiphoton excitation observation apparatus 1 and the multiphoton excitation observation light source apparatus 5 according to the first embodiment described above. Omitted.

The multiphoton excitation observation apparatus 100 and the multiphoton excitation observation light source apparatus 101 according to this embodiment are the multiphoton excitation type according to the first embodiment in the optical path selection device 103 provided in the introduction optical system 102. This is different from the observation device 1 and the multi-photon excitation observation light source device 5.
In the present embodiment, the optical path selection device 103 is a merging of the half mirror 104 disposed at the branch point between the first optical path 20 and the second optical path 22, and the first optical path 20 and the second optical path 22. A polarization beam splitter 105 disposed at a point, a rotator 106 that rotates the polarization plane of the ultrashort pulse laser light L in the first optical path 20 by 90 °, and an acoustooptic device 31 provided in the first optical system 21. And a mechanical shutter 50 provided in the second optical system 23.

  According to the multiphoton excitation observation device 100 and the multiphoton excitation observation light source device 101 according to the present embodiment configured as described above, the ultrashort pulse laser light L emitted from the infrared pulse laser light source 2 is The group velocity dispersion is compensated in advance by the dispersion compensation optical system 18, and the first optical path 20 and the second optical path are adjusted in a state in which the angle and position of the optical axis are accurately adjusted to a certain position by the alignment adjusting device 19. The light is branched at a predetermined light quantity ratio by the half mirror 104 arranged at a branching point with 22.

  The ultrashort pulse laser beam L branched to the first optical path 20 by the half mirror 104 leaks into the effective range X of the crystal 34 of the acoustooptic device 31 with the beam diameter reduced by the beam shaping optical system 32. The acoustooptic device 31 is subjected to fine on / off or output adjustment. After that, the ultrashort pulse laser beam L is corrected again by the first collimating optical system 47 with the light beam diameter and the beam divergence in a state where the position and angle of the optical axis are adjusted with accuracy again by the alignment adjusting device 46. Then, the pupil position of the objective lens 9 of the fluorescence microscope main body 3 is corrected so as to have a light beam diameter substantially equal to the pupil diameter.

  Then, the ultrashort pulsed laser light L that has passed through the first collimating optical system 47 is passed through the rotator 106, so that its polarization plane is rotated by 90 ° and is incident on the polarization beam splitter 105. The polarization beam splitter 105 is set to reflect the ultrashort pulse laser beam L whose polarization plane is rotated by 90 ° and transmit the ultrashort pulse laser beam L whose polarization plane is not rotated. Therefore, the ultrashort pulse laser beam L that has passed through the first optical path 20 is reflected by the polarization beam splitter 105 and introduced into the fluorescence microscope main body 3.

  On the other hand, the ultrashort pulsed laser light L branched to the second optical path 22 by the half mirror 104 is turned on / off by the mechanical shutter 50, the intensity is adjusted by the ND filter 53, and then by the second collimating optical system 51. The light beam diameter and the beam divergence are corrected so that the pupil position of the objective lens 9 of the fluorescence microscope main body 3 has a light beam diameter substantially equal to the pupil diameter. Then, the light passes through the polarization beam splitter 105 as it is and is introduced into the fluorescence microscope main body 3.

  Thus, according to the multiphoton excitation observation device 100 and the multiphoton excitation observation light source device 101 according to the present embodiment, the half mirror 104 always polarizes both the first optical path 20 and the second optical path 22. An ultrashort pulse laser beam which is supplied with the short pulse laser beam L and has passed through both the optical paths 20 and 22 by the acoustooptic device 31 provided in the first optical path 20 and the mechanical shutter 50 provided in the second optical path. L can be introduced into the fluorescent microscope main body 3 alternatively or simultaneously. Therefore, in addition to the effects of the first embodiment, the ultrashort pulse that has passed through the second optical path 22 while giving a light stimulus to the sample A with high accuracy by the ultrashort pulse laser light L that has passed through the first optical path 20. There is an advantage that a bright and clear fluorescent image of a deep part of the sample A can be acquired by the laser light L.

Next, a multiphoton excitation observation apparatus 110 and a multiphoton excitation observation light source apparatus 111 according to a third 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 given to portions having the same configurations as those of the multiphoton excitation observation apparatus 100 and the multiphoton excitation observation light source apparatus 101 according to the second embodiment described above, and description thereof is omitted. To do.

  In the multiphoton excitation observation apparatus 110 according to the present embodiment, the fluorescence microscope main body 112 includes two galvanometer mirrors 6 and 113, and the ultrashort pulse laser light L that has passed through the first and second optical paths 20 and 22 is generated. It is different from the multiphoton excitation observation apparatus 100 according to the second embodiment in that it is introduced into separate galvanometer mirrors 6 and 113. Further, the multi-photon excitation observation light source device 111 according to the present embodiment is different in that the ultrashort pulse laser light L that has passed through the two optical paths 20 and 22 is output separately without being combined. This is different from the multi-photon excitation observation light source device 101 according to the second embodiment. In the figure, reference numeral 114 denotes a second pupil projection lens, reference numeral 115 denotes a polarization beam splitter, and reference numeral 116 denotes an introduction optical system.

  The operation of the thus configured multiphoton excitation observation device 110 and multiphoton excitation observation light source device 111 according to this embodiment will be described below. When the ultrashort pulse laser beam L that has passed through the first optical path 20 is introduced into the fluorescence microscope main body 112, it is scanned two-dimensionally by the first galvanometer mirror 113. On the other hand, when the ultrashort pulse laser beam L that has passed through the second optical path 22 is introduced into the fluorescence microscope main body 112, it is scanned two-dimensionally by the second galvanometer mirror 6. Since the first optical path 20 is provided with a rotator 106 that rotates the plane of polarization by 90 °, two extremely short pulses that are separately two-dimensionally scanned by the first and second galvanometer mirrors 6 and 113 are provided. The laser light L is combined by the polarization beam splitter 115 provided in the fluorescence microscope main body 112 and irradiated onto the sample A through the same objective lens 9.

  Therefore, according to the multiphoton excitation observation device 110 and the multiphoton excitation observation light source device 111 according to the present embodiment, in addition to the effects of the second embodiment, an ultrashort pulse laser that has passed through the first optical path 20 While the light L is applied to the desired region of the sample A with high accuracy, the ultrashort pulse laser light L that has passed through the second optical path 22 causes the sample A disposed in a region different from the light stimulation region. There is an advantage that a bright and clear fluorescent image of a deep part can be acquired.

Next, a multiphoton excitation observation apparatus 120 and a multiphoton excitation observation light source apparatus 121 according to a fourth 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 given to portions having the same configurations as those of the multiphoton excitation observation apparatus 110 and the multiphoton excitation observation light source apparatus 111 according to the third embodiment described above, and the description is omitted. To do. Reference numeral 122 denotes an introduction optical system.

  The multiphoton excitation observation device 120 and the multiphoton excitation observation light source device 121 according to the present embodiment include two infrared pulse laser light sources 2, and each infrared pulse laser light source 2 includes a first optical path 20 and a first optical path 20. The second optical path 22 is separately connected to the multiphoton excitation observation device 110 and the multiphoton excitation observation light source device 111 according to the third embodiment. Therefore, the multiphoton excitation observation device 120 and the multiphoton excitation observation light source device 121 according to the present embodiment are the same as the dispersion compensation optical system 18 and the alignment adjustment device 19 provided in common in the third embodiment. The optical path 20 is provided separately for each optical path 20, 22, while the half mirror 104 that branches the two optical paths 20, 22 is not provided.

  The ultrashort pulse laser beam L emitted from the first infrared pulse laser light source 2 passes through the first optical path 20 and is scanned by the first galvanometer mirror 113. Further, the ultrashort pulse laser beam L emitted from the second infrared pulse laser light source 2 passes through the second optical path 22 and is scanned by the second galvanometer mirror 6.

  According to the multiphoton excitation type observation device 120 and the multiphoton excitation type observation light source device 121 according to the present embodiment configured as described above, an extremely short wavelength from the infrared pulse laser light source 2 is not branched by the half mirror 104. The pulse laser beam L can be passed through the second optical path 22 and introduced into the fluorescence microscope main body 112 as it is. Accordingly, it is possible to irradiate the sample A with the ultrashort pulse laser beam L having higher intensity. As a result, there is an effect that a bright and clear fluorescent image of a deeper part of the sample A can be acquired.

  In the present embodiment, the case where the two optical paths 20 and 22 are separately provided with the dispersion compensation optical system 18 has been described. However, the second optical system 23 that does not include the acoustooptic device 31 has a small amount of dispersion. Therefore, the dispersion compensation optical system 18 may be omitted.

1 is an overall configuration diagram schematically 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 shows typically the dispersion compensation optical system with which the multiphoton excitation type observation apparatus and multiphoton excitation type observation light source apparatus of FIG. 1 were equipped. It is a perspective view which shows the alignment adjustment apparatus with which the multiphoton excitation observation apparatus of FIG. 1 and the multiphoton excitation observation light source apparatus were equipped. It is a figure which expands and shows the beam shaping optical system and acousto-optical apparatus which are used for the multiphoton excitation observation device and the multiphoton excitation observation light source device of FIG. FIG. 2 is a perspective view illustrating an example of an acoustooptic device used in the multiphoton excitation observation apparatus and the multiphoton excitation observation light source apparatus of FIG. 1. It is a longitudinal cross-sectional view which shows the internal structure of the acoustooptic device of FIG. It is a figure which expands and shows the collimation optical system used for the multiphoton excitation observation device of FIG. 1 and the light source device for multiphoton excitation observation. It is a figure which shows the modification of the beam shaping optical system of FIG. It is a figure which shows the other modification of the beam shaping optical system of FIG. FIG. 5 is a diagram showing a modification of the beam shaping optical system of FIG. 4 and including a switching mechanism for exchanging a plurality of correction optical units adjusted in advance. It is a whole block diagram which shows typically the multiphoton excitation observation apparatus and the multiphoton excitation observation light source device which concern on the 2nd Embodiment of this invention. It is a whole block diagram which shows typically the multiphoton excitation observation apparatus and the multiphoton excitation observation light source device which concern on the 3rd Embodiment of this invention. It is a whole block diagram which shows typically the multiphoton excitation type observation apparatus and multiphoton excitation type observation light source device which concern on the 4th Embodiment of this invention.

Explanation of symbols

A Sample F Fluorescence L Ultrashort pulse laser beam 1,100,110,120 Multiphoton excitation observation device 2 Pulse laser light source 3,112 Observation device body 4,102,116,122 Introduction optical system 5,101,111,121 Multiphoton excitation light source device for observation 6,113 Galvano mirror (scanning device)
DESCRIPTION OF SYMBOLS 9 Objective lens 20 1st optical path 22 2nd optical path 24 Optical path selection apparatus 31 Acousto-optic apparatus 32 Beam shaping optical system (incident correction apparatus)
33 Microscope incidence correction device (incident adjustment device)
42, 43, 48 Lens 44, 45 Lens (correction movement mechanism)
46 Alignment adjustment device (incident adjustment device)
47 First collimation optical system (incident adjustment device)
49 Lens movement mechanism (adjustment movement mechanism)
50 Mechanical shutter (shutter)
51 Second collimation optical system (incident adjustment device)
54,55 Total reflection mirror 58 Aperture (spatial filter)

Claims (12)

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;
An introduction optical system that is disposed between the pulse laser light source and the observation apparatus main body, adjusts an ultrashort pulse laser beam emitted from the pulse laser light source and introduced into the observation apparatus main body,
The introduction optical system includes in parallel a first optical path including an acoustooptic device that performs on / off or output adjustment of an ultrashort pulse laser beam emitted from a pulsed laser light source and a second optical path that bypasses the acoustooptic device. And a multi-photon excitation observation device including an optical path selection device that allows ultrashort pulse laser light to pass through at least one of these optical paths.   The multi-photon excitation observation device according to claim 1, wherein the optical path selection device includes a shutter disposed in the second optical path.   The multi-photon excitation observation device according to claim 1, wherein the optical path selection device includes a total reflection mirror that is detachably disposed at a branch point and a junction point between the first optical path and the second optical path. Two pulsed laser light sources that emit ultrashort pulsed laser light;
An observation apparatus body for irradiating a sample with an ultrashort pulse laser beam emitted from each pulse laser light source and observing fluorescence emitted from the sample;
An introduction optical system that is disposed between the pulse laser light source and the observation apparatus main body, adjusts an ultrashort pulse laser beam emitted from the pulse laser light source and introduced into the observation apparatus main body,
The introduction optical system includes a first optical path including an acoustooptic device that performs on / off or output adjustment of an ultrashort pulse laser beam emitted from one pulse laser light source, and an ultrashort pulse laser beam emitted from the other pulse laser light source. A multi-photon excitation observation device provided in parallel with a second optical path that does not include an acousto-optic device that passes the light.   2. An incident adjusting device for adjusting a beam diameter and beam divergence of an ultrashort pulse laser beam emitted from a pulse laser light source is provided in the first optical path and the second optical path of the introduction optical system. 4. The multiphoton excitation observation apparatus according to 4. The observation apparatus body includes an objective lens that focuses an ultrashort pulse laser beam on a sample;
The multi-photon excitation type according to claim 5, wherein the incidence adjustment device adjusts the beam diameter and beam divergence so that an ultrashort pulse laser beam having a beam diameter substantially equal to the pupil diameter is incident on the entrance pupil position of the objective lens. Observation device.   The multi-photon excitation observation device according to claim 5 or 6, wherein the incident adjustment device includes a plurality of lenses and an adjustment movement mechanism that moves at least one of the lenses in the optical axis direction. .   An incident correction device is provided between the acoustooptic device in the first optical path and the pulse laser light source, and reduces the beam diameter of the ultrashort pulse laser light so that it can enter the effective range of the acoustooptic device. The multiphoton excitation observation apparatus according to any one of claims 1 to 7.   The multi-photon excitation observation device according to claim 8, wherein the incident correction device includes a plurality of lenses and a correction movement mechanism that moves at least one of the lenses in the optical axis direction.   The multi-photon excitation observation apparatus according to claim 8, wherein the incident correction device is configured by a spatial filter such as a diaphragm, a pinhole, or a slit.   The multiphoton excitation observation according to any one of claims 1 to 10, wherein the observation device main body includes two scanning devices that respectively scan ultrashort pulse laser beams that have passed through two optical paths of the introduction optical system. apparatus. A pulse laser light source that emits ultrashort pulse laser light;
An introduction optical system for adjusting the ultrashort pulse laser beam emitted from the pulse laser light source,
The introduction optical system includes in parallel a first optical path including an acoustooptic device that performs on / off or output adjustment of an ultrashort pulse laser beam emitted from a pulsed laser light source and a second optical path that bypasses the acoustooptic device. And a multi-photon excitation observation light source device including an optical path selection device that allows ultrashort pulse laser light to pass through at least one of these optical paths.

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