JP4642525B2 - Multiphoton excitation observation device - Google Patents

Description

The present invention relates to multiphoton-excitation observation equipment.

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 an acousto-optic element such as a Modulator.
Japanese Patent Laid-Open No. 10-512959

  The acousto-optic element is generally a crystal made of tellurium dioxide or molybdate with a square of about 1 cm, and the effective range in which incident laser light can be modulated is relatively narrow. On the other hand, the ultrashort pulse laser beam incident on the acoustooptic device is generally considered to be substantially parallel light, but strictly speaking, a beam waist having the smallest luminous flux diameter at a specific position along the optical axis direction. And a beam divergence that increases the beam diameter at a predetermined spread angle as the distance from the position of the beam waist increases.

Therefore, if the position of the beam waist can be made coincident with the position of the acoustooptic device, the entire ultrashort pulse laser beam can be incident within the effective range of the acoustooptic device.
However, if the position of the ultrashort pulse laser beam incident on the acousto-optic element is far away from the position of the beam waist, the diameter of the light beam becomes larger and falls outside the effective range of the acousto-optic element. There is a problem that the beam shape deteriorates. 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, the position of the beam waist of the ultrashort pulse laser light greatly varies depending on individual differences of the pulse laser light source, the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source, the amount of dispersion compensation by the dispersion compensation optical system, and the like. Therefore, there is a disadvantage that it is difficult to make the position coincide with the acoustooptic device.

The present invention has been made in view of the above-described circumstances, and prevents the light flux of the ultrashort pulse laser light from the pulse laser light source from deviating from the effective range of the acousto-optic device, thereby providing a multiphoton excitation effect. efficiently generate, and its object is to provide a multiphoton-excitation observation equipment that it is possible to obtain a clear multiphoton fluorescence image.

In order to achieve the above object, the present invention provides the following means.
The present invention comprises a pulsed laser source for emitting a wavelength tunable ultrashort pulsed laser light, an objective lens which the ultrashort pulsed laser light emitted from said pulsed laser light source is focused on the sample, the objective of the sample An observation device main body for observing fluorescence emitted by multiphoton excitation at the focal position of the lens, and the on / off of the ultrashort pulse laser light emitted from the pulse laser light source, disposed between the pulse laser light source and the observation device main body, or An acoustooptic device that performs output adjustment, and an incident correction device that is disposed between the acoustooptic device 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. If the acousto-optic device and the disposed between the observation apparatus main body, quasi-optical vision to adjust the beam diameter of the ultrashort pulsed laser light emitted from the acousto-optic device With the door, the collimation optical system, the beam diameter of the ultrashort pulsed laser light, and adjusted according to the wavelength of the ultrashort pulsed laser light emitted from the pulsed laser light source, adapted to the pupil of the objective lens A multiphoton excitation observation apparatus is provided.

  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. Since the ultrashort pulse laser beam passes through the acoustooptic device before entering the observation device main body, it is turned on / off or output adjusted by the acoustooptic device.

  In this case, according to the present invention, since the incident correction device is provided between the acousto-optic device and the pulse laser light source, an extremely short pulse propagated from the pulse laser light source by the operation of the incident correction device. The beam diameter of the laser light is reduced and is incident on the acoustooptic 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, it is preferable that the incident correction device includes a plurality of lenses and a moving mechanism that moves at least one of the lenses in the optical axis direction.
In the above invention, the incident correction device may include a plurality of concave mirrors and a moving mechanism that adjusts the distance and angle between the concave mirrors.

  With this configuration, the distance between the lenses is adjusted by the operation of the moving mechanism, or a plurality of lenses are moved, or the distance and angle between the plurality of concave mirrors are adjusted, thereby reducing the reduction ratio of the light beam diameter and The beam divergence can be adjusted. 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, the reduction mechanism and / or beam divergence of the beam diameter can be adjusted by the operation of the moving mechanism, and the multi-photon excitation effect is efficiently achieved by ensuring that it is incident within the effective range of the acousto-optic device. Can be generated.

In the said invention, it is preferable that the said incident correction apparatus is provided with the control apparatus which operates a moving mechanism according to the wavelength of the ultrashort pulse laser beam emitted from a pulse laser light source.
Furthermore, in the above invention, the control device includes a memory device that stores the wavelength of the ultrashort pulse laser beam and the operating state of the moving mechanism in association with each other, and according to the wavelength of the ultrashort pulse laser beam, The moving mechanism may be adjusted to the operating state stored in the memory device.

  With this configuration, even if the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source fluctuates, the movement mechanism is activated by the operation of the control device, and the distance between lenses or the distance and angle between the concave mirrors. Is automatically adjusted. Further, by obtaining the wavelength of the ultrashort pulse laser beam by the memory device, it can be adjusted to the stored operating state of the moving mechanism, and it is not necessary to adjust while detecting the operating state of the moving mechanism. A clear multiphoton fluorescence image can be easily obtained.

In the above invention, the incident correction device switches the correction optical unit to a plurality of correction optical units each composed of a plurality of lenses or a plurality of concave mirrors, which are respectively adjusted for ultrashort pulse laser beams having a plurality of wavelengths. It is good also as providing a switching mechanism.
With this configuration, when the wavelength of the ultrashort pulse laser beam is changed, the correction optical unit corresponding to the ultrashort pulse laser beam having the set wavelength is selected by the operation of the switching mechanism, and the pulse It arrange | positions between a laser light source and an acoustooptic device. As a result, the ultrashort pulse laser light emitted from the pulse laser light source is allowed to pass through the selected correction optical unit, so that the light beam diameter is reduced at a reduction rate suitable for the wavelength, and does not protrude from the effective range. Incident to the acousto-optic device. Therefore, deterioration of PSF due to mixing of stray light or the like can be prevented, and a multiphoton excitation effect can be efficiently generated.

  In the above invention, the incident correction device includes a control device that operates a switching mechanism in accordance with the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source, and the control device includes the ultrashort pulse laser beam. And a correction optical unit are stored in association with each other, and the correction optical unit corresponding to each wavelength stored in the memory device is selected according to the wavelength of the ultrashort pulse laser beam. Alternatively, the switching mechanism may be adjusted.

  With this configuration, even if the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source is changed, the switching mechanism is operated by the operation of the control device, and the correction optics suitable for the changed wavelength is obtained. The unit is automatically selected. At this time, by obtaining the wavelength of the ultrashort pulse laser beam, the correction optical unit corresponding to the changed wavelength stored in the memory device is selected, and a clear multiphoton fluorescence image can be easily obtained.

Furthermore, 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.

Furthermore, in the said invention, it is arrange | positioned between the said pulsed laser light source and the said acoustooptic device, It equips with the alignment apparatus which adjusts the optical axis of the ultrashort pulse laser beam which injects into the said acoustooptic device, The said alignment device is The optical axis may be adjusted according to the wavelength of the ultrashort pulse laser beam.

  According to the present invention, since the ultrashort pulse laser beam that is allowed to pass through the acoustooptic device is corrected by the incident correction device so as to enter the effective range, on / off of the ultrashort pulse laser beam by the acoustooptic device is achieved. Alternatively, it is possible to prevent the stray light from being generated during output modulation, and the PSF at the condensing position of the sample of the ultrashort pulse laser light from being deteriorated. Therefore, it is possible to prevent the focus shift due to the variation of the beam waist position in the sample, and also to prevent the decrease of the photon density at the beam waist position of the sample, thereby efficiently generating the multi-photon excitation effect, and the clear multi-photon fluorescence image. There is an effect that can be obtained.

The multiphoton excitation observation apparatus according to the first embodiment of the present invention will be described below 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 ultrashort pulse laser light, a dispersion compensation optical system 5 that compensates for group velocity dispersion of the ultrashort pulse laser light L emitted from the pulse laser light source 4, and dispersion compensation. The acousto-optic device 6 for on / off or modulating the intensity of the ultra-short pulse laser beam L, and the ultra-short pulse laser beam L disposed between the acousto-optic device 6 and the pulse laser light source 4 and incident on the acousto-optic device 6 And a microscope incident correction device 8 for adjusting the ultrashort pulse laser beam L emitted from the acousto-optic device 6 and incident on the fluorescence microscope main body 3.

  The dispersion compensation optical system 5 includes, for example, a pair of prisms 9 and 10 and a mirror 11. Or you may be comprised by the grating pair. By adjusting the distance between the prisms 9 and 10 or the distance between the gratings, the optical path length for each wavelength is adjusted, and 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 body 3 is achieved. Is to compensate.

The incident correction device 7 includes an alignment adjusting device 12 and a beam shaping optical system 13.
The alignment adjusting device 12 includes, for example, two mirrors whose positions and angles can be adjusted, adjusts the position and deflection of the optical axis of the ultrashort pulse laser light L, and is in an appropriate position with respect to the acoustooptic device 6. It is configured so that it can be accurately incident at an angle. In the figure, reference numeral 14 denotes an ultrashort pulse laser beam L output from the dispersion compensation optical system 5 while reflecting and directing the ultrashort pulse laser beam L emitted from the pulse laser light source 4 to the dispersion compensation optical system 5. This mirror is arranged at a position deviated from the optical axis in the direction perpendicular to the paper.

Further, the beam shaping optical system 13 narrows the beam diameter of the ultrashort pulse laser light L that has passed through the dispersion compensation optical system 5 and is aligned by the alignment adjusting device 12, and the crystal 15 of the acoustooptic device 6 (see FIG. 2). ) To enter the effective range X without leakage.
In the present embodiment, the beam shaping optical system 13 is configured by, for example, a Galilean beam expander in which a biconvex lens 16 and a plano-concave lens 17 are combined. The beam shaping optical system 13 is preferably disposed between the pulse laser light source 4 and the acoustooptic device 6 and closer to the acoustooptic device 6 than the center. The beam shaping optical system 13 is more preferably disposed at a position 100 to 1000 mm in front of the acoustooptic device 6.

  2 to 4, the acoustooptic device 6 is, for example, an AOM, which is bonded to the crystal 15 made of tellurium dioxide, a case 18 for fixing the crystal 15, and the crystal 15. A transducer 19 made of a piezoelectric element, an acoustic wave generating electric circuit 20 for transmitting an electric signal E inputted to the transducer 19, and a heat sink (heat radiating member) 21 bonded to the crystal 15 are provided. Between the crystal 15 and the case 18, and between the crystal 15 and the heat sink 21, an interface material 22 made of, for example, a thermal compound such as silicone grease or an indium foil is interposed. In FIG. 4, reference numeral 23 denotes a sound absorbing material that absorbs an acoustic wave emitted from the transducer 19 and propagated through the crystal 15.

An opening 24 is provided in each of the pair of side walls of the case 18 facing each other with the crystal 15 interposed therebetween, and the ultrashort pulse laser beam L that has entered the ultrashort pulse laser beam L through the single opening 24 and passed through the crystal 15 is provided. The light L is emitted from the other opening 24.
The heat sink 21 is made of a material having high thermal conductivity such as aluminum or aluminum alloy, for example, and includes a plurality of plate-like fins 21a arranged at parallel intervals as shown in FIG. .

The microscope incident correction device 8 includes an alignment adjustment device 25 and a collimation optical system 26.
The alignment adjusting device 25 has the same structure as the alignment adjusting device 12, and can accurately adjust the position and deflection of the optical axis of the ultrashort pulse laser beam L incident on the fluorescence microscope main body 3. Yes.

  The collimating optical system 26 includes, for example, a biconvex lens 27 and a plano-concave lens 28, and is configured by a Galilean beam expander similar to the beam shaping optical system 13. The collimating optical system 26 has its beam diameter and beam divergence corrected so as to pass through the effective range X of the acoustooptic device 6 without omission by the beam shaping optical system 13, and the position and position of the optical axis are adjusted by the alignment adjusting device 25. The ultrashort pulse laser beam L whose deflection is adjusted is emitted so as to form a beam waist at a predetermined position (predetermined position of the sample A) in front of the objective lens 3a of the fluorescent microscope body 3. The beam diameter and beam divergence of the laser beam L are corrected. More specifically, the collimating optical system 26 adjusts the beam diameter and beam divergence so that the ultrashort pulse laser beam L has a beam diameter substantially equal to the pupil diameter at the pupil position of the objective lens 3a. It is to be corrected.

  The fluorescence microscope main body 3 includes a pair of galvanometer mirrors 29 that two-dimensionally scans the ultrashort pulse laser light L, and a pupil projection lens 30 that focuses the scanned ultrashort pulse laser light L to form an intermediate image. , An imaging lens 31 for converting the ultrashort pulse laser beam L formed with the intermediate image into a substantially parallel beam, and an objective lens 3a for focusing the ultrashort pulse laser beam L converted into a substantially parallel beam to form an image on the sample A The dichroic mirror 32 that branches the multiphoton fluorescence F emitted from the sample A and collected by the objective lens 3a from the ultrashort pulse laser light L, the condenser lens 33 that collects the branched multiphoton fluorescence F, And a photomultiplier tube (PMT) 34 for detecting the generated multiphoton fluorescence F. Reference numerals 35 and 36 are mirrors.

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, the group velocity dispersion is compensated by passing the ultrashort pulse laser light L emitted from the pulse laser light source 4 through the dispersion compensation optical system 5. Then, the light is incident on the incident correction device 7.

  In the incident correction device 7, first, the alignment adjusting device 12 adjusts the position and deflection of the optical axis of the ultrashort pulse laser beam L. Next, the light beam diameter is reduced by the beam shaping optical system 13 and enters the acoustooptic device 6 from one opening 24.

In this case, the beam shaping optical system 13 is configured to reduce the beam diameter of the ultrashort pulsed laser light L so as to be incident within the effective range X of the crystal 15 of the acoustooptic device 6. The pulse laser beam L is allowed to pass through the crystal 15 without leakage.
In the acoustooptic device 6, an electrical signal E having a predetermined frequency is input to the transducer 19 by the operation of the acoustic wave generating electric circuit 20, and an ultrashort pulse laser in the crystal 15 is generated by the acoustic wave generated by the transducer 19. The acoustooptic effect of the light L occurs. Further, by changing the amplitude of the acoustic wave generated by the transducer 19, the diffraction intensity of the ultrashort pulse laser beam L in the crystal 15 is changed, and the intensity of the ultrashort pulse laser beam L emitted from the acoustooptic device 6 is changed. Is modulated at a predetermined ratio according to the amplitude of the acoustic wave.

As a result, the incident ultrashort pulsed laser light L passes through the crystal 15 and is emitted from the opening 24 of the case 18 in a state modulated to a predetermined intensity.
In this case, since the ultrashort pulse laser beam L is incident on the crystal 15 by the beam shaping optical system 13 without leaking, the ultrashort pulse laser beam L not incident on the crystal 15 or the side surface of the crystal 15 is incident. 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 15.

  As a result, the occurrence of wavefront disturbance in the ultrashort pulse laser beam L emitted from the crystal 15 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.

  Further, by turning on and off the acoustic wave input to the crystal 15 by the transducer 19, 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 29 of the fluorescence microscope main body 3, the sample A can be brought into only 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 15 of the acoustooptic device 6. At this time, since an acoustic wave is input to the crystal 15 from the transducer 19, the crystal 15 locally generates heat due to the energy.

  According to the multiphoton excitation observation apparatus 1 according to the present embodiment, the heat capacity of the crystal 15 itself is small, but since the heat sink 21 having a large heat capacity is bonded to the crystal 15, the heat generated in the crystal 15 is the heat sink 21. Is absorbed by. Further, since the heat sink 21 is provided with a plurality of fins 21a, the heat absorbed from the crystal 15 is efficiently dissipated to the outside air through these fins 21a. Thereby, the local temperature rise of the crystal | crystallization 15 is suppressed and the fluctuation | variation of the refractive index in the crystal | crystallization 15 is suppressed significantly.

  When the refractive index of the crystal 15 varies, the diffraction of the ultrashort pulse laser beam L in the crystal 15 changes, and the emission direction from the acoustooptic device 6 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 12 and 25, the acoustic wave input from the transducer 19 is used. As a result, the temperature of the crystal 15 changes with time, and the emission direction from the crystal 15 shifts, and the crystal 15 is not emitted to an appropriate position.

Further, since the amplitude of the acoustic wave input to the crystal 15 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, in the case of AOM, no measures are taken due to the high drive power input to the transducer 19 for generating acoustic waves and the small effective area for the incidence of the ultrashort pulse laser beam L. In some cases, the inside of the crystal 15 is locally heated.

According to the present embodiment, since the temperature change of the crystal 15 is suppressed by the heat sink 21 having a large heat capacity, even if the driving power input to the transducer 19 to generate an acoustic wave is high, local heating in the crystal 15 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 15 with time, or to adjust the alignment adjusting device 25 based on the detected temperature of the crystal 15 each time. In the aligned state, fluorescence observation can be performed by irradiating the appropriate irradiation range of the sample A with the ultrashort pulse laser beam L.
In the present embodiment, the heat sink 21 is illustrated as having the flat fins 21a, but any other heat sink 21 can be employed instead.

The modulated ultrashort pulse laser beam L emitted from the acoustooptic device 6 is incident on the microscope incident correction device 8. The microscope incident correction device 8 is incident on the collimating optical system 26 with the position and deflection of the optical axis adjusted by the operation of the alignment adjustment device 25.
In the collimation optical system 26, an ultrashort pulse laser emitted from the pulse laser light source 4 and passed through the dispersion compensation optical system 5, the alignment adjustment device 12, the beam shaping optical system 13, the acoustooptic device 6, and the alignment adjustment device 25. By correcting the light beam diameter and beam divergence of the light L again, the pupil position of the objective lens 3a 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 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 multiphoton excitation observation apparatus 1 and the multiphoton excitation observation light source apparatus 2 according to the present embodiment, the Galilean beam shaping optical system 13 and the collimation optical system 26 are employed. A Kepler type beam shaping optical system 13 and a collimating optical system 26 having a plurality of biconvex lenses may be employed. Further, as shown in FIG. 5, the beam shaping optical system 13 may be configured by arranging a pair of concave mirrors 37 and 38 to face 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 13, and the pulse width is increased. Can be prevented.

  Further, as shown in FIG. 6, a diaphragm 39 (or a spatial filter such as a slit or a pinhole) may be employed as the beam shaping optical system 13. 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.

  Further, as shown in FIG. 7, the biconvex lenses 16 and 27 and the plano-concave lenses 17 and 28 constituting the beam shaping optical system 13 and / or the collimation optical system 26 are provided with moving mechanisms 40 and 41, respectively. The convex lenses 16, 27 or the plano-concave lenses 17, 28 may be moved in the optical axis direction. With this configuration, individual differences due to replacement of the pulse laser light source 4, change of the wavelength of the ultra-short pulse laser light L emitted from the pulse laser light source 4, or extremely short when switching a plurality of pulse laser light sources 4 are performed. The beam diameter and beam divergence of the ultrashort pulsed laser light L incident on the beam shaping optical system 13 or the collimating optical system 26 by changing the wavelength of the pulsed laser light L or changing the dispersion compensation amount by these changes. , The moving mechanisms 40 and 41 are operated to move at least one of the biconvex lenses 16 and 27 or the plano-concave lenses 17 and 28 in the optical axis direction. Can be shaped to be constant.

  Therefore, even if the wavelength or the like is changed, the ultrashort pulsed laser light L emitted from the beam shaping optical system 13 is incident on the effective range X of the crystal 15 of the acoustooptic device 6 without leaking, so A photon excitation effect can be generated. Even if the wavelength or the like is changed, the ultrashort pulse laser beam L emitted from the collimation optical system 26 is condensed at a predetermined position in front of the objective lens 3a, and the multiphoton excitation effect is efficiently obtained. Can be generated. Further, it is possible to prevent the beam waist position of the ultrashort pulse laser beam L having a different wavelength from being shifted in the optical axis direction, and it is possible to focus on the same position of the sample A regardless of the wavelength.

When the beam shaping optical system 13 is composed of two or more lenses, the moving mechanisms 40 and 41 may be configured to move the at least one lens in the optical axis direction.
When the beam shaping optical system 13 is constituted by two concave mirrors 37 and 38, the moving mechanisms 40 and 41 not only change the distance between the two concave mirrors 37 and 38 but also the two concave mirrors 37 and 38. It is necessary that the angle of 38 is also changed. By configuring in this way, the same effects as described above can be obtained.

Furthermore, when adjusting the positions of the lenses 16, 17, 27, 28 and the positions and angles of the concave mirrors 37, 38 by the operation of the moving mechanisms 40, 41, it may be adjusted manually. You may decide to provide the control apparatus 42 which adjusts the operating state of the moving mechanisms 40 and 41 based on the wavelength of the light L. FIG.
The example shown in FIG. 8 includes a tunable pulsed laser light source 4, and when the wavelength of the ultrashort pulsed laser light L emitted from the pulsed laser light source 4 is changed, the dispersion compensation optical system 5 and alignment adjustment are performed. This is a multi-photon excitation observation device 1 that adjusts each adjustment device such as the devices 12 and 25, the beam shaping optical system 13, the acousto-optic device 6, and the collimation optical system 26 by a control device 42.

  The control device 42 includes the wavelength λ of the ultrashort pulse laser light L emitted from the pulse laser light source 4 and the optimum operating state Y of each adjusting device when the wavelength λ is selected, for example, a dispersion compensation optical system. 5 dispersion compensation amount (space between prisms and gratings), positions and angles of mirrors constituting the alignment adjusting devices 12 and 25, lenses 16, 17, 27, constituting the beam shaping optical system 13 and collimating optical system 26, 28, a memory device 43 for storing the position and angle of the concave mirrors 37 and 38, the frequency of the acoustic wave input to the acoustooptic device 6 and the like in association with each other. Since the operating state Y of each adjusting device may be related to each other, it is preferable that the wavelength λ and the operating state Y are set for all the adjusting devices.

  The wavelength λ of the ultrashort pulse laser light L emitted from the pulse laser light source 4 is, for example, wavelength information output from the pulse laser light source L (or wavelength information of the ultrashort pulse laser light input from the outside). When acquired by λ, the control device 42 searches the memory device 43 based on the acquired wavelength information λ, and automatically adjusts each adjusting device so as to be in the operation state Y corresponding to the wavelength λ. Thereby, regardless of the change of the wavelength λ of the pulsed laser light source 4, the ultrashort pulsed laser light L can be passed through the effective range X of the crystal 15 of the acoustooptic device 6 without leaking easily or accurately, or the objective lens 3a. 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.

  When the wavelength λ that can be adopted is determined in advance, as described above, the wavelength λ and the operating state Y are stored in the memory device 43 in association with each other, so that the control can be performed most simply and accurately. it can. However, the present invention is not limited to this, and the beam diameter of the ultrashort pulse laser beam L input to the beam shaping optical system 13 or the ultrashort pulse laser beam L output from the beam shaping optical system 13 is set. Based on the measurement result, the moving mechanism 40, 41 may be automatically adjusted by the control device 42 so that the beam diameter reduction ratio and beam divergence by the beam shaping optical system 13 can be achieved.

  In addition, the adjustment by the control device 42 detects an alignment shift, a beam waist position shift, and a beam divergence variation caused by a change in the wavelength λ, and each of the adjustments can be performed so that a desired alignment, beam waist position, and beam divergence can be achieved. It may be performed on the adjusting device.

  Furthermore, when the wavelength λ that can be used by the ultrashort pulse laser beam L is limited to a finite number, as shown in FIG. 9, the wavelength λ is fixed in a state adjusted in advance to a distance and angle suitable for each wavelength λ. A plurality of correction optical units 13A, 13B, and 13C made up of lens groups or concave mirrors, and a switching mechanism 44 that switches the correction optical units 13A, 13B, and 13C may be provided. Even in this case, the wavelength λ and the correction optical units 13A, 13B, and 13C are stored in the memory device 43 in association with each other, and the memory device 43 is searched based on the obtained wavelength information λ to perform the corresponding correction. If the optical units 13A, 13B, and 13C are selected and switched by the switching mechanism 44, a highly accurate multiphoton fluorescence image can be easily acquired as described above.

Similarly, the collimating optical system 26 may have a structure having a plurality of correction optical units and a switching mechanism for switching them.
Further, the lens configurations of the collimating optical system 26 and the beam shaping optical system 13 are not limited to the combination of the biconvex lens and the plano-concave lens as shown in the figure, and any lens having a positive or negative power is used. It may be configured by a combination.

1 is an overall configuration diagram illustrating a multiphoton excitation observation apparatus and a multiphoton excitation observation light source apparatus according to an embodiment of the present invention. 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 apparatus of FIG. It is a perspective view which shows an example of the acousto-optic device used for the multiphoton excitation observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the internal structure of the acoustooptic device of FIG. 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. It is a modification of the beam shaping optical system of FIG. 2, Comprising: It is a figure which shows a beam shaping optical system provided with the moving mechanism which moves each lens to an optical axis direction. It is a whole block diagram which shows the modification of the multiphoton excitation observation apparatus of FIG. It is a modification of the beam shaping optical system of FIG. 2, and is a diagram showing a beam shaping optical system and an acousto-optic device having a switching mechanism for exchanging a plurality of correction optical units adjusted in advance.

Explanation of symbols

A Sample F Fluorescence L Ultrashort pulse laser beam X Effective range Y Operating state λ Wavelength 1 Multiphoton excitation type observation device 2 Multiphoton excitation type light source device 3 Fluorescence microscope main body (observation device main body)
4 Pulse laser light source 6 Acousto-optic device 7 Incident correction device 13 Beam shaping optical system (incident correction device)
13A, 13B, 13C Correction optical unit 16, 17 Lens 37, 38 Concave mirror 39 Diaphragm (spatial filter)
40, 41 Movement mechanism 42 Control device 43 Memory device 44 Switching mechanism

Claims (9)

A pulsed laser light source that emits an ultrashort pulsed laser beam having a variable wavelength ;
An observation apparatus main body having an objective lens for condensing an ultrashort pulse laser beam emitted from the pulse laser light source on a sample, and observing fluorescence emitted by multiphoton excitation at a focal position of the objective lens in the sample; ,
An acousto-optic device that is disposed between the pulse laser light source and the observation device main body and performs on / off or output adjustment of the ultrashort pulse laser light emitted from the pulse laser light source;
An incident correction device disposed between the acoustooptic device and the pulsed laser light source, and configured to reduce the beam diameter of the ultrashort pulse laser beam so that the beam diameter can enter the effective range of the acoustooptic device;
A collimating optical system that is arranged between the acoustooptic device and the observation device main body and adjusts the beam diameter of the ultrashort pulse laser beam emitted from the acoustooptic device ,
Multi-photon excitation in which the collimating optical system adjusts the beam diameter of the ultrashort pulse laser light according to the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source and adapts it to the pupil of the objective lens Mold observation device.   The multi-photon excitation observation device according to claim 1, wherein the incident correction device includes a plurality of lenses and a moving mechanism that moves at least one of the lenses in the optical axis direction.   The multi-photon excitation observation device according to claim 1, wherein the incident correction device includes a plurality of concave mirrors and a moving mechanism that adjusts a distance and an angle between the concave mirrors.   The multi-photon excitation observation device according to claim 2 or 3, wherein the incident correction device includes a control device that operates a moving mechanism in accordance with a wavelength of an ultrashort pulse laser beam emitted from a pulse laser light source. Wherein the controller, the provided with ultrashort pulsed laser light wavelengths, the memory device stores an association with operation states of the moving mechanism, according to the wavelength of the ultrashort pulsed laser light, is stored in the memory device The multiphoton excitation observation apparatus according to claim 4, wherein the moving mechanism is adjusted to an operating state.   The incident correction device includes a plurality of correction optical units each composed of a plurality of lenses or a plurality of concave mirrors adjusted for ultrashort pulse laser beams having a plurality of wavelengths, and a switching mechanism for switching the correction optical units. 2. The multiphoton excitation observation apparatus according to 1. Said incident compensation device, a control device for actuating the switching mechanism according to the wavelength of the ultrashort pulsed laser light emitted from the pulsed laser light source,
The control device includes a memory device that stores the wavelength of the ultrashort pulse laser beam and the correction optical unit in association with each other, and stores each of the memory devices stored in the memory device according to the wavelength of the ultrashort pulse laser beam. The multiphoton excitation observation apparatus according to claim 6, wherein the switching mechanism is adjusted so as to select a correction optical unit corresponding to a wavelength.   The multi-photon excitation observation apparatus according to claim 1, wherein the incident correction device includes a spatial filter such as a diaphragm, a pinhole, or a slit. An alignment device that is arranged between the pulse laser light source and the acoustooptic device and adjusts the optical axis of the ultrashort pulse laser beam incident on the acoustooptic device;
The multiphoton excitation observation apparatus according to claim 1, wherein the alignment apparatus adjusts an optical axis according to a wavelength of the ultrashort pulse laser beam.

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