JP4685489B2 - 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).
Although the ultrashort pulse laser beam used in such a multi-photon excitation type observation apparatus is generally considered to be substantially parallel light, strictly speaking, the beam diameter is the most at a specific position along the optical axis direction. It has a beam waist that decreases, and also has a beam divergence that increases the diameter of the light beam at a predetermined spread angle as it moves away from the position of the beam waist. Also, based on the geometric optical concept, it is considered that the light is condensed at the focal position determined by the focal length of the lens and the diameter of the light beam becomes the smallest at that position. The beam diameter becomes the smallest at the beam waist position determined by the beam divergence.
Japanese Patent Laid-Open No. 10-512959

  However, the beam diameter, beam divergence, and beam waist position of the ultrashort pulse laser light vary depending on the individual difference of the pulse laser light source that emits the ultrashort pulse laser light and the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source. To do. When the beam waist position fluctuates in the optical axis direction, the position where the multiphoton excitation effect occurs in the sample fluctuates in the optical axis direction, and the obtained multiphoton fluorescence image differs from the image at a position different in the sample depth direction. There is an inconvenience of becoming. In particular, when irradiating a sample with ultrashort pulse laser beams of a plurality of different wavelengths and observing fluorescence excited by the ultrashort pulse laser beams of each wavelength, multiphoton excitation images at different depth positions for each wavelength are obtained. There is a problem that it will be acquired.

This invention has been made in view of the above circumstances, and even if the individual difference of the pulse laser light source that emits the ultrashort pulse laser light, the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source changes, and its object is to provide a multiphoton-excitation observation equipment which can be placed a beam waist at the same depth position of the sample.

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 body for observing fluorescence emitted by multiphoton excitation at the focal position of the lens, and a light beam diameter of an ultrashort pulse laser beam emitted between the pulse laser light source and the observation device body and emitted from the pulse laser light source And an incident adjustment device for adjusting beam divergence, wherein the pulse laser light source changes the beam divergence and / or beam waist position of the ultra-short pulse laser light by changing the wavelength of the ultra-short pulse laser light, The incidence adjusting device has the same depth of the sample according to the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source. As the beam waist is located at a position to provide a multiphoton-excitation observation apparatus for adjusting the beam divergence of the ultrashort pulsed laser beam incident on the objective lens.

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.
In this case, according to the present invention, since the incident adjustment device is provided between the pulse laser light source and the observation device main body, an extremely short pulse propagated from the pulse laser light source by the operation of the incident adjustment device. The beam diameter and beam divergence of the laser light are adjusted and incident on the observation apparatus 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 it enters the laser beam, so that the multiphoton excitation effect can be generated accurately at a certain depth position of the sample. Is possible. 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 for condensing an ultrashort pulse laser beam on a sample, and the incidence adjustment apparatus has a pole having a beam diameter substantially equal to the pupil diameter at the entrance pupil position of the objective lens. It is preferable to adjust the beam diameter and beam divergence so that the short pulse laser beam is incident.
By configuring 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 above invention, the incident adjustment device may include a plurality of lenses and a moving 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 moving mechanism, or by moving a plurality of lenses. Therefore, the position of the beam waist of the ultra-short pulse laser light is changed in the optical axis direction due to individual differences in the pulse laser light source, the wavelength of the ultra-short pulse laser light emitted from the pulse laser light source, the dispersion compensation amount by the pre-chirp optical system Even if it fluctuates, it is possible to adjust the beam diameter reduction ratio and / or beam divergence by operating the moving mechanism, and to efficiently generate a multiphoton excitation effect at a certain beam waist position in front of the objective lens. It becomes.

Moreover, in the said invention, it is good also as providing the control apparatus which operates the said moving mechanism according to the wavelength of the ultra-short pulse laser beam emitted from the said 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 when the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source fluctuates, the movement mechanism is operated by the operation of the control device, and the distance between the lenses 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.

  Further, in the above invention, the incident adjustment device includes a plurality of correction optical units each composed of a plurality of lenses adjusted for ultrashort pulse laser beams having a plurality of wavelengths, and a switching mechanism for switching the correction optical units. It is good also as providing.

  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 observation apparatus main body. As a result, the ultrashort pulse laser beam emitted from the pulse laser light source is adjusted to have a light beam diameter and / or beam divergence suitable for the wavelength by passing through the selected correction optical unit. Therefore, a beam waist can be formed at a certain position in front of the objective lens, and a multiphoton excitation effect can be efficiently generated.

  Further, in the above invention, a control device that operates a switching mechanism according to the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source is provided, and the control device includes the wavelength of the ultrashort pulse laser beam and the correction optics. A memory device that stores the unit in association with each other, and adjusts the switching mechanism so as to select a correction optical unit corresponding to each wavelength stored in the memory device according to the wavelength of the ultrashort pulse laser beam; It is good as well.

  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.

The present invention further includes a pulse laser light source that emits an ultrashort pulse laser beam, and an objective lens that focuses the ultrashort pulse laser beam emitted from the pulse laser light source onto the sample, and the objective lens in the sample An observation apparatus body for observing fluorescence emitted by multiphoton excitation at the focal position of the light source, and a beam diameter of an ultrashort pulsed laser beam emitted between the pulse laser light source and the observation apparatus body, An objective adjusting device for adjusting beam divergence, and the objective adjusting lens is arranged such that when the pulse laser light source is replaced, the beam waist is arranged at the same depth position of the sample before and after the replacement. Provided is a multiphoton excitation observation apparatus that adjusts the beam divergence of an ultrashort pulse laser beam incident on the beam .
In the above invention, the incident adjustment device may adjust the beam diameter and beam divergence so that an extremely short pulse laser beam having a beam diameter substantially equal to the pupil diameter is incident on the entrance pupil position of the objective lens. Good.

  According to the present invention, even if the individual difference of the pulse laser light source that emits the ultrashort pulse laser light, the wavelength of the ultrashort pulse laser light emitted from the pulse laser light source, the amount of dispersion in the dispersion compensation optical system, and the like change, The beam waist can be arranged at the same depth position, and the observation position can be prevented from fluctuating in the optical axis direction due to a change in wavelength or the like, and a multiphoton excitation effect can be efficiently generated. There is an effect.

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 dispersion compensation amount is adjusted for each wavelength, and the group velocity in the entire optical system from the pulse laser light source 4 to the objective lens 3a of the fluorescence microscope main body 3 is adjusted. Dispersion is compensated.

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. The mirror is arranged at a position deviated from the optical axis in the direction perpendicular to the paper surface.

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 arranged 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 arranged at a position of 100 to 1000 mm 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 Galilean beam expander similar to the beam shaping optical system 13 composed of a biconvex lens 27 and a plano-concave lens 28, a biconvex lens 27, and a flat as shown in FIG. It is comprised by the moving mechanisms 40 and 41 which each support the concave lens 28 so that a movement to an optical axis direction is possible. Due to the operation of the moving mechanisms 40 and 41, the ultrashort pulse laser beam L is emitted so that the ultrashort pulse laser beam L enters the pupil position of the objective lens 3a of the fluorescent microscope body 3 with a light beam diameter substantially equal to the pupil diameter. The beam diameter and beam divergence of the laser beam L can be adjusted. The moving mechanisms 40 and 41 are driven manually or by a motor (not shown), for example, so that the biconvex lens 27 and the plano-concave lens 28 can be arranged at desired positions, respectively.

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 fluorescent microscope main body 3 scans the ultrashort pulse laser light L in a two-dimensional manner (for example, a galvano mirror such as a proximity galvanometer mirror), condenses the scanned ultrashort pulse laser light L, and forms an intermediate image. A pupil projection lens 30 for focusing the light, an imaging lens 31 for condensing the ultrashort pulse laser light L formed on the intermediate image to make it substantially parallel light, and an ultrashort pulse laser light L made substantially parallel light. The objective lens 3a that forms an image on the sample A by light, 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, and the branched multiphoton fluorescence A condensing lens 33 for condensing F and a photomultiplier tube (PMT) 34 for detecting the condensed multiphoton fluorescence F are provided. 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 pulse laser beam 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 is suppressed. That is, at the beam waist position of 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 present embodiment, the biconvex lens 27 and the plano-concave lens 28 constituting the collimation optical system 26 are supported by the moving mechanisms so that the position of the collimating optical system 26 can be adjusted in the optical axis direction. In the case where the wavelength of the ultrashort pulse laser light L emitted from the pulse laser light source 4 can be changed, the pulse laser light source 4 includes a plurality of pulse laser light sources that emit ultrashort pulse laser light L having different wavelengths. The biconvex lens 27 and the plano-concave lens 28 can be moved in the optical axis direction by operating the moving mechanism when the dispersion compensation amount is changed by the dispersion compensation optical system in accordance with the change of the wavelength or the change of the optical system. .

  Therefore, even if the wavelength is changed, the ultrashort pulse laser beam L can be adjusted so that the pupil position of the objective lens 3a of the fluorescence microscope body 3 has a light beam diameter substantially equal to the pupil diameter. . As a result, the ultrashort pulse laser beam L can always be focused on the sample A efficiently without waste, and the multiphoton excitation effect can be efficiently 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.

In the multiphoton excitation observation device 1 and the multiphoton excitation observation light source device 2 according to the present embodiment, the Galilean beam shaping optical system 13 and the collimation optical system 26 are employed. The Kepler type beam shaping optical system 13 and collimating optical system 26 having a plurality of convex lenses may be employed.
Further, as shown in FIG. 6, 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. 7, 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, also in the beam shaping optical system 13, the moving mechanisms 40 and 41 are provided on the biconvex lens 16 and the plano-concave lens 17 constituting the beam shaping optical system 13, respectively. It is good also as moving to an axial direction. With this configuration, the beam diameter and beam divergence of the ultrashort pulse laser light L incident on the beam shaping optical system 13 by changing the wavelength of the ultrashort pulse laser light L emitted from the pulse laser light source 4 and the like. Is changed, the moving mechanism 40, 41 is operated to move at least one of the biconvex lens 16 or the plano-concave lens 17 in the optical axis direction, so that the emitted light beam diameter and the beam divergence become constant. Can be shaped.

  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.

When the beam shaping optical system 13 is composed of two or more lenses, the moving mechanism 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 acoustooptic 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, as the collimating optical system 26, as shown in FIG. 10, a structure having a plurality of correction optical units 26A, 26B, 26C and a switching mechanism 45 for switching them is adopted. Also good. Also in this case, the wavelength λ and the correction optical units 26A, 26B, and 26C 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 26A, 26B, and 26C are selected and switched by the switching mechanism 45, a highly accurate multiphoton fluorescence image can be easily acquired as described above. In addition, about the correction | amendment optical units 13A-13C and 26A-26C, the number may be two or more.

Further, the collimation optical system 26 shown in FIG. 5 is optically complicated because it is necessary to control both the beam divergence and the beam diameter by moving the lens system. Therefore, in order to configure the collimation optical system 26 of a simple optical system, an optical path adjustment mechanism 50 may be provided as shown in FIG.
The optical path adjusting mechanism 50 includes, for example, two fixed mirrors 51 and 52 and two movable mirrors 53 and 54, and adjusts the optical path length by moving the movable mirrors 53 and 54 in the direction of the arrow. As a result, the light beam diameter at the entrance pupil position of the objective lens 3a, that is, the position of the galvano mirror 29 can be adjusted.

  Therefore, the beam divergence of the ultrashort pulse laser beam L can be adjusted exclusively by the collimating optical system 26, and the beam diameter at the entrance pupil position of the objective lens 3a can be adjusted by the optical path adjusting mechanism 50. As a result, it is not necessary to adjust the beam diameter by the collimating optical system 26. For example, the collimating optical system 26 can be simply configured by only a combination of two lenses 27 and 28 'as shown in the figure. it can. The configuration of the optical path adjustment mechanism 50 is not limited to the configuration of FIG.

  The lens configurations of the collimating optical system 26 and the beam shaping optical system 13 are not limited to the combination of a biconvex lens and a 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 expands and shows the collimation optical system used for the multiphoton excitation observation apparatus 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 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. FIG. 6 is a diagram showing a collimation optical system including a switching mechanism for exchanging a plurality of correction optical units adjusted in advance, which is a modification of the collimation optical system of FIG. 5. It is a figure which shows the modification of the collimation optical system of FIG.

Explanation of symbols

A Sample F Fluorescence L Ultra-short pulse laser light λ Wavelength Y Operating state 1 Multiphoton excitation observation device 2 Multiphoton excitation light source device 3 Fluorescence microscope body (observation device body)
3a Objective lens 4 Pulsed laser light source 8 Microscope incidence adjustment device (incidence adjustment device)
26 Collimation optics (incident adjustment device)
26A, 26B, 26C Correction optical unit 27 Biconvex lens (lens)
28 Plano-concave lens (lens)
40, 41 Movement mechanism 42 Control device 43 Memory device 45 Switching mechanism

Claims (11)

A pulsed laser light source that emits an ultrashort pulsed laser beam with 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 incident adjusting device that is disposed between the pulse laser light source and the observation device main body and adjusts the beam diameter and beam divergence of the ultrashort pulse laser light emitted from the pulse laser light source;
The pulse laser light source changes the wavelength divergence and / or beam waist position of the ultrashort pulse laser light by changing the wavelength of the ultrashort pulse laser light,
The ultrashort pulse that is incident on the objective lens so that the beam waist is arranged at the same depth position of the sample according to the wavelength of the ultrashort pulse laser beam emitted from the pulse laser light source. A multiphoton excitation observation device that adjusts the beam divergence of laser light . Entering-morphism adjusting device, multi-photon excitation of claim 1 for adjusting the beam diameter and beam divergence to be incident ultrashort pulsed laser light of pupil diameter substantially equal to the beam diameter on the entrance pupil position of the objective lens Mold observation device.   The multi-photon excitation observation device according to claim 1 or 2, wherein the incident adjustment device includes a plurality of lenses and a moving mechanism that moves at least one of the lenses in the optical axis direction.   The multiphoton excitation observation apparatus according to claim 3, further comprising a control device that operates the moving mechanism in accordance with a wavelength of an ultrashort pulse laser beam emitted from the 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 said incident adjustment apparatus is provided with the several correction | amendment optical unit which consists of several lenses each adjusted with respect to the ultra-short pulse laser beam of a some wavelength, and the switching mechanism which switches this correction | amendment optical unit. Item 3. The multiphoton excitation observation apparatus according to Item 2. 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 multiphoton excitation observation apparatus according to any one of claims 1 to 3, further comprising a control device that adjusts the incident adjustment device in accordance with a wavelength of an ultrashort pulse laser beam emitted from the pulse laser light source. The control device includes a memory device that stores the wavelength of the ultrashort pulse laser beam and the operating state of the incident adjustment device in association with each other, and stores the memory device in the memory device according to the wavelength of the ultrashort pulse laser beam. The multi-photon excitation type observation device according to claim 8 , wherein the incident adjustment device is adjusted to an activated state. A pulse laser light source that emits ultrashort pulse laser light;
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 incident adjusting device that is disposed between the pulse laser light source and the observation device main body and adjusts the beam diameter and beam divergence of the ultrashort pulse laser light emitted from the pulse laser light source;
When the incident adjustment device replaces the pulse laser light source, the beam divergence of the ultrashort pulse laser light incident on the objective lens is set so that the beam waist is disposed at the same depth position of the sample before and after the replacement. Multiphoton excitation observation device to be adjusted . The multi-photon excitation type according to claim 10, 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.

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