JP4406108B2 - Multiphoton excitation laser microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a laser microscope of a type that irradiates a specimen to be observed with a pulsed laser beam, and more particularly to a multiphoton excitation scanning laser microscope for detecting a chemical reaction and fluorescence due to multiphoton absorption of a specimen.
[0002]
[Prior art]
The multiphoton excitation method is a method in which excitation performed by a normal one photon (single photon) is performed by multiphotons. For example, in the two-photon excitation method, fluorescence excitation performed at a wavelength of 400 nm (single photon) is performed at a double wavelength of 800 nm. At this wavelength of 800 nm, fluorescence excitation is performed using two photons.
[0003]
Normally, a mercury lamp or a continuous wave laser used in a fluorescence microscope has a low photon density per unit time, and therefore, a huge light intensity is required to cause a multiphoton excitation phenomenon. Furthermore, it is not suitable for practical use unless problems such as damage to the optical system and specimen are increased.
[0004]
For this reason, as the light source of the multiphoton excitation method, for example, a light source capable of oscillating a sub-picosecond pulse laser beam is used. This is because the multiphoton excitation phenomenon occurs with a probability almost proportional to the square of the unit area and the photon density per unit time. For this reason, with a sub-picosecond pulse laser beam, the probability of multiple photons is high.
[0005]
For example, Japanese Laid-Open Patent Publication No. 5-503149 discloses a laser light source that emits a sub-picosecond pulse laser beam and scanning optics for scanning a sample surface (focal plane) with the pulse laser beam emitted from the laser light source. A two-photon excitation scanning laser microscope combined with a unit is described.
[0006]
On the other hand, a sub-picosecond pulse laser beam emitted from a laser light source used for multiphoton excitation is not completely monochromatic but has a certain wavelength width correlated with the pulse width. In general, when light passes through an optical system, the shorter the wavelength, the slower the speed in the medium, and the longer the wavelength, the faster the speed in the medium. Therefore, when the pulse laser beam has a wavelength width as described above, the transit time varies depending on the wavelength when the pulse laser beam passes through the optical system. As a result, the pulse width after passing through the optical system expands in the time axis direction compared to the pulse width before entering the optical system.
[0007]
Since the probability that the multiphoton excitation phenomenon occurs depends on the photon density, the spread of the pulse width on the sample surface (focal plane) of the optical system decreases the probability that the multiphoton excitation phenomenon occurs. Therefore, it is desirable to make the pulse width as small as possible on the sample surface.
[0008]
As a general method for solving such problems, so-called pre-chirp compensation is known. Pre-chirp compensation is a method in which a pulsed laser beam is passed through a prism pair or a grating pair, so that light on the short wavelength side is emitted first, in other words, light on the long wavelength side is delayed.
[0009]
This method is described in, for example, the document `` Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation; GJ Brakenhoff, M. Muller & J. Squier; J. of Microscopy, Vol. 179, Pt. 3, September 1995, pp. 253. -260 ". Although detailed explanation is omitted, this document describes that the pulse width of the pulse laser beam on the specimen surface can be arbitrarily varied by moving and adjusting a prism pair or a graded pair used as a pre-chirp compensator. .
[0010]
[Problems to be solved by the invention]
However, when the multiphoton excitation scanning laser microscope has a plurality of objective lenses that are used alternatively, the objective lenses have different optical path lengths, and therefore the degree of correction by the pre-chirp compensator depends on each objective lens. Come different. That is, even if the pre-chirp compensator is adjusted so that the pulse width of the pulse laser beam on the sample surface is minimized in accordance with one objective lens, the pulse width on the sample surface is expanded in the case of other objective lenses. End up. Here, in this specification, the term “optical path length” means not the geometric length of the optical element, but the optical length of the optical path formed by the optical element.
[0011]
In this scanning laser microscope, similarly to the microscope observation, first, an object to be observed in a specimen is searched using a low-magnification objective lens capable of wide-range observation, and then a high-magnification objective for observing details. In general, it is used by switching to a lens. At this time, if the pulse width of the pulse laser beam on the sample surface is changed by switching the objective lens, the multiphoton excitation phenomenon cannot be caused under the optimum conditions.
[0012]
If an attempt is made to deal with this problem by adjusting the pre-chirp compensator, there arises a problem that the fluorescence emitted from the specimen fades in proportion to the time spent for the adjustment. As a countermeasure for preventing the fading of the fluorescence of the specimen, a method of moving the specimen from the observation field when adjusting the pre-chirp compensator is conceivable. In this method, the specimen must be moved again into the observation field after adjustment of the pre-chirp compensator. However, this method is not practical for observation and measurement because it is very difficult to return the specimen to the exact same position as before the movement.
[0013]
An object of the present invention is to easily observe under optimum conditions according to the optical path length of an optical member that can be selectively arranged on the optical path in a multi-photon excitation laser microscope using a pulsed laser beam having a wavelength width. Is to make it possible.
[0014]
[Means for Solving the Problems]
  According to a first aspect of the present invention, in a multi-photon excitation laser microscope, an arrangement unit for arranging a specimen to be observed and a pulse laser beam for exciting the specimen to emit fluorescence by a multi-photon excitation phenomenon are provided. A laser light source for emitting; a detector for detecting fluorescence of the specimen; and an optical system for forming an optical path of the pulse laser beam to guide the pulse laser beam from the laser light source to the specimen. And the optical system comprises:IncidentThe pulse laser beamHas the effect of emitting light in order of wavelength so that the shorter the wavelength,A pre-chirp compensator, and an optical member that can be selectively disposed on the optical path;This is caused by the change of the optical member arranged on the optical path.Optical path length of the optical pathchange ofA correction mechanism having an optical correction means for correctingNo need to adjust the action of the pre-chirp compensator according to the change of the optical memberIt is characterized by that.
[0015]
According to a second aspect of the present invention, in the multi-photon excitation laser microscope according to the first aspect, the optical system further includes a scanning mechanism for scanning the specimen with the pulse laser beam.
[0016]
According to a third aspect of the present invention, in the multiphoton excitation laser microscope according to the first aspect, the optical member is selectively placed on the optical path so as to focus the pulse laser beam on the specimen. A plurality of objective lenses that can be arranged.
[0017]
According to a fourth aspect of the present invention, in the multiphoton excitation laser microscope according to the first aspect, the optical member is selectively placed on the optical path so as to focus the pulsed laser beam on the specimen. A plurality of objective lenses that can be arranged, and a flat optical element that is removably provided between the pre-chirp compensator and the objective lens.
[0018]
According to a fifth aspect of the present invention, in the multiphoton excitation laser microscope according to the fourth aspect, the flat optical element is an optical element for observing transmitted light of a Nomarski type, and the microscope includes the pulse laser. It further comprises an optical system and a detector for detecting light transmitted through the specimen of the beam.
[0019]
According to a sixth aspect of the present invention, in the multiphoton excitation laser microscope according to the first aspect, the optical correction unit is switched in conjunction with the switching of the optical member.
[0020]
According to a seventh aspect of the present invention, in the multi-photon excitation laser microscope according to the first aspect, the optical correction unit is configured so that the pulse laser beam on the optical path is a parallel light beam and the angle of the light beam does not change. It is characterized by being arranged.
[0021]
  Eighth of the present inventionOr 7In the multi-photon excitation laser microscope of the first viewpoint, the optical correction means is selected on the optical path so that the optical path length of the optical path is constant according to the optical path length of the optical member. A plurality of optical correction elements that can be arranged in a single pattern.
[0022]
  The ninth of the present inventionOr 7In the multi-photon excitation laser microscope of the first viewpoint, the optical correction means applies a different voltage according to the optical path length of the optical member, so that the optical path length of the optical path becomes constant. The optical correction element can be adjusted as described above.
[0023]
  Tenth of the present inventionOr 7In the multi-photon excitation laser microscope of the first viewpoint, the optical correction means applies a different pressure according to the optical path length of the optical member, so that the optical path length of the optical path becomes constant. The optical correction element can be adjusted as described above.
  According to an eleventh aspect of the present invention, in the multiphoton excitation laser microscope according to the first or seventh aspect, the optical path length of the optical path is the same regardless of which optical member is used. Thus, the correction plate is alternatively arranged as a pair with the optical member.
  According to a twelfth aspect of the present invention, in the multiphoton excitation laser microscope according to the ninth or tenth aspect, the optical correction element is a parallel flat plate whose optical path length is changed by application of voltage or pressure. To do.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, components having substantially the same function and configuration are denoted by the same reference numerals, and redundant description will be given only when necessary.
[0025]
FIG. 1 is a block diagram showing a multiphoton excitation scanning laser microscope 1 according to an embodiment of the present invention.
[0026]
The microscope 1 includes a laser light source 4 for emitting a pulsed laser beam for irradiating the specimen S, and an optical system 3 for forming an optical path for guiding the laser beam between the laser light source 4 and the specimen S. Have. The optical system 3 includes a large number of optical elements including an optical element in the microscope main body 2 having a stage 15 for arranging the specimen S.
[0027]
The microscope 1 is disposed on a vibration isolation table 26 for eliminating vibration from the floor. Two mounts 27 and 28 are disposed on the vibration isolation table 26. On the gantry 27, a laser light source 4, a beam collimator 5, and a pre-chirp compensator 6 are disposed. On the gantry 28, the scanning optical unit 7 and the correction mechanism 22 are disposed. The scanning optical unit 7 and a photomultiplier 21 described later are connected to a computer 30 having an operation panel 31.
[0028]
The laser light source 4 oscillates a sub-picosecond ultrashort pulse QP at a wavelength in the near infrared region. Since the width of the wavelength width is inversely proportional to the size of the pulse width, the ultrashort pulse QP used here has a wavelength width of about several nanometers.
[0029]
A beam collimator 5 and a pre-chirp compensator 6 are disposed on the optical path of the laser beam Q from the laser light source 4. The beam collimator 5 collimates the laser beam Q emitted from the laser light source 4 into a parallel light beam. The pre-chirp compensator 6 is used to adjust in advance so as to cancel the spread of the pulse width caused by the wavelength width of the pulse when the laser beam Q passes through the optical system 3. Specifically, the pre-chirp compensator 6 is composed of optical elements 6a to 6d such as prisms, for example, and the pre-chirp compensator in order of wavelength so that the shorter the wavelength the incident collimated laser beam Q is. 6 has the effect of emitting light.
[0030]
The scanning optical unit 7 is for scanning the cross section (focal plane) S ′ of the specimen S with the laser beam Q emitted from the pre-chirp compensator 6. On the exit side of the scanning optical unit 7, the microscope body 2 is disposed via a relay lens 8 and an optical path bending unit 9. Note that the relay lens 8, the optical path bending unit 9, and the microscope main body 2 are arranged so that their optical axes coincide.
[0031]
The microscope main body 2 has a U-shaped casing 11 fixed on a vibration isolation table 26. An imaging lens 12 is disposed on the upper portion of the U-shaped casing 11, and a plurality of objective lenses having different magnifications, for example, three objective lenses 14a, 14b, and 14c are attached to the lower side of the imaging lens 12. A revolver 13 is rotatably arranged. The objective lenses 14a, 14b, and 14c are configured to be alternatively arranged on the optical axis. A stage 15 for placing the specimen S is disposed below the revolver 13. The stage 15 is attached to the housing 11 so that the position can be adjusted up and down.
[0032]
When the sample S is irradiated with the laser beam Q of the ultrashort pulse QP, the sample S emits fluorescence due to the multiphoton excitation phenomenon. The scanning optical unit 7 is provided with a dichroic mirror 16 for separating the fluorescence generated in the sample S and the laser beam Q emitted from the pre-chirp compensator 6.
[0033]
On the optical path of the laser beam Q that passes through the dichroic mirror 16, a pair of galvanometer mirrors 17 and 18 for scanning and driving the laser beam Q in directions orthogonal to each other, and the laser beam Q that has been scanned and driven are guided to the microscope body. A relay lens 8 is disposed.
[0034]
On the other hand, a condenser lens 19, a pinhole 20, and a photomultiplier 21 are arranged on the optical path for fluorescence detection separated by the dichroic mirror 16. The condensing lens 19 is used to condense the fluorescence from the specimen S into the pinhole 20. The pinhole 20 is disposed at a position conjugate with the sample S, and removes miscellaneous light other than the focal plane included in the light from the sample. In the multiphoton excitation scanning laser microscope, the multiphoton excitation phenomenon occurs only on the focal planes of the objective lenses 14a, 14b, and 14c, that is, the cross section S ′ of the specimen S. Therefore, the pinhole 20 is not necessarily provided in the scanning optical unit 7. Not necessary.
[0035]
The objective lenses 14a, 14b, and 14c have different optical path lengths. For this reason, even if the pre-chirp compensator 6 is adjusted so that the pulse width of the ultrashort pulse QP in the cross section S ′ (focal plane of the optical system 3) is adjusted to one of the objective lenses, the objective lens is In the case of switching, the pulse width in the cross section S ′ may be widened. In order to cope with this problem, the optical path length of the optical system 3 is corrected so that the pulse width in the cross section S ′ is constant and minimum when any of the objective lenses 14a, 14b, and 14c is selected. A correction mechanism 22 is provided. As described above, in the present specification, the term “optical path length” means not the geometric length of the optical element but the optical length of the optical path formed by the optical element.
[0036]
Specifically, in the present embodiment, the correction mechanism 22 is disposed in a common housing with the scanning optical unit 7. The correction mechanism 22 includes correction plates 23a, 23b, and 23c that can be alternatively arranged on the optical path of the laser beam Q collimated to a parallel light beam. The correction plates 23a, 23b, and 23c are made of the same glass material having an optical path length set corresponding to the objective lenses 14a, 14b, and 14c, respectively. The optical path length from the laser light source 4 to the cross section S ′ (focal plane of the optical system 3) is the same regardless of which of the pair of the corresponding objective lenses 14a, 14b, 14c and the correction plates 23a, 23b, 23c is used. Is set to be
[0037]
As shown in FIG. 2, the correction plates 23a, 23b, and 23c are attached to a rotatable turret 24. By rotating the turret 24, the correction plates 23a, 23b, and 23c can be alternatively arranged on the optical axis of the optical system 3.
[0038]
Next, the operation of the multiphoton excitation scanning laser microscope shown in FIG. 1 will be described.
[0039]
When a laser beam Q having an extremely short pulse QP is emitted from the laser light source 4, the pulse laser beam Q is first converted into a parallel beam by the beam collimator 5. Next, the laser beam Q is incident on the pre-chirp compensator 6, adjusted so that the pulse width of the laser beam Q is minimized in the cross section S ′, and introduced into the scanning optical unit 7.
[0040]
Next, the laser beam Q introduced into the scanning optical unit 7 is applied to any one of the correction plates 23a, 23, 23c corresponding to the objective lenses 14a, 14b, 14c, for example, the correction plate 23a corresponding to the objective lens 14a. And is shaped so that the pulse width of the laser beam Q in the cross section S ′ is constant corresponding to the objective lens 14a.
[0041]
Next, the laser beam Q is incident on the dichroic mirror 16 of the scanning optical unit 7. The laser beam Q that has passed through the dichroic mirror 16 is reflected by the pair of galvanometer mirrors 17 and 18, and then enters the optical system of the microscope body 2 through the relay lens 8 and the optical path bending unit 9.
[0042]
The laser beam Q incident on the microscope body 2 is converted by the imaging lens 12 into a light beam diameter that satisfies one of the objective lenses 14a, 14b, and 14c, for example, the pupil diameter of the objective lens 14a, and is applied to the objective lens 14a. be introduced. Next, the laser beam Q thus introduced into the objective lens 14a is condensed by the objective lens 14a onto the cross section S ′ placed on the stage 15.
[0043]
The specimen S is partially excited by the two-photon excitation phenomenon in the cross section S ′ by irradiation with the pulsed laser beam Q. Thereby, fluorescence corresponding to the stained fluorescent dye is emitted from the cross section S ′ of the specimen S. This fluorescence is again taken into the objective lens 14a, enters the dichroic mirror 16 through the imaging lens 12, the optical path bending unit 9, the relay lens 8, and the pair of galvanometer mirrors 18 and 17, and is separated from the pulse laser beam Q.
[0044]
The fluorescent light is reflected by the dichroic mirror 16 and collected by the condenser lens 19 in the pinhole 20. Only the fluorescence that has passed through the pinhole 20 enters the photomultiplier 21. The photomultiplier 21 receives fluorescence and outputs an electrical signal corresponding to the amount of light to the computer 30.
[0045]
Along with the above operation, the pair of galvanometer mirrors 17 and 18 of the scanning optical unit 7 are driven in the XY directions, and the cross section S ′ is scanned in the XY directions by the laser beam Q. The computer 30 can construct a two-dimensional image of the cross section S ′ of the specimen S by synchronizing the output signal of the photomultiplier 21 obtained by this scanning with the scanning drive of the galvanometer mirrors 17 and 18.
[0046]
In this way, when obtaining a two-dimensional image of the specimen S, the revolver 13 may be rotated in accordance with the application, and the objective lens 14a may be switched to the objective lens 14c (or objective lens 14b). In this case, the correction plate 23c corresponding to the objective lens 14c is arranged on the optical path of the pulsed laser beam Q collimated into a parallel light beam by rotating the turret 24 of the correction mechanism 22 almost simultaneously with the switching of the objective lens.
[0047]
As described above, the correction plates 23a, 23b, and 23c are set so that the pulse width in the cross section S 'is constant when combined with the corresponding objective lenses 14a, 14b, and 14c. Therefore, for example, if the pulse width of the laser beam Q in the cross section S ′ is adjusted to the minimum in the initial stage (at the time of apparatus setup) using the objective lens 14a and the correction plate 23a, then other objective lenses and correction plates are used. Even when the pair is used, adjustment of the pre-chirp compensator 6 is not necessary. In other words, the pulse width in the cross section S ′ can always be constant and minimized regardless of which pair of the corresponding objective lenses 14a, 14b, 14c and the correction plates 23a, 23b, 23c is used.
[0048]
Therefore, according to the multi-photon excitation scanning laser microscope shown in FIG. 1, the multi-photon excitation phenomenon can be caused under optimum conditions when any of the objective lenses 14a, 14b, and 14c is switched. In addition, since observation can be started in an optimum state instantly, the fading of fluorescence can be minimized and work efficiency is improved. Further, since the correction plates 23a, 23b, and 23c are inserted on the optical path of the laser beam Q collimated into parallel light beams, the correction plates 23a, 23b, and 23c have the performance of the objective lenses 14a, 14b, and 14c. The optical performance of the entire system can be easily maintained without deterioration.
[0049]
The correction plates 23a, 23b, and 23c can be switched in conjunction with switching of the objective lenses 14a, 14b, and 14c electrically or mechanically in addition to switching manually. In this case, the operation of the apparatus is simplified, work efficiency is improved, and erroneous insertion of the correction plates 23a, 23b, and 23c does not occur. For example, a mechanism as shown by a broken line in FIG.
[0050]
That is, in this interlocking mechanism, the revolver 13 and the turret 24 are driven by the step motors 32 and 33, respectively. Also, encoders 34 and 35 are connected to the rotation shafts of the step motors 32 and 33, respectively. These members 32 to 35 are connected to the computer 30. The computer 30 is provided with an operation panel 31 for selectively inputting which objective lens 14a, 14b, or 14c is used. Thereby, based on the input from the operation panel 31, the objective lenses 14a, 14b, 14c and the correction plates 23a, 23b, 23c can be automatically switched in conjunction with each other.
[0051]
In addition to the switching mechanism described above, for example, the position and magnification of the objective lens attached to the revolver are set in advance, and when the revolver is manually rotated, the correction plate corresponding to the switched objective lens is determined and corrected. You may make it switch a board.
[0052]
The correction plates 23a, 23b, and 23c are desirably arranged on the optical path at a position where the laser beam Q is a parallel light beam and the angle of the light beam does not change, for example, a position between the laser light source 4 and the dichroic mirror 16. For this reason, in addition to the position between the pre-chirp compensator 6 and the scanning optical unit 7 (position shown in FIG. 1), for example, it can be installed between the beam collimator 5 and the pre-chirp compensator 6.
[0053]
Further, as shown in FIG. 3, the correction plates 23a, 23b, and 23c can be incorporated in the revolver 13 or the objective lens if the optical systems of the objective lenses 14a, 14b, and 14c are infinite systems. Alternatively, a mechanically interlocking mechanism can be substituted. According to this configuration, the device configuration becomes simple and inexpensive, and there is no fear of malfunction.
[0054]
In the above embodiment, the same glass material is used for the correction plate and the optical path length is changed by changing the plate thickness. However, the present invention is not limited to this. For example, the optical path length can be set by changing the refractive index by changing the glass material of the correction plate formed to have the same thickness. In this case, the correction plates 23a, 23b, and 23c have the same geometric dimensions, so that components to be attached to the correction mechanism 22 can be shared.
[0055]
4 to 6 are schematic views showing correction mechanisms 41, 46, and 51 according to a modified example of the correction mechanism 22.
[0056]
A correction mechanism 41 shown in FIG. 4 is an example in which correction plates 23a, 23b, and 23c are attached to rectangular blocks 42, respectively. The rectangular block 42 is detachably fixed to a rectangular rotor 43. In this case, the correction plates 23 a, 23 b, and 23 c can be alternatively arranged on the optical axis of the optical system 3 by the rotation of the rotor 43.
[0057]
The correction mechanism 46 shown in FIG. 5 is an example in which the correction plates 23a, 23b, and 23c are attached to a slider 47 that can slide. In this case, the correction plates 23 a, 23 b and 23 c can be alternatively arranged on the optical axis of the optical system 3 by the linear operation of the slider 47.
[0058]
In the correction mechanism 51 shown in FIG. 6, a single optical correction element 52 that can selectively provide different optical path lengths on the optical path by an electrical or physical external action by the drive unit 53 is used. . As the optical correction element 52, a parallel flat plate (EOD element) whose optical path length is changed by application of voltage, or a parallel flat plate whose optical path length is changed by utilizing photoelasticity by application of pressure can be used. .
[0059]
FIG. 7 is a schematic view showing a modified example when a Nomarski-type transmitted light observation function is added to the microscope 1 shown in FIG.
[0060]
In this modification, a birefringent element 62 typified by a polarizer 61 and a Nomarski prism is attached to a slider (not shown), and is located inside the scanning optical unit 7 and closer to the pre-chirp compensator 6 side than the dichroic mirror 16. It can be inserted and removed integrally on the optical path. In this case, when only the fluorescence is detected by the photomultiplier 21, if the polarizer 61 and the birefringent element 62 are removed from the optical path, the optical path length changes. In order to cope with this problem, a correction plate 23 d corresponding to the optical path lengths of the polarizer 61 and the birefringent element 62 is attached to a slider 24 a that is separate from the turret 24 and forms a part of the correction mechanism 22. The correction plate 23d is inserted into the optical path in conjunction with the polarizer 61 and the birefringent element 62 being removed from the optical path.
[0061]
A birefringent element (Nomarski prism) 65 and a polarizer 66 are attached to the lower part of the housing 11 of the microscope main body 2 so as to be positioned below the opening 15 a of the stage 15. Further, a mirror 67 and a detector 68 for reflecting and detecting transmitted light that has passed through the polarizer 66 are attached to the housing 11 of the microscope body 2.
[0062]
The polarizer 61, the birefringent elements 62 and 65, and the polarizer 66 are phase information that cannot be detected by the eyes, such as a colorless transparent object having a slightly different refractive index and a surface of an opaque object having a slight step. Visualize with interference of polarization color by polarization interference. Therefore, in addition to the information obtained from the fluorescence detected by the photomultiplier 21 described above, the structure of the specimen S can be captured three-dimensionally from the information obtained from the transmitted light detected by the detector 68.
[0063]
If only the fluorescence detection by the photomultiplier 21 is required, the upper polarizer 61 and the birefringent element 62 may be removed from the optical path. In conjunction with this, a correction plate 23d corresponding to the optical path length of the polarizer 61 and the birefringent element 62 is inserted into the optical path. Thereby, even in a microscope that switches between the Nomarski-type transmitted light observation function and the fluorescence detection by the photomultiplier 21, the pulse width in the cross section S ′ can always be constant and minimized. Instead of the illustrated configuration, a correction plate (not shown) that takes into account the optical path lengths of the polarizer 61 and the birefringent element 62 is disposed on the turret 24 in addition to the correction plates 23a, 23b, and 23c. May be.
[0064]
In the microscope shown in FIG. 7, the correction plate 23d corresponding to the flat polarizer 61 and the birefringent element 62 for performing Nomarski-type transmitted light observation is described, but the present invention is not limited to this. Instead, other flat optical elements can be applied when a correction plate corresponding to this is provided.
[0065]
In the above-described embodiment and modification, the scanning optical unit having a pair of galvanometer mirrors 17 and 18 for running the laser beam Q as a scanning mechanism for scanning the sample S with the pulse laser beam Q is used. 7 is used. However, since scanning of the sample S can be achieved by relatively moving the sample S and the laser beam Q, various mechanisms can be employed for this scanning. For example, a scanning mechanism that stops the laser beam Q and drives the stage 15 of the microscope main body 2 for scanning may be used.
[0066]
【The invention's effect】
As described above, according to the present invention, in a multiphoton excitation laser microscope that uses a pulse laser beam and has a plurality of objective lenses, according to the optical path length of an optical member that can be selectively disposed on the optical path, Observation can be easily performed under optimum conditions.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing a multiphoton excitation scanning laser microscope according to an embodiment of the present invention.
FIG. 2 is a schematic plan view showing an optical path length correction mechanism used in the microscope shown in FIG.
FIG. 3 is a schematic side view showing a modified example of an optical path length correction mechanism.
FIG. 4 is a schematic plan view showing another modification of the optical path length correction mechanism.
FIG. 5 is a schematic plan view showing still another modified example of the optical path length correction mechanism.
FIG. 6 is a schematic side view showing still another modification of the optical path length correction mechanism.
7 is a schematic diagram showing a modification example when a Nomarski-type transmitted light observation function is added to the microscope shown in FIG. 1;
[Explanation of symbols]
1: Microscope,
2: Microscope body
3: Optical system
4: Laser light source
5: Beam collimator
6: Pre-chirp compensator
7: Scanning optical unit
12: Imaging lens
13: Revolver
14a, 14b, 14c: objective lens
15: Stage
16: Dichroic mirror
17, 18: Galvano mirror
19: Condensing lens
20: Pinhole
21: Photomultiplier
22, 41, 46, 51: Correction mechanism
23a, 23b, 23c, 23d: correction plates
24: Turret
30: Computer
31: Operation panel
32, 33: Step motor
34, 35: Encoder
52: Optical correction element
61: Polarizer
62, 65: Birefringent elements
66: Polarizer
67: Mirror
68: Detector
S: Specimen

Claims (12)

A placement section for placing the specimen to be observed;
A laser light source for emitting a pulsed laser beam for exciting the specimen to emit fluorescence by a multiphoton excitation phenomenon;
A detector for detecting fluorescence of the specimen;
An optical system for forming an optical path of the pulsed laser beam to guide the pulsed laser beam from the laser light source to the specimen;
The optical system comprises:
A pre-chirp compensator having the effect of emitting the incident pulsed laser beam in order of wavelength so that the shorter the wavelength is,
An optical member that can be selectively disposed on the optical path;
Have a, a correction mechanism having a optical correction means for correcting the variation of the optical path length of the optical path caused by a change of the optical members disposed in the optical path,
A multi-photon excitation laser microscope characterized in that adjustment of the action of the pre-chirp compensator according to the change of the optical member is unnecessary .   The multi-photon excitation laser microscope according to claim 1, wherein the optical system further includes a scanning mechanism for scanning the specimen with the pulse laser beam.   2. The multi-purpose lens according to claim 1, wherein the optical member is a plurality of objective lenses that can be alternatively arranged on the optical path so as to focus the pulse laser beam on the specimen. Photon excitation laser microscope.   The optical member includes a plurality of objective lenses that can be alternatively arranged on the optical path so as to focus the pulsed laser beam on the specimen, and between the pre-chirp compensator and the objective lens. The multi-photon excitation laser microscope according to claim 1, wherein the multi-photon excitation laser microscope is a flat optical element detachably provided.   The flat optical element is a Nomarski-type optical element for observing transmitted light, and the microscope further includes an optical system and a detector for detecting transmitted light transmitted through the specimen of the pulse laser beam. The multi-photon excitation laser microscope according to claim 4, wherein the multi-photon excitation laser microscope is provided.   The multi-photon excitation laser microscope according to claim 1, wherein the optical correction unit is switched in conjunction with switching of the optical member.   2. The multi-photon excitation laser microscope according to claim 1, wherein the optical correction unit is arranged at a position where the pulse laser beam on the optical path is a parallel light beam and the angle of the light beam does not change. The optical correction means is a plurality of optical correction elements that can be alternatively arranged on the optical path such that the optical path length of the optical path is constant according to the optical path length of the optical member. The multiphoton excitation laser microscope according to claim 1 or 7 . The optical correction means is an optical correction element that can be adjusted so that the optical path length of the optical path is constant by applying different voltages according to the optical path length of the optical member. The multiphoton excitation laser microscope according to claim 1 or 7 . The optical correction means is an optical correction element that can be adjusted so that the optical path length of the optical path is constant by applying different pressures according to the optical path length of the optical member. The multiphoton excitation laser microscope according to claim 1 or 7 .   The optical correction means is a correction plate that is alternatively arranged as a pair with the optical member so that the optical path length of the optical path is the same regardless of which optical member is used. The multiphoton excitation laser microscope according to claim 1 or 7.   11. The multi-photon excitation laser microscope according to claim 9, wherein the optical correction element is a parallel flat plate whose optical path length is changed by application of voltage or pressure.

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