JP2008281708A - Laser scanning type microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform more adequate fluorescence observation by relieving the burden of a user. <P>SOLUTION: A laser scanning type microscope 1 is equipped with: a multiphoton excitation illumination optical system 3 which includes a pulse laser light source 2 emitting ultrashort pulse laser light; a one-photon excitation illumination optical system 5 which includes a continuous laser light source 4 emitting continuous laser light; a scanning optical system 6 which two-dimensionally scans the laser light emitted from these illumination optical systems 3 and 5 and irradiates a specimen A with the laser light; a multiphoton fluorescence observation optical system 8 and a confocal observation optical system 9 which detect fluorescence generated from the specimen A; an input section 11 for inputting fluorochrome and an observation method; a storage section 10 which stores the setting of the illumination optical systems 3 and 5 and the observation optical systems 8 and 9, in association with the fluorochrome and the observation method; and a setting control section 12 which sets the illumination optical systems 3 and 5 and the observation optical systems 8 and 9 to the setting stored in the storage section 10, in accordance with the fluorochrome and the observation method inputted by the input section 11. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a laser scanning microscope.

Conventionally, a laser scanning microscope that automatically selects a wavelength of laser light by selecting a fluorescent dye is known (see, for example, Patent Document 1).
According to this laser scanning microscope, there is an advantage that even a user who has no knowledge of the relationship between the excitation wavelength with respect to the fluorescent dye and the adjustment of the oscillation wavelength of the laser beam can be used easily.

JP 2003-15045 A

  However, in the laser scanning microscope, when a fluorescent dye is selected, the item to be adjusted is not only the wavelength selection of the laser beam, but various settings need to be adjusted. In particular, in a laser scanning microscope that can perform both confocal observation performed by irradiating continuous laser light and multiphoton excitation observation performed by irradiating ultrashort pulse laser light, the adjustment is complicated. Thus, there is a problem that proper observation cannot be performed simply by automatically adjusting the wavelength of the laser beam.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a laser scanning microscope that can further reduce the burden on the user and perform more appropriate fluorescence observation.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a multi-photon excitation illumination optical system including a pulse laser light source that emits ultrashort pulse laser light, a one-photon excitation illumination optical system including a continuous laser light source that emits continuous laser light, and emission from these illumination optical systems. Scanning optical system that scans the laser beam in two dimensions and irradiates the specimen, a multiphoton fluorescence observation optical system that detects fluorescence generated from the specimen by irradiating the specimen with laser light, and a confocal system An input unit for inputting an observation optical system, a fluorescent dye and an observation method, a storage unit for storing settings of the illumination optical system and the observation optical system in association with the fluorescent dye and the observation method, and an input by the input unit A laser scanning microscope comprising: a setting controller configured to set the illumination optical system and the observation optical system in settings stored in the storage unit corresponding to the fluorescent dye and the observation method performed To provide.

  According to the present invention, the setting of the illumination optical system and the observation optical system corresponding to the fluorescent dye and the observation method input from the input unit is read from the storage unit by the operation of the setting control unit, and the illumination optical system and the observation optical system are read out. The system is set. As the setting of the illumination optical system, the wavelength of the laser light emitted from the laser light source, in the multi-photon excitation observation optical system, the alignment optical system, the wavefront adjustment optical system, the beam system adjustment optical system, the negative chirp, the light control means, etc. Setting etc. are mentioned. As for the setting of the observation optical system, in the confocal observation optical system, the wavelength characteristics of the excitation dichroic mirror that separates the detected fluorescence and the spectral dichroic mirror that separates the fluorescence for each wavelength, the aperture diameter of the confocal pinhole The characteristics of the barrier filter, the detection circuit setting of the photodetector, and the like.

In the above invention, the confocal observation optical system includes a confocal pinhole capable of changing an aperture, and when the multiphoton fluorescence observation is selected as an observation method from the input unit, the multiphoton fluorescence observation optical The fluorescence generated from the specimen may be detected by a system and a confocal observation optical system in which the confocal pinhole is opened.
By doing in this way, multiphoton fluorescence observation can be performed using a confocal observation optical system. Thus, when detecting multiphoton fluorescence having a number of wavelengths exceeding the detection channel of the multiphoton fluorescence observation optical system, the detection channel of the confocal observation optical system can be assigned and detected.

In the above invention, the storage unit stores excitation efficiency profile information with respect to the excitation wavelength of the fluorescent dye, and when the plurality of fluorescent dyes having overlapping excitation wavelengths are input from the input unit, the storage is performed. The wavelength of the laser beam may be set to a wavelength at which the profile information of each fluorescent dye stored in the section intersects.
By doing so, the plurality of fluorescent dyes can be excited most efficiently, and the plurality of fluorescent dyes can be simultaneously excited and observed by laser light of one wavelength.

  According to the present invention, it is possible to further reduce the burden on the user and perform more appropriate fluorescence observation.

A laser scanning microscope 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the laser scanning microscope 1 according to the present embodiment includes a multi-photon excitation illumination including a pulse laser light source 2 that emits an ultrashort pulse laser beam (hereinafter referred to as a pulse laser beam or a laser beam). Laser light emitted from the optical system 3, a one-photon excitation illumination optical system 5 including a continuous laser light source 4 that emits continuous laser light, and the multi-photon excitation illumination optical system 3 or the one-photon excitation illumination optical system 5. Is generated from the specimen S by being irradiated with the laser light, the scanning optical system 6 for two-dimensionally scanning, the objective lens 7 for condensing the laser light scanned by the scanning optical system 6 on the specimen S, and the like. A multiphoton fluorescence observation optical system 8 and a confocal observation optical system 9 for detecting fluorescence, a storage unit 10 for storing various settings, an input unit 11 for inputting the type and observation method of a fluorescent dye, and the optical systems 3 , 8 And a control unit 12 for controlling the 9.

  The multi-photon excitation illumination optical system 3 performs a pulse laser light source 2, an alignment mirror 13 that adjusts the optical axis of the pulse laser light emitted from the pulse laser light source 2, and wavelength selection and intensity adjustment of the pulse laser light. A dimming element 14 composed of an acousto-optic element, an alignment mirror 15 for adjusting the optical axis of the pulsed laser light emitted from the dimming element 14, and a dispersion compensation optical system 16 for providing negative dispersion of the pulsed laser light A collimating optical system 17 that adjusts the beam diameter by making the laser light substantially parallel light, and an alignment mirror 18 that performs final optical axis adjustment of the pulsed laser light that is output from the multiphoton excitation illumination optical system 3. I have.

  The dispersion compensation optical system 16 includes a λ / 2 plate 19, a pair of gratings 20 a and 20 b provided so that the distance between them can be adjusted, and alignment for adjusting the emission position of the laser light emitted from the pair of gratings 20 a and 20 b. A mirror 20c and a λ / 4 plate 21 are provided. The pair of gratings 20a and 20b can adjust the amount of negative dispersion imparted to the laser light by adjusting the distance between the pair of gratings 20a and 20b. The mirror 20c is adjusted so that the emission position and angle of the laser beam emitted from the dispersion compensation optical system 16 do not change even if the optical path fluctuates due to the movement of the pair of gratings 20a and 20b.

The one-photon excitation illumination optical system 5 includes a continuous laser light source 4.
The optical paths of the multiphoton excitation illumination optical system 3 and the one-photon excitation illumination optical system 5 are joined by a dichroic mirror 22.

  The scanning optical system 6 is scanned by, for example, a scanner 6a composed of a so-called proximity galvanometer mirror in which two galvanometer mirrors (not shown) that are swung around mutually intersecting axes are opposed to each other, and the scanner 6a. And a pupil projection lens 6b for condensing the laser light.

The multiphoton fluorescence observation optical system 8 includes an epi-illumination observation optical system 8A and a transmission observation optical system 8B.
The epi-illumination observation optical system 8A is reflected in a state where the dichroic mirror 23 provided in the optical path between the objective lens 7 and the pupil projection lens 6b is detachable, and the dichroic mirror 23 is inserted in the optical path. It includes a dichroic mirror 24 that branches multiphoton fluorescence for each wavelength, a barrier filter 25 that blocks pulsed laser light contained in the multiphoton fluorescence, and a photodetector 26 that detects multiphoton fluorescence.

  Further, the transmission observation optical system 8B is inserted into and removed from the condenser lens 27 disposed at a position facing the objective lens 7 with the sample A interposed therebetween, and the optical path that reflects the multiphoton fluorescence condensed by the condenser lens 27. A possible mirror 28, a dichroic mirror 29 for branching the multiphoton fluorescence reflected by the mirror 28 for each wavelength, a barrier filter 30 for blocking the pulsed laser light contained in the multiphoton fluorescence, and detecting the multiphoton fluorescence. And a photodetector 31.

  The confocal observation optical system 9 is disposed between the one-photon excitation illumination optical system 5 and the scanner 6a, and condenses the branched fluorescence. The dichroic mirror 32 branches the fluorescence returning through the scanner 6a. A confocal lens 33, a confocal pinhole 34 having an adjustable aperture through which light collected by the confocal lens 33 passes, and a condensing lens that condenses the fluorescence that has passed through the confocal pinhole 34 35, a dichroic mirror 36 and a mirror 37 for branching fluorescence for each wavelength, a barrier filter 38, and a photodetector 39.

  In the storage unit 10, a multiphoton excitation illumination optical system 3, a one-photon excitation illumination optical system 5, a multi-photon excitation illumination optical system 3, a multi-photon excitation illumination optical system 3, and a multi-photon excitation illumination optical system 3 Various settings of the photon fluorescence observation optical system 8 and the confocal observation optical system 9 are stored.

Regarding the multiphoton excitation illumination optical system 3, the setting stored in the storage unit 10 is selected by the wavelength of the pulsed laser light emitted from the pulsed laser light source 2, the angles of the alignment mirrors 13 and 15, and the light control element 14. Wavelength and intensity of pulse laser beam, adjustment of λ / 2 plate 19, adjustment of position of gratings 20a and 20b pair of dispersion compensation optical system 16 and alignment mirror 20c, adjustment of λ / 4 plate 21, beam diameter by collimating optical system 17 Adjustment values and setting values such as the angle of the alignment mirror 18 are included.
Regarding the one-photon excitation illumination optical system 5, the setting stored in the storage unit 10 includes a setting value of the wavelength of continuous laser light emitted from the continuous laser light source 4.

Regarding the multiphoton fluorescence observation optical system 8, the settings stored in the storage unit 10 include the type of the dichroic mirrors 23 and 28, the switching of the barrier filters 25 and 30, and the detection circuit settings of the photodetectors 26 and 31. ing.
The confocal observation optical system 9 includes the types of dichroic mirrors 32 and 36, the aperture diameter of the confocal pinhole 34, the switching of the barrier filter 38, and the detection circuit setting of the photodetector 39.

  The type of fluorescent dye input from the input unit 11 and the observation method are performed, for example, by selecting icons P1 to P5 displayed on the screen as shown in FIG. In the example shown in FIG. 2, icons P1 to P5 are prepared for each observation method for the same fluorescent dye, so that the user can select the type of fluorescent dye and the observation method simply by selecting the icons P1 to P5. You can enter it. In the figure, “2P” means for two-photon excitation observation.

  When the type and the observation method of the fluorescent dye are input from the input unit 11, the control unit 12 searches the storage unit 10 using the type of the fluorescent dye and the observation method as a key, and the various types stored correspondingly. The setting is read out. The control unit 12 applies the read setting to the multiphoton excitation illumination optical system 3, the one photon excitation illumination optical system 5, the multiphoton fluorescence observation optical system 8, or the confocal observation optical system 9.

The operation of the laser scanning microscope 1 according to the present embodiment configured as described above will be described below.
In order to perform fluorescence observation of the specimen A using the laser scanning microscope 1 according to the present embodiment, first, as shown in FIG. 3, the user operates the input unit 11 to determine the type and observation method of the fluorescent dye. Are selected (step S1).

  The control unit 12 searches the database in the storage unit 10 based on the fluorescent dye input from the input unit 11 and the observation method, and reads the setting data of each unit stored correspondingly (step S2). In addition to the setting data of each part, the read data includes the wavelength of the laser beam suitable for exciting the input fluorescent dye and the wavelength of the fluorescence emitted when the fluorescent dye is excited by the laser light. ing.

  Then, the control unit 12 determines whether the input observation method is confocal observation or multiphoton excitation observation (step S3). If the result of determination is confocal observation, the control unit 12 determines one photon. When the excitation illumination optical system 5 and the confocal observation optical system 9 are set (step S4) and multiphoton excitation observation is input, the multiphoton excitation illumination optical system 3 and the multiphoton fluorescence observation optical system 8 are set. Setting is performed (step S5).

Here, the case where the input observation method is confocal observation will be described in more detail with reference to FIG.
First, the one-photon excitation illumination optical system 5 is set (step S11).
That is, the wavelength of the continuous laser light read from the storage unit 10 in step S2 and suitable for exciting the input fluorescent dye is set in the continuous laser light source 4 (step S11). Thereby, the setting for the one-photon excitation illumination optical system 5 is completed.

Next, the confocal observation optical system 9 is set (steps S12 to S17).
First, a dichroic mirror (excitation dichroic mirror) 32 adapted to the fluorescence wavelength for branching the fluorescence generated from the specimen A is set on the optical path (step S12). Next, the aperture diameter of the confocal pinhole 34 is narrowed down sufficiently to adjust the optical axis (step S13).

  Then, the dichroic mirror (spectral dichroic mirror) 36 necessary for detecting the read fluorescence wavelength is switched (step S14), and the barrier filter 38 that can effectively block the laser beam having the read wavelength is obtained. Switching (step S15). Thereafter, detection circuit setting of the photodetector 39 suitable for detecting the read fluorescence wavelength is performed (step S16). Thereby, the setting of each part for confocal observation is completed.

On the other hand, the case where the input observation method is multiphoton excitation observation will be described in more detail with reference to FIG.
First, the multiphoton excitation illumination optical system 3 is set (steps S21 to S31).
That is, the wavelength of the pulse laser light read from the storage unit 10 in step 2 and suitable for exciting the input fluorescent dye is set in the pulse laser light source 2 (step S21). When the wavelength of the pulse laser light source 2 is changed, the next setting is performed after the output is stabilized (step S22).

  When the wavelength of the pulsed laser light emitted from the pulsed laser light source 2 is changed, the optical axis of the pulsed laser light from the pulsed laser light source 2 fluctuates. Therefore, the alignment mirror 13 is swung to adjust the optical axis. (Step S23). Then, by adjusting the dimming element 14, the wavelength of the pulsed laser beam is selected and its intensity is adjusted (step S24). Since the light control element 14 is composed of an acousto-optic element, the optical axis of the emitted pulsed laser light varies by performing wavelength selection and intensity adjustment of the pulsed laser light.

Therefore, the optical axis is adjusted by swinging the alignment mirror 15 (step S25).
Thereafter, the polarization direction of the pulsed laser light is adjusted by adjusting the λ / 2 plate 19 (step S26), and the pair of gratings 20a and 20b of the dispersion compensation optical system 16 is adjusted to give the pulsed laser light. The negative dispersion amount is adjusted (step S27), and the optical axis shifted by adjusting the pair of gratings 20a and 20b is adjusted by moving the alignment mirror 20c in translation (step S28). Then, by adjusting the λ / 4 plate 21, the pulse laser beam is converted into circularly polarized light (step S29).

Next, by adjusting the collimating optical system 17, the beam diameter of the pulsed laser light is adjusted so as to fill the pupil of the objective lens 7 (step S 30), and finally the multiphoton excitation illumination is adjusted by adjusting the alignment mirror 18. The optical axis of the pulse laser beam emitted from the optical system 3 is adjusted (step S31).
Thereby, the setting of each part of the multiphoton excitation illumination optical system 3 is completed.

Next, the multiphoton fluorescence observation optical system 8 is set (steps S32 to S36).
Since the multiphoton fluorescence observation optical system 8 includes the epi-illumination observation optical system 8A and the transmission observation optical system 8B, a case where both are used will be described.

  First, a dichroic mirror (excitation dichroic mirror) 32 adapted to the fluorescence wavelength for branching the multiphoton fluorescence generated from the specimen A is set on the optical path (step S32). Accordingly, in some cases, the photodetector 39 of the confocal observation optical system 9 can be used for multiphoton fluorescence observation.

Next, the dichroic mirror 23 adapted to the fluorescence wavelength for branching the multiphoton fluorescence generated from the specimen A is set on the optical path (step S33).
Then, the dichroic mirrors (spectral dichroic mirrors) 24 and 29 necessary for detecting the fluorescence wavelength read from the storage unit 10 are switched (step S34), and the laser light having the read wavelength is effectively used. The barrier filters 25 and 30 that can be blocked are switched (step S35). Thereafter, detection circuit settings of the photodetectors 26 and 31 suitable for detecting the read fluorescence wavelength are performed (step S36). Thereby, the setting of each part for multiphoton fluorescence observation is completed.

Thus, after each part is set, either confocal observation or multiphoton excitation observation is performed.
When confocal observation is performed, the continuous laser light emitted from the continuous laser light source 4 is two-dimensionally scanned by the scanner 6 a, condensed by the pupil projection lens 6 b, and collected on the sample A by the objective lens 7. Lighted. Since the wavelength of the continuous laser light emitted from the continuous laser light source 4 is set by reading from the storage unit 10 one suitable for the type of the input fluorescent dye, the fluorescent dye contained in the specimen A Can be efficiently excited to generate fluorescence of a desired wavelength.

  Fluorescence generated in the specimen A by irradiation with the continuous laser light returns through the objective lens 7, the pupil projection lens 6b, and the scanner 6a, is branched by the dichroic mirror 32, and enters the confocal observation optical system 9. . Since the dichroic mirror 32 is read and set from the storage unit 10 for the fluorescence wavelength corresponding to the type of the input fluorescent dye, the fluorescence can be efficiently branched and detected.

  The fluorescence branched by the dichroic mirror 32 is condensed by the confocal lens 33, and the light that has passed through the confocal pinhole 34 is condensed by the condenser lens 35 and branched by the dichroic mirror 36 for each wavelength. It is detected by the photodetector 39. Since the aperture diameter of the confocal pinhole 34 is set to be sufficiently small for confocal observation, only the fluorescence generated at the condensing position of the continuous laser light in the specimen A is allowed to pass through the confocal pinhole 34. It is detected by the photodetector 39.

  Thereby, a slice image of the specimen A spreading on the focal plane of the objective lens 7 can be acquired with high resolution, and a clear fluorescent image can be observed. In this case, according to the present embodiment, since the wavelength setting of the dichroic mirror 32 and the detection circuit setting of the photodetector 39 suitable for the type of the input fluorescent dye are performed, the confocal pinhole 34 is passed. The weak fluorescence can be detected with high accuracy by branching for each wavelength with no waste.

  When the resolution of the fluorescence image can be designated as an input from the input unit 11, the opening diameter of the confocal pinhole 34 may be adjusted in accordance with this. In this case, when the aperture diameter of the confocal pinhole 34 is increased, the resolution is lowered, but a bright fluorescent image can be acquired.

  When multiphoton fluorescence observation is performed, the pulse laser light emitted from the pulse laser light source 2 is adjusted in optical axis by the alignment mirror 13, adjusted in light by the dimming element 14, and optical axis in the alignment mirror 15. After adjustment, negative dispersion is applied by the dispersion compensation optical system 16, the beam diameter is adjusted by the collimating optical system 17, the optical axis is adjusted by the alignment mirror 18, and the light is directed to the scanner 6 a by the dichroic mirror 22. The wavelength of the pulsed laser light emitted from the pulsed laser light source 2 is set by reading from the storage unit 10 one that is suitable for the type of the input fluorescent dye, so that the fluorescent dye contained in the specimen A Can be efficiently excited to generate multiphoton fluorescence of a desired wavelength.

  By adjusting the λ / 2 plate 19 before entering the dispersion compensation optical system 16, negative dispersion can be efficiently imparted. Since the pair of gratings 20a and 20b of the dispersion compensation optical system 16 is adjusted according to the wavelength of the pulse laser beam, an ultrashort pulse laser beam having a sufficiently short pulse width can be emitted from the tip of the objective lens 7. Further, the collimating optical system 17 sets the beam diameter of the pulsed laser light so as to fill the pupil of the objective lens 7, so that the pulsed laser light having a high numerical aperture that fully exhibits the performance of the objective lens 7 is obtained from the sample A. Can be irradiated. Thereby, the length in the optical axis direction in which the multi-photon excitation phenomenon is generated by the pulse laser beam can be shortened, and a multi-photon fluorescence image with high resolution can be acquired.

  Of the multiphoton fluorescence generated from the position where the pulse laser beam is collected in the specimen A, the multiphoton fluorescence emitted in the direction of the objective lens 7 is immediately after being collected by the objective lens 7 and immediately after being collected by the objective lens 7. Therefore, it is decomposed for each wavelength and detected by the photodetector 26. Since the dichroic mirrors 23 and 24 are set by reading from the storage unit 10 those suitable for the fluorescence wavelength according to the type of the input fluorescent dye, the multi-photon fluorescence is efficiently branched and detected. be able to.

  On the other hand, among the multiphoton fluorescence generated from the position where the pulse laser beam is collected in the sample A, the multiphoton fluorescence transmitted through the sample A and emitted toward the condenser lens 27 is collected by the condenser lens 27. Immediately thereafter, the light is reflected by the mirror 28, decomposed for each wavelength by the dichroic mirror 29, and detected by the photodetector 31.

  Thereby, a slice image of the specimen A spreading on the focal plane of the objective lens 7 can be acquired with high resolution, and a clear multiphoton fluorescence image can be observed. In this case, according to the present embodiment, the wavelength setting of the dichroic mirrors 23, 24 and 29, the setting of the barrier filters 25 and 30 and the detection circuit setting of the photodetectors 26 and 31 suitable for the type of the input fluorescent dye are performed. Therefore, multiphoton fluorescence can be accurately branched for each wavelength and detected without waste.

  In addition, when multiple types of fluorescent dyes are input from the input unit 11 and multiphoton fluorescence observation is selected, the detected fluorescence wavelength increases. In some cases, detection is performed only with the multiphoton fluorescence observation optical system 8. Sometimes it ca n’t be done. In such a case, the confocal pinhole 34 of the confocal observation optical system 9 is opened to the maximum, and the settings of the dichroic mirrors 32 and 36 and the barrier filter 38 are adjusted. In this way, multi-photon fluorescence having a number of wavelengths that cannot be detected only by the photodetectors 26 and 31 of the multi-photon fluorescence observation optical system 8 can be detected using the confocal observation optical system 9. .

  As described above, according to the laser scanning microscope 1 according to the present embodiment, the setting of each unit stored in the storage unit 10 is read and set only by inputting the fluorescent dye and the observation method. The fluorescence generated from the fluorescent dye can be efficiently detected at the wavelength of the laser light suitable for the fluorescent dye, and a clear and bright fluorescent image can be obtained. Therefore, even if the user does not have detailed knowledge about the setting of the fluorescent dye and the laser scanning microscope 1, appropriate fluorescence observation can be performed. As a result, there is an effect that the burden on the user can be further reduced.

  In the present embodiment, the type of the fluorescent dye is input, and the laser beam having the excitation wavelength suitable for this is set to be irradiated. Of course, this setting includes irradiating laser light with different excitation wavelengths for each fluorescent dye, but when multiple fluorescent dyes can be excited with a single excitation wavelength. The excitation wavelength may be selected.

In this case, for example, as illustrated in FIG. 6, when different fluorescent dyes D <b> 1 and D <b> 2 have wavelength characteristics (profile information) of excitation efficiency that overlap each other, the wavelength characteristics of each excitation efficiency are stored in the storage unit 10. In addition, instead of selecting the excitation wavelengths λ ₁ and λ ₂ having the maximum excitation efficiency indicated by the chain line in the figure, the excitation wavelength λ _{3 at} which the excitation efficiency characteristics intersect as shown by the solid line is selected. do it. By doing so, a laser beam of a single excitation wavelength lambda _3, it is possible to excite simultaneously several fluorescent dyes D1, D2 with relatively high excitation efficiency. Therefore, it is possible to easily perform fluorescence observation that requires simultaneity.

In the present embodiment, the laser scanning microscope 1 having the multi-photon excitation illumination optical system 3 and the one-photon excitation illumination optical system 5 has been described as an example. However, instead of this, as shown in FIG. In addition, a laser scanning microscope 1 ′ having a multiphoton excitation stimulation optical system 3 ′ having the same configuration as the multiphoton excitation illumination optical system 3 may be employed. In the figure, in the multiphoton excitation stimulation optical system 3 ′, portions having the same configuration as the multiphoton excitation illumination optical system 3 are denoted by “′ (dash)”, the same reference numerals are used, and the description thereof is omitted.
In this case, the setting of each part of the multiphoton excitation stimulation optical system 3 ′ including the wavelength of the stimulation laser beam having a wavelength suitable for the input fluorescent dye may be stored in the storage unit 10. .

  Moreover, in this embodiment, although the method of selecting the icons P1-P5 prepared for every observation method with respect to the fluorescent dye as an input method of the type and the observation method of the fluorescent dye from the input unit 11, Alternatively, the fluorescent dye and the observation method may be input by assigning a fluorescent dye icon not including information on the observation method to a box prepared for each observation method. Further, an icon including a fluorescent dye and an observation method may be assigned to the photodetector.

1 is a block diagram illustrating an overall configuration of a laser scanning microscope according to an embodiment of the present invention. It is a figure which shows an example of the input of the fluorescent dye and observation method by the input part of the laser scanning microscope of FIG. It is a flowchart explaining the setting procedure of each part by the laser scanning microscope of FIG. It is a flowchart which shows the setting procedure for confocal observation among the setting procedures of FIG. It is a flowchart which shows the setting procedure for multiphoton fluorescence observation among the setting procedures of FIG. It is a graph which shows the wavelength characteristic of excitation efficiency explaining the selection method of the excitation wavelength in the case of exciting many fluorescent dyes with a single excitation wavelength. FIG. 5 is a block diagram showing a general configuration of a laser scanning microscope having a stimulation illumination optical system, which is a modification of the laser scanning microscope of FIG. 1.

Explanation of symbols

A Specimen 1,1 ′ Laser scanning microscope 2 Pulse laser light source 3 Multiphoton excitation illumination optical system 4 Continuous laser light source 5 1 photon excitation illumination optical system 6 Scanning optical system 8 Multiphoton fluorescence observation optical system 9 Confocal observation optical system 10 Storage unit 11 Input unit 12 Control unit (setting control unit)
34 Confocal pinhole

Claims (3)

A multiphoton excitation illumination optical system including a pulsed laser light source that emits ultrashort pulsed laser light;
A one-photon excitation illumination optical system including a continuous laser light source that emits continuous laser light;
A scanning optical system that two-dimensionally scans the laser light emitted from these illumination optical systems and irradiates the specimen;
A multiphoton fluorescence observation optical system and a confocal observation optical system for detecting fluorescence generated from the sample by irradiating the sample with laser light;
An input unit for inputting a fluorescent dye and an observation method;
A storage unit for storing settings of the illumination optical system and the observation optical system in association with the fluorescent dye and the observation method;
A laser scanning microscope comprising: a setting control unit that sets the illumination optical system and the observation optical system to settings stored in the storage unit corresponding to the fluorescent dye and the observation method input by the input unit. The confocal observation optical system includes a confocal pinhole capable of changing an aperture amount,
When multiphoton fluorescence observation is selected as an observation method from the input unit, fluorescence generated from a specimen is detected by the multiphoton fluorescence observation optical system and a confocal observation optical system in which the confocal pinhole is opened. 2. A laser scanning microscope according to 1. The storage unit stores excitation efficiency profile information with respect to the excitation wavelength of the fluorescent dye,
The wavelength of the laser beam is set to a wavelength at which profile information of each fluorescent dye stored in the storage unit intersects when a plurality of fluorescent dyes having overlapping excitation wavelengths are input from the input unit. 2. A laser scanning microscope according to 1.

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