JP4818634B2 - Scanning fluorescence observation system - Google Patents

Description

  The present invention relates to a scanning fluorescence observation apparatus.

Conventionally, in order to correct various aberrations generated in an objective lens of a microscope for performing white light observation, an adaptive mirror having a variable reflecting surface shape has been used (for example, see Patent Document 1). According to the adaptive mirror, it is possible to prevent the focal position of the objective lens from fluctuating due to aberration by adjusting the wavefront of the light incident on the objective lens.
Japanese Patent Application Laid-Open No. 11-101942

However, the microscope of Patent Document 1 is for performing white light observation, and its wavelength range is limited to a relatively narrow range.
On the other hand, when the wavelength band of the laser light incident on the objective lens covers a wide range as in a laser scanning confocal fluorescence microscope, the wavefront of the light incident on the objective lens is changed as in Patent Document 1. Only by adjusting, it is difficult to sufficiently suppress a decrease in resolution due to chromatic aberration, and there is a problem that a clear fluorescent image cannot be obtained.

  The present invention has been made in view of the above-described circumstances, and is capable of obtaining a clear fluorescent image even when a sample is irradiated with excitation light having a wide wavelength band from the ultraviolet region to the infrared region. An object of the present invention is to provide a type fluorescence observation apparatus.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a light source device capable of emitting a plurality of excitation light beams having different wavelengths, a scanning device for scanning excitation light emitted from the light source device, and condensing the excitation light scanned by the scanning device on a sample. An objective optical system, a condensing optical system that collects the fluorescence generated in the sample irradiated with excitation light and returned through the objective optical system and the scanning device, and the fluorescence collected by the condensing optical system are detected. And a first wavefront adjusting device arranged on the optical path of the excitation light and adjusting a wavefront of the excitation light, and a second wavefront arranged on the optical path of the fluorescence and adjusting the wavefront of the fluorescence An adjustment device; and a control device that interlocks the first and second wavefront adjustment devices based on the wavelength of the excitation light emitted from the light source device , the first wavefront adjustment device and the second wavefront adjustment device The wavefront adjusting device is a deformable mirror or liquid crystal optical element. Providing consisting of either scanning fluoroscopy apparatus.

  According to the present invention, the excitation light emitted from the light source device is condensed by the sample through the objective optical system after being scanned by the scanning device. From the sample irradiated with the excitation light, fluorescence corresponding to the fluorescent substance contained in the sample and the wavelength of the excitation light is generated. The fluorescence generated in the sample returns through the objective optical system and the scanning device, is condensed by the condensing optical system, and is detected by the light detection device. Thereby, the fluorescence image of a sample is acquirable.

  In this case, since the first wavefront adjusting device is disposed on the optical path of the excitation light, the wavefront of the excitation light is adjusted by the operation of the first wavefront adjusting device. When the wavelength of the excitation light emitted from the light source device is different, the condensing position of the excitation light irradiated on the sample varies based on the chromatic aberration of various optical elements from the light source device to the objective optical system. By operating the first wavefront adjusting device according to the wavelength of the excitation light, it is possible to collect light at the same position even if the wavelength of the excitation light changes.

  In addition, since the second wavefront adjusting device is disposed on the fluorescence optical path, the fluorescence wavefront is adjusted by the operation of the second wavefront adjusting device. When the fluorescence emitted from the sample changes based on the difference in the wavelength of the excitation light, the intensity of the fluorescence detected by the light detection device varies based on the chromatic aberration of various optical elements from the sample to the light detection device. By operating the second wavefront adjusting device in accordance with the fluorescence wavelength, the fluorescence can be condensed at the same position even if the fluorescence wavelength changes.

  For example, in the case of a confocal observation apparatus having a confocal pinhole at the condensing position of the condensing optical system, the fluorescence can be condensed at the position of the confocal pinhole regardless of the wavelength of the fluorescence. Therefore, it is possible to prevent inconvenience that the fluorescence generated from the same position of the sample is not detected depending on the wavelength of the fluorescence, and it is possible to accurately acquire the fluorescence images of a plurality of colors at the same position of the sample. .

Further, according to the present invention, the first wavefront adjusting device and the second wavefront adjusting device are interlocked by the operation of the control device. The wavefront of the excitation light is adjusted by the first wavefront adjusting device, and the wavefront of the fluorescence is adjusted by the second wavefront adjusting device. However, since the wavelength of the generated fluorescence differs depending on the wavelength of the excitation light, the control device uses the excitation light. By linking the first and second wavefront adjusting devices based on the wavelength of the light, both the condensing position of the excitation light in the sample and the condensing position of the fluorescence in the photodetector are automatically adjusted. .
In addition, by configuring the first wavefront adjusting device and the second wavefront adjusting device from either a deformable mirror or a liquid crystal optical element, the wavefronts of the excitation light and the fluorescence can be easily obtained based on the wavelength of the excitation light. Can be adjusted .

  In the above invention, a fluorescence branching unit that branches fluorescence directed to the light detection device from the optical path of the excitation light is provided, and the first wavefront tuning device is disposed between the light source device and the fluorescence branching unit. Preferably, the second wavefront tuning device is disposed between the fluorescence branching unit and the light detection device.

  With this configuration, optical paths for only excitation light and only fluorescence are provided between the light source device and the fluorescence branching unit and between the fluorescence branching unit and the light detection device, respectively. Thereby, the first wavefront adjusting device can adjust the wavefront of the excitation light in the optical path of only the excitation light, and the second wavefront adjusting device can adjust the wavefront of the fluorescence in the optical path of only the fluorescence. Therefore, the wavefront adjustment of excitation light and the wavefront adjustment of fluorescence can be performed independently, and the adjustment of both can be facilitated. As the fluorescence branching means, for example, a dichroic mirror is used.

Moreover, in the said invention, the fluorescence wavelength branching means which branches the said fluorescence for every wavelength is provided, and the said 2nd wavefront adjustment apparatus is arrange | positioned between the said fluorescence wavelength branching means and the said optical detection apparatus. It is good.
With this configuration, when multiple wavelengths of fluorescence are generated from the sample, the fluorescence can be branched for each wavelength by the operation of the fluorescence wavelength branching unit. Therefore, the fluorescence of a plurality of wavelengths can be condensed at the same position by adjusting the wavefront for each wavelength. In this case, the second wavefront adjusting device may be arranged for all the fluorescence, but the second wavefront adjusting device may be arranged for the fluorescence having a wavelength other than the reference wavelength. In addition, the second wavefront adjusting device may be shared for the fluorescence having the same wavelength of chromatic aberration. As the fluorescence wavelength branching means, for example, a dichroic mirror is used.

Further, in the above-described invention, the pumping light wavelength branching unit that branches the pumping light for each wavelength is provided, and the first wavefront tuning device is disposed between the pumping light wavelength branching unit and the fluorescence branching unit. It is good to be.
With this configuration, when excitation light with a plurality of wavelengths is emitted from the light source device, the excitation light is branched for each wavelength by the operation of the excitation light wavelength branching unit, and for each wavelength by the first wavefront tuning device. The wavefront can be adjusted. In this case, the first wavefront tuning device may be arranged for all wavelengths of excitation light, but the first wavefront tuning device is arranged for excitation light of a wavelength other than the reference wavelength. Also good. In addition, the first wavefront adjusting device may be shared for the excitation light having the same wavelength as the chromatic aberration. As the excitation light wavelength branching means, for example, a dichroic mirror is used.

Moreover, in the said invention, it is preferable that the said 1st wavefront adjustment apparatus and / or the said 2nd wavefront adjustment apparatus are arrange | positioned in the entrance pupil position of the said objective lens, or a conjugate position with this.
With this configuration, it is possible to minimize fluctuations in the diameters of the excitation light and / or fluorescent light beams incident on the first wavefront adjusting device and / or the second wavefront adjusting device that adjust the wavefront. Therefore, wavefront adjustment of excitation light and / or fluorescence can be facilitated.

  According to the present invention, even when excitation light having different wavelengths is emitted from the light source device, the excitation light can be condensed at the same position of the sample regardless of the wavelength of the excitation light, and the same light detection device can be used. Fluorescence can be collected at a position. As a result, even when excitation light having different wavelengths is emitted from the light source device, fluctuations in the condensing position based on chromatic aberration of the optical element are prevented, and a clear fluorescent image at the same position of the sample can be obtained with high accuracy. There is an effect.

Hereinafter, a scanning fluorescence observation apparatus 1 according to the present invention will be described with reference to FIG.
As shown in FIG. 1, the scanning fluorescence observation apparatus 1 according to the present embodiment includes an optical unit 2, an observation apparatus body 3, and a control apparatus 4 that controls these.

The optical unit 2 includes a light source device 5, a light detection device 6, and a dichroic mirror 7 that branches an optical path from the light source device 5 and an optical path to the light detection device 6.
The light source device 5 includes, for example, three laser light sources 8 having different wavelengths, a collimator lens 9 that makes the laser light (excitation light) L emitted from each laser light source 8 substantially parallel light, and the three laser light sources 8. There are provided a mirror 10 and a dichroic mirror 11 for joining the emitted laser beams L of three types of wavelengths into the same optical path.

  The light detection device 6 includes a condensing lens 12 that condenses the fluorescence F branched by the dichroic mirror 7, a pinhole 13 that is disposed at a condensing position of the fluorescence F by the condensing lens 12, and a pinhole 13. And a light detector 14 for detecting the fluorescence F that has passed through. The condensing lens 12 includes a moving device 12a that moves the condensing lens 12 along the optical axis direction. The photodetector 14 is, for example, a photomultiplier tube (PMT).

  The observation device body 3 condenses the laser light L emitted from the light source device 5 and forms a first intermediate image, and a laser that forms the first intermediate image. The second relay lens 16 that condenses the light L into substantially parallel light, the scanning device 17 that two-dimensionally scans the laser light L that is made substantially parallel light, and the laser light that is two-dimensionally scanned. The pupil projection lens 18 that focuses L and forms a second intermediate image, the imaging lens 19 that converts the laser light L formed on the second intermediate image into substantially parallel light, and the substantially parallel light. And an objective lens 20 that focuses the laser beam L and irradiates the sample A. In the figure, reference numeral 21 denotes a mirror, and reference numeral 22 denotes a stage on which a sample A such as a biological sample accommodated in the petri dish 23 is placed.

  In addition, the observation apparatus body 3 includes a shape variable mirror 24 disposed between the dichroic mirror 7 and the first relay lens 15. The deformable mirror 24 includes a plurality of actuators (not shown), and a desired reflecting surface shape can be achieved by driving each actuator in accordance with a given electric signal. Thereby, the wave front of the reflected laser beam L can be adjusted. Further, the variable shape mirror 24 is arranged in a positional relationship substantially conjugate with the entrance pupil position of the objective lens 20.

  The scanning device 17 is constituted by a pair of galvanometer mirrors, for example. By changing the angles of the pair of galvanometer mirrors, the laser beam L can be scanned two-dimensionally.

  The control device 4 stores the wavelength of the laser light L emitted from the light source device 5 in association with the shape of the reflecting surface of the variable shape mirror 24. When the wavelength is given, the shape of the reflecting surface is stored. An electric signal for achieving the above is input to the deformable mirror 24. Further, the control device 4 stores the wavelength of the laser light L emitted from the light source device 5 in association with the position of the condenser lens 12, and when the wavelength of the laser light L is given, The moving device 12a is driven to move the condenser lens 12 to a position.

The operation of the thus configured scanning fluorescence observation apparatus 1 according to this embodiment will be described below.
When the sample A is subjected to fluorescence observation using the scanning fluorescence observation apparatus 1 according to the present embodiment, when the wavelength of the laser light L emitted from the light source device 5 is selected, the controller 4 operates the shape variable mirror 24. The shape of the reflecting surface and the position of the condenser lens 12 are set. In this state, when the laser light L having the selected wavelength is emitted from the laser light source 8, the laser light L is reflected by the dichroic mirror 7 and then reflected by the variable shape mirror 24.

  Thereafter, the laser light L relayed through the first relay lens 15 and the second relay lens 16 is two-dimensionally scanned by the scanning device 17. The scanned laser light L is condensed on the sample A through the pupil projection lens 18, the imaging lens 19 and the objective lens 20. In the sample A, when the laser light L is irradiated, the fluorescent substance contained therein is excited, and fluorescence F having a wavelength corresponding to the wavelength of the laser light L is emitted.

  The generated fluorescence F returns through the objective lens 20, the imaging lens 19, the pupil projection lens 18, the scanning device 17, the second relay lens 16, and the first relay lens 15, and is reflected by the variable shape mirror 24. The laser beam L is branched by being transmitted through the dichroic mirror 7 later. Then, the light is condensed at the position of the pinhole 13 by the condenser lens 12 and then detected by the photodetector 14.

  Next, when the laser light source 8 that generates the laser light L of another wavelength is selected, the variable shape mirror 24 is formed in a reflecting surface shape corresponding to the wavelength of the laser light L by the operation of the control device 4 in the same manner. In addition to being set, the position of the condenser lens 12 is set in conjunction with this.

As a result, even if the wavelength of the laser beam L changes, the wavefront of the laser beam L is adjusted by the operation of the variable shape mirror 24, so that the laser beam L remains at the same position on the sample A regardless of the wavelength. It is condensed.
Further, even if the wavelength of the fluorescence F generated in the sample A changes due to the change of the wavelength of the laser light L, the position of the condenser lens 12 is adjusted by the operation of the moving device 12a. Regardless of the wavelength, the light is passed through the pinhole 13 arranged at the same position and detected by the photodetector 14.

  In the scanning fluorescence observation apparatus 1 according to the present embodiment, since the focal position of the sample A and the position of the pinhole 13 are arranged in a conjugate relationship with each other, only the fluorescence F generated at the focal position of the sample A is obtained. Passes through the pinhole 13 and is detected by the photodetector 14. Therefore, it is possible to acquire a fluorescence image having a high spatial resolution.

In this case, according to the scanning fluorescence observation apparatus 1 according to the present embodiment, even if the wavelength of the laser beam L irradiated to the sample A is changed by switching the laser light source 8, Since the chromatic aberration is corrected by the operation, the laser beam L can be condensed at the same position in the depth direction of the sample A. Further, when the wavelength of the laser light L is changed, the wavelength of the fluorescence F generated in the sample A also changes. However, even if chromatic aberration occurs in the optical system up to the photodetector 14, the variable shape mirror 24 and Correction is performed by the condenser lens 12.
Therefore, it is possible to acquire fluorescent images at the same observation position in the depth direction of the sample A using the laser beams L having a plurality of wavelengths.

  In particular, in the scanning fluorescence observation apparatus 1 according to the present embodiment, since the variable shape mirror 24 is disposed at a position conjugate with the entrance pupil position of the objective lens 20, the laser beam L at the position of the variable shape mirror 24 is not affected. Changes in the beam diameter are suppressed. Therefore, the adjustment of the wavefront of the laser light L by the variable shape mirror 24 can be performed easily and accurately.

In the present embodiment, the light source device 5 including the three laser light sources 8 is illustrated, but the present invention is not limited to this, and two or more laser light sources capable of emitting two or more different wavelengths of the laser light L. 8 may be provided. Moreover, you may employ | adopt the wavelength-variable laser light source 8 which can change the wavelength of the emitted laser beam L continuously or intermittently. In this case, the wavelength of the laser light L emitted from the laser light source 8 may be instructed to the laser light source 8 and input to the control device 4, and the wavelength of the laser light L emitted from the laser light source 8 may be changed. It is also possible to actually measure and input the measured wavelength to the control device 4.
Further, although the variable shape mirror 24 and the condensing lens 12 movable in the optical axis direction are exemplified as the wavefront adjusting device, a liquid crystal optical element may be employed instead.

Next, a scanning fluorescence observation apparatus 30 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, portions having the same configuration as those of the scanning fluorescence observation apparatus 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 2, the scanning fluorescence observation apparatus 30 according to the present embodiment includes a coupling lens 31 that condenses the laser light L combined in the same optical path by the dichroic mirror 11, and the coupling lens 31. An optical fiber 32 having one end disposed at the condensing position, and a collimating lens 33 that makes the laser light L propagated by the optical fiber 32 and emitted from the other end substantially parallel light are provided.

  In the present embodiment, the variable shape mirror 34 that adjusts the wavefront of the laser light L is disposed between the collimating lens 33 and the dichroic mirror 7. A variable shape mirror 35 for adjusting the wavefront of the fluorescence F is disposed between the dichroic mirror 7 and the condenser lens 12.

  According to the scanning fluorescence observation apparatus 30 according to the present embodiment configured as described above, the variable shape mirrors 34 and 35 are arranged on the optical paths of only the laser beam L and only the fluorescence F, respectively. The wavefronts of L and F can be adjusted independently. Therefore, the condensing position in the sample A and the condensing position to the pinhole 13 can be individually adjusted, and the adjustment can be performed easily and accurately.

In the scanning fluorescence observation apparatus 30 according to the present embodiment, the optical fiber 32 that propagates the laser light L is used. However, due to the occurrence of chromatic aberration due to the change in the wavelength of the laser light L, the optical fiber 32 is collected. The light position may also vary. For this reason, as shown in FIG. 3, in order to prevent fluctuations in the condensing position on the end face of the optical fiber 32, a variable shape mirror 36 is disposed between the end face of the optical fiber 32 and the laser light source 8. The wavefront of the laser light L incident on the optical fiber 32 may be adjusted based on the wavelength of the laser light L.
In particular, as in a CARS microscope (Coherent Anti-Stokes Raman Spectroscopy), two laser beams of different wavelengths are irradiated onto the sample A, and the Stokes-Raman scattering that interacts in the sample A coherently causes a single It is suitable for applications where strong fluorescence is obtained at a non-Stokes wavelength.

  Further, as shown in FIG. 4, the correction of chromatic aberration is not required for the laser light L1 having a specific wavelength among the laser light L having a plurality of wavelengths, and the wavefront is adjusted only for the laser light L2 having other wavelengths. Therefore, the laser beam L2 may be branched for each wavelength by the dichroic mirror 37, and the wavefront may be adjusted by the shape variable mirror 38 only for the laser beam L2 having a wavelength that requires correction of chromatic aberration.

  Further, as shown in FIG. 5, in an application in which the sample A is irradiated with the laser light L having a plurality of wavelengths at the same time and the fluorescence F1 and F2 having a plurality of wavelengths generated in the sample A are simultaneously acquired, the laser light L The fluorescence F branched from the light is further branched into the fluorescence F1 and F2 for each wavelength by the dichroic mirror 39, and the wavefront of the branched fluorescence F1 and F2 is adjusted by the separate variable shape mirror 35 for each wavelength. Good.

[First embodiment]
Next, a first embodiment of the scanning fluorescence observation apparatus according to the present invention will be described below with reference to FIGS. 6 to 12 and Tables 1 to 10.
The scanning fluorescence observation apparatus according to the present example substantially corresponds to the first embodiment. As shown in FIG. 6, the scanning fluorescence observation apparatus is the first in that a variable shape mirror DMF2 is used instead of the condenser lens 12. This is different from the first embodiment. In FIG. 6, the light source device 5 is omitted.

The distance between each optical element is as shown in Table 1.

Lens data of the objective lens is shown in FIG.

Further, lens data of the imaging lens is shown in FIG.

The lens data of the pupil projection lens is shown in FIG.

Further, lens data of the relay lenses R1 and R2 are shown in FIG. The relay lenses R1 and R2 have the same lens data.

Further, lens data of the condenser lens is shown in FIG.

Examples of the shape variable data are shown in FIGS. In the present embodiment, the light is incident and emitted in the YZ plane. A solid line B in the figure indicates a region where the light hits.
In the present embodiment, the shape Z (x, y) of the reflecting surface of the variable shape mirror is defined as the following equation (1). Further, the deformation parameters Cj (j = 4, 6, 11, 13, 15) in the number 1 of the two deformable mirrors DFM1, DFM2 are defined as shown in Tables 7 and 8.

  According to the scanning fluorescence observation apparatus according to the present embodiment configured as described above, as shown in Table 9, even if the wavelength of the laser light L is changed by the action of the variable shape mirror DFM1, the sample A It can be seen that the shift amount ΔZ of the focal position can be kept extremely small. Table 9 also shows a case where correction by the deformable mirror DFM1 is not performed as a comparative example.

  Further, according to the scanning fluorescence observation apparatus according to the present embodiment, as shown in Table 10, even if the wavelength of the laser beam L is changed mainly by the action of the deformable mirror DFM2, the detection position in the photodetector is detected. It can be seen that the amount of fluorescence shift ΔZ in can be minimized. Table 10 also shows a case where correction by the deformable mirror DFM2 is not performed as a comparative example. In the comparative example, numerical values based on the wavelength of 542 nm are shown.

[Second Embodiment]
Next, a second embodiment of the scanning fluorescence observation apparatus according to the present invention will be described below with reference to FIG. 15 and Tables 11 to 13.
This example corresponds to the second embodiment described above. The lens data for each lens is the same as in the first embodiment. The deformation parameters of the deformable mirror DFM1 are also the same as those in the first embodiment. In FIG. 15, the distance between each optical element is shown in Table 11. The distance not shown is the same as in the first embodiment.

Table 12 shows deformation parameters of the deformable mirror DFM2.

  According to the scanning fluorescence observation apparatus according to the present embodiment, as shown in Table 13, even when the wavelength of the laser beam L is changed by the action of the variable shape mirror DFM2, the fluorescence at the detection position in the photodetector is detected. It can be seen that the amount of deviation ΔZ can be kept extremely small. Table 13 also shows a case where correction by the variable shape mirror DFM2 is not performed as a comparative example. In the comparative example, numerical values based on the wavelength of 542 nm are shown.

1 is an overall configuration diagram showing a scanning fluorescence observation apparatus according to a first embodiment of the present invention. It is a whole block diagram which shows the scanning fluorescence observation apparatus which concerns on the 2nd Embodiment of this invention. FIG. 3 is an overall configuration diagram showing a first modification of the scanning fluorescence observation apparatus in FIG. 2. FIG. 6 is an overall configuration diagram showing a second modification of the scanning fluorescence observation apparatus in FIG. 2. It is a whole block diagram which shows the 3rd modification of the scanning fluorescence observation apparatus of FIG. 1 is an overall configuration diagram showing a first embodiment of a scanning fluorescence observation apparatus according to the present invention. It is a figure which shows the lens structure of the objective lens of the scanning fluorescence observation apparatus of FIG. It is a figure which shows the lens structure of the imaging lens of the scanning fluorescence observation apparatus of FIG. It is a figure which shows the lens structure of the pupil projection lens of the scanning fluorescence observation apparatus of FIG. It is a figure which shows the lens structure of the relay lens of the scanning fluorescence observation apparatus of FIG. It is a figure which shows the lens structure of the condensing lens of the scanning fluorescence observation apparatus of FIG. It is a perspective view which shows the example of a shape of the reflective surface of the shape variable mirror of the scanning fluorescence observation apparatus of FIG. It is a graph which shows XY cross-sectional shape of the reflective surface of the shape variable mirror of FIG. It is a graph which shows the YZ cross-sectional shape of the reflective surface of the shape-variable mirror of FIG. It is a whole block diagram which shows 2nd Example of the scanning fluorescence observation apparatus which concerns on this invention.

Explanation of symbols

A Sample F Fluorescence L, L1, L2 Laser light (excitation light)
DESCRIPTION OF SYMBOLS 1,30 Scanning fluorescence observation apparatus 4 Control apparatus 5 Light source apparatus 6 Photodetection apparatus 7 Dichroic mirror (fluorescence branching means)
12 Condensing optical system 12a Moving device (second wavefront adjusting device)
17 Scanning device 20 Objective lens (objective optical system)
24, 34 variable shape mirror (first wavefront tuning device)
35 Shape variable mirror (second wavefront adjusting device)
37 Dichroic mirror (excitation light wavelength branching means)
39 Dichroic mirror (fluorescence wavelength branching means)

Claims (5)

A light source device capable of emitting excitation light having a plurality of different wavelengths;
A scanning device that scans excitation light emitted from the light source device;
An objective optical system that focuses the excitation light scanned by the scanning device on the sample;
A condensing optical system that collects fluorescence generated in the sample irradiated with the excitation light and returned through the objective optical system and the scanning device;
A photodetection device for detecting fluorescence collected by the condensing optical system;
A first wavefront adjusting device that is disposed on the optical path of the excitation light and adjusts the wavefront of the excitation light;
A second wavefront adjusting device disposed on the fluorescent light path for adjusting the wavefront of the fluorescence;
A control device for interlocking the first and second wavefront tuning devices based on the wavelength of the excitation light emitted from the light source device ;
The scanning fluorescence observation apparatus, wherein the first wavefront adjusting device and the second wavefront adjusting device are either a variable shape mirror or a liquid crystal optical element . A fluorescence branching means for branching fluorescence from the optical path of the excitation light toward the light detection device;
The first wavefront adjusting device is disposed between the light source device and the fluorescence branching means;
The scanning fluorescence observation apparatus according to claim 1, wherein the second wavefront adjusting device is disposed between the fluorescence branching unit and the light detection device. Fluorescence wavelength branching means for branching the fluorescence for each wavelength,
The scanning fluorescence observation apparatus according to claim 2, wherein the second wavefront tuning device is disposed between the fluorescence wavelength branching unit and the light detection device. An excitation light wavelength branching means for branching the excitation light for each wavelength;
The scanning fluorescence observation apparatus according to any one of claims 1 to 3, wherein the first wavefront tuning device is disposed between the excitation light wavelength branching unit and the fluorescence branching unit.   The said 1st wavefront adjustment apparatus and / or the said 2nd wavefront adjustment apparatus are arrange | positioned in the entrance pupil position of the said objective lens, or a position conjugate with this. Scanning fluorescence observation device.

Images