JP2004309785A - Microscope system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope system in which a confocal observation using a scanning type laser microscope and a fluorescent observation using evanescent illumination can be simultaneously or switchably performed, and especially even when a focal position in an optical direction on the scanning type laser microscope side is positively changed, the usability for the fluorescent observation using the evanescent illumination is excellent. <P>SOLUTION: A laser beam from an Ar laser 2 is converged onto a sample 25 through a scanning unit 10 and an objective lens 24 so as to be two-dimensionally scanned, evanescent illumination is generated on the sample 25 by a laser beam from a green helium neon laser 3 and a wave front conversion element 14 is arranged on the optical path of the laser beam from the Ar laser 2. Then mechanical positional relation between the objective lens 24 and the sample 25 is fixed on the sample 25 so that a condition for performing evanescent illumination is satisfied and the converged position of the laser beam on the surface of the sample 25 can be controlled by the wave front conversion element 14. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microscope system that can support confocal observation with a scanning laser microscope and fluorescence observation with evanescent illumination.
[0002]
[Prior art]
In recent years, functional analysis of living cells has been actively performed.
[0003]
In such functional analysis of living cells, in particular, a fluorescent substance such as a fluorescent protein is localized in a certain part of a thick living cell, and its fluorescence is observed to elucidate the three-dimensional structure. A scanning laser microscope is used.
[0004]
For example, as disclosed in Patent Document 1, a scanning laser microscope focuses laser light from a laser light source on a sample using an objective lens, optically two-dimensionally scans the focus point, and scans the sample from the sample. (Especially fluorescence) passes through the objective lens again, is detected by a photodetector through a confocal pinhole, and two-dimensional information is obtained. In other words, it is known that a scanning laser microscope has a resolution in the optical axis direction because it can cut light from a position other than the focal point by using a confocal pinhole. A plurality of two-dimensional slice images are obtained by varying the relative positional relationship between the sample and the electric focusing mechanism such as a stepping motor, and these two-dimensional images are three-dimensionally constructed. An image can be obtained.
[0005]
On the other hand, as a biological microscope used for functional analysis of living cells, a microscope called a TIRFM (Total Internal Reflection Fluo-rescense Microscopy) has been attracting attention.
[0006]
This microscope excites a fluorescent substance by using light called evanescent light that infiltrates a small area of a few hundred nm or less on the sample side when the illumination light is totally reflected at the interface between the cover glass and the sample. Since only a small range of fluorescence near the cover glass is observed, the background is very dark and faint fluorescence can be observed.
[0007]
A total internal reflection fluorescence microscope using the evanescent light is disclosed in, for example, Patent Document 2. The evanescent illumination here differs from the conventional coaxial epi-illumination, which irradiates the entire sample with excitation light such as a mercury lamp, because the irradiation range on the sample is extremely shallow in the depth direction of the sample. The feature is that information can be obtained with high sensitivity.
[0008]
[Patent Document 1]
JP 2001-356272 A
[0009]
[Patent Document 2]
JP 2001-272606 A
[0010]
[Patent Document 3]
JP-A-11-101942
[0011]
[Patent Document 4]
JP 2001-166213 A
[0012]
[Patent Document 5]
Japanese Patent No. 2848952
[0013]
[Problems to be solved by the invention]
Thus, in recent years, a scanning laser microscope and a total internal reflection fluorescence microscope using evanescent illumination have been used for fluorescence observation of biological cells. However, a microscope system having the functions of these microscopes simultaneously exists. Absent.
[0014]
The reason for this is that the observation with a scanning laser microscope is based on the fact that the relative positional relationship between the three-dimensionally thick sample and the objective lens is positively changed in the optical axis direction by an electric focusing stage or the like, and the three-dimensional Although dimensional observation is possible, if the focus position of the objective lens is set inside the sample, fluorescence observation by evanescent illumination limited to observation near the cell surface near the cover glass cannot be performed. It is.
[0015]
Thus, compatibility between a scanning laser microscope for performing three-dimensional observation of a sample and a total reflection fluorescence microscope using evanescent illumination for observing the surface of the sample is not practical in view of the inconvenience of the system.
[0016]
The present invention has been made in view of the above circumstances, and confocal observation by a scanning laser microscope and fluorescence observation by evanescent illumination can be performed simultaneously or by switching. In particular, focus in the optical axis direction on the scanning laser microscope side can be achieved. It is an object of the present invention to provide a microscope system that is easy to use for fluorescence observation by evanescent illumination even when the position is positively changed.
[0017]
[Means for Solving the Problems]
The invention according to claim 1 includes a first light source that generates a laser beam, an objective lens that focuses the laser beam from the first light source on a sample, and two-dimensional scanning of the laser beam on the sample. A light scanning unit for emitting light for performing evanescent illumination on the sample, and a light source provided on an optical path of laser light from the first light source, and condensing the laser light on the sample. Focusing position control means for optically variably controlling the position in the optical axis direction of the objective lens, and imaging means for imaging fluorescence generated by the evanescent illumination, and a condition for performing evanescent illumination on the sample is provided. In a state where the mechanical positional relationship between the objective lens and the sample is fixed so as to satisfy the condition, the focus position of the laser light on the sample surface can be controlled by the focus position control means. That.
[0018]
According to a second aspect of the present invention, in the first aspect of the present invention, confocal observation means for confocal observation of light emitted from a focusing position on the sample on which the laser light from the first laser light source is focused. Is further provided.
[0019]
According to a third aspect of the present invention, in the first aspect of the present invention, the laser light from the first laser light source causes a photochemical reaction at a condensing position on the sample.
[0020]
According to a fourth aspect of the present invention, in the first aspect of the present invention, the second light source comprises a laser light source that generates a laser beam, and a single laser is used together with the first light source. Forming a light source unit, wherein the first and second light sources are respectively provided with first and second optical fibers for deriving laser light, respectively;
A light source for condensing the laser light passing through the first optical fiber on the sample by the objective lens and a light source for performing evanescent illumination on the sample with the laser light passing through the second optical fiber It is characterized in that it is used.
[0021]
According to a fifth aspect of the present invention, in the fourth aspect of the present invention, the laser light source unit sets the first and second light sources to have the same wavelength of light, and the first and second light sources have the same wavelength. It is characterized in that the light source is constituted by one light source.
[0022]
According to a sixth aspect of the present invention, in the first aspect of the present invention, the first and second light sources have different wavelengths of generated light.
[0023]
According to a seventh aspect of the present invention, in any one of the first to sixth aspects, the light condensing position control means is a wavefront conversion element.
[0024]
According to an eighth aspect of the present invention, in the invention according to any one of the first to sixth aspects, the light-collecting position control means converts a beam diameter of the laser light focused on the sample to a beam diameter. And means for changing the degree of collimation of the laser light emitted from the beam diameter converting means.
[0025]
According to a ninth aspect of the present invention, in the first, third, sixth and eighth aspects, the first laser light source generates a two-photon excitation pulse laser.
[0026]
According to a tenth aspect of the present invention, in the invention according to any one of the first to ninth aspects, the focus position of the laser light on the sample surface is controlled by the focus position control means and the light scanning means is controlled by the light scanning means. The method is characterized in that at least one of two-dimensional scanning control of the laser beam on the sample is controlled in synchronization with the imaging of the fluorescence by the imaging unit.
[0027]
According to an eleventh aspect of the present invention, in the invention of the tenth aspect, the confocal observation means has a light detection means for detecting light emitted from a condensing position on the sample. It is characterized in that the detection and the imaging of the fluorescence by the imaging means are controlled in synchronization.
[0028]
According to a twelfth aspect of the present invention, in the tenth and eleventh aspects, the timing of irradiating the sample with the laser beam from the first laser light source and the timing of irradiating the sample with the second light source are described. It is characterized in that the timing is controlled in synchronization.
[0029]
According to a thirteenth aspect of the present invention, in the sixth, tenth, and eleventh aspects, the first light source focuses the light on the sample on an imaging optical path of an imaging unit for imaging the fluorescence generated by the evanescent illumination. A filter means for cutting the wavelength of the laser light to be emitted.
[0030]
According to a fourteenth aspect of the present invention, in the first aspect of the invention, the evanescent illumination is performed via a condenser lens disposed on the opposite side of the objective lens with the sample interposed therebetween. Features.
[0031]
According to a fifteenth aspect of the present invention, there is provided a first light source for generating laser light, an objective lens for condensing the laser light from the first laser light source on a sample, and a two-dimensional scanning of the laser light on the sample. Optical scanning means, a second light source for generating light for illuminating the sample via the objective lens, and an imaging means for imaging light emitted from the sample by illumination from the second light source Optical path synthesizing means for coaxially guiding respective optical axes on the way of the laser light from the light scanning means and the light emitted from the second light source toward the objective lens; and a laser from the first light source. A focusing position control means provided in an optical path of light, for optically variably controlling a focusing position of the laser light on the sample in an optical axis direction of the objective lens, wherein the objective lens and the sample Fixed the mechanical positional relationship of In state, it is characterized in that the controllable light condensing position on the sample surface of the laser beam by the condensing position control means.
[0032]
As a result, according to the present invention, the scanning laser microscope including the variable control of the observation cross section in the optical axis direction with the mechanical positional relationship between the objective lens and the sample satisfying the condition for performing the evanescent illumination on the sample fixed. At the same time as performing confocal observation by using the evanescent illumination.
[0033]
Further, according to the present invention, it is possible to perform fluorescence observation by evanescent illumination while using a two-photon excitation pulse laser to release caged at any three-dimensional position in the thickness and plane directions of the sample. As a result, it is possible to observe changes over time on the sample surface.
[0034]
Further, according to the present invention, the evanescent illumination is performed via the condenser lens arranged on the opposite side of the objective lens with the sample interposed therebetween, so that the restriction of the objective lens (high NA for generating the evanescent illumination) is obtained. , Oil immersion objective) and the degree of freedom of observation can be expanded.
[0035]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0036]
(First Embodiment)
FIG. 1 shows a schematic configuration of a microscope system to which the present invention is applied.
[0037]
In this case, in the present embodiment, a single laser light source unit having a first laser light source that irradiates the sample with laser light through the optical scanning unit and a second laser light source that performs evanescent illumination is provided. The laser light source unit uses two single-mode fibers to provide laser light to two ports, specifically, an epi-illumination light pipe for performing evanescent illumination, and a scanning unit having optical scanning means and confocal observation means. Light is supplied.
[0038]
In the figure, reference numeral 1 denotes a laser light source unit, which has an Ar laser 2 oscillating 488 nm laser light and a green helium neon laser 3 oscillating 543 nm laser light.
[0039]
A reflection mirror 4 is arranged on the optical path of the laser light from the green helium neon laser 3. A dichroic mirror 5 is disposed on the optical path of the laser beam from the Ar laser 2 at the intersection with the laser beam reflected by the reflection mirror 4. The dichroic mirror 5 combines these two laser light paths, and transmits the laser light from the Ar laser 2 and reflects the laser light reflected by the reflection mirror 4. In other words, the dichroic mirror 5 here has such characteristics that it reflects 543 nm laser light and transmits 488 nm laser light.
[0040]
A beam splitter 7 is arranged on the optical path of the laser light synthesized by the dichroic mirror 5. The beam splitter 7 divides the optical path synthesized by the dichroic mirror 5 into two, an optical path 6a for scanning laser microscope illumination and an optical path 6b for evanescent illumination. Here, the beam splitter 7 reflects 30% with respect to the optical path 6a for illumination of the scanning laser microscope and transmits 70% with respect to the optical path 6b for evanescent illumination in both the wavelength ranges of 488 nm and 543 nm. Has characteristics. The ratio of reflection and transmission in the beam splitter 7 is merely an example, and a configuration in which the ratio can be switched to another ratio may be adopted.
[0041]
On the optical paths 6a and 6b separated by the beam splitter 7, wavelength-selective acousto-optic elements (AOTFs) 8a and 8b are separately arranged.
[0042]
An incident end of a single mode fiber 9a as a first optical fiber is disposed on an optical path 6a for illumination of the scanning laser microscope, and a laser beam for illumination of the scanning laser microscope is sent to the scanning unit 10 via the single mode fiber 9a. Is to lead to.
[0043]
On the other hand, an incident end of a single mode fiber 9b as a second optical fiber is arranged on the optical path 6b for evanescent illumination, and guides the laser light for evanescent illumination to the epi-illumination tube 11 via the single mode fiber 9b. It has become.
[0044]
The scanning unit 10 is provided with a visible laser light introduction port 10a for introducing laser light emitted from the single mode fiber 9a. A collimator lens 12 and an excitation dichroic mirror 13 are arranged on the optical path of laser light emitted from the visible laser light introduction port 10a.
[0045]
The collimating lens 12 converts laser light emitted from the visible laser light introduction port 10a into collimated light. The excitation dichroic mirror 13 has such characteristics that it reflects the wavelength of laser light (488 nm, 543 nm) and transmits the wavelength range of fluorescence (500 to 530 nm and 560 to 650 nm) emitted from the sample 25 described later.
[0046]
On the reflected light path of the excitation dichroic mirror 13, a wavefront conversion element 14 as a light condensing position control means and a galvano mirror unit 15 as an optical scanning means are arranged.
[0047]
The wavefront conversion element 14 is capable of converting the wavefront of the laser light as disclosed in, for example, Patent Document 3 and Patent Document 4. That is, the reflection surface can be minutely changed by applying a voltage to the element itself. By changing the reflection surface and changing the optical power, the optical axis in the sample 25 can be changed as described later. It is possible to move the beam condensing position in the direction without moving the mechanical positional relationship between the objective lens 24 and the sample 25. In addition, it is also possible to cancel the aberration generated at that time.
[0048]
FIG. 3 illustrates the operation state of the wavefront conversion element 14. When the reflection surface 14a is flat as shown by the solid line in FIG. 3, there is no optical power, and the laser light reflected from the reflection surface 14a When the reflecting surface 14a is minutely deformed (curved) by applying a voltage as shown by a broken line in the drawing, the laser light reflected by the deformed reflecting surface 14b is The light is emitted as slightly divergent light as indicated by a broken line 14d.
[0049]
A galvanomirror unit 15 is arranged on the reflected light path of the wavefront conversion element 14. The galvanomirror unit 15 has two galvanomirrors 15a and 15b for deflecting light in two orthogonal directions. The galvanomirrors 15a and 15b scan laser light in a two-dimensional direction. I have.
[0050]
On the other hand, a confocal lens 16, a reflecting mirror 17, a confocal pinhole 18, which constitutes a confocal observation means, and a fluorescence wavelength region to be detected by cutting the excitation laser light are extracted on the transmission optical path of the excitation dichroic mirror 13 described above. The barrier filter 19 and the light detector 20 are arranged. The photodetector 20 here uses, for example, a photomultiplier.
[0051]
The scanning unit 10 is connected to a microscope body, here an inverted microscope body 22, via a pupil projection lens unit 21. The inverted microscope main body 22 has a bottom port 22a formed in a hole 50a formed on the table 50, and the scanning unit 10 is connected to the bottom port 22a via the pupil projection lens unit 21. .
[0052]
A pupil projection lens 21a in the pupil projection lens unit 21, an imaging lens 23 in the inverted microscope main body 22, and an objective lens 24 are arranged on the optical path of the laser light emitted from the galvanometer mirror unit 15 of the scanning unit 10. Have been.
[0053]
A fluorescently labeled sample 25 is arranged at the light-converging position of the tip lens 24b of the objective lens 24. The sample 25 is fixed to a cover glass 28 attached to a petri dish 27 arranged on a stage 26 as shown in FIG. In this case, the petri dish 27 is filled with a liquid for protecting the sample 25, for example, water. Immersion oil is filled between the tip lens 24b of the objective lens 24 and the cover glass 28 so as to secure a high NA and cause total reflection by evanescent illumination.
[0054]
On the other hand, the above-mentioned incident light projection tube 11 is provided with a laser light introduction port 11a for introducing laser light emitted from the single mode fiber 9b. A collimator lens 31 and a condenser lens 32 are arranged on the optical path of the laser light emitted from the laser light introduction port 11a.
[0055]
The collimating lens 31 converts laser light emitted from the laser light introduction port 11a into collimated light. The condenser lens 32 condenses the collimated light from the collimator lens 31 to the pupil position 24a of the objective lens 24.
[0056]
An evanescent excitation dichroic mirror 33 is arranged in an optical path between the condenser lens 32 and the objective lens 24 (between the imaging lens 23 and the objective lens 24). The evanescent excitation dichroic mirror 33 combines the optical paths such that the respective optical axes lead coaxially while the laser light from the scanning unit 10 and the laser light from the epi-illumination light pipe 11 are on their way to the objective lens 24. Then, the laser beam for the evanescent illumination from the condenser lens 32 is reflected, the fluorescence emitted from the sample 25 is transmitted by the evanescent illumination, and the wavelength of the laser beam for the scanning laser microscope illumination from the other scanning unit 10 and its wavelength It has a property of transmitting the fluorescence emitted from the sample 25 by the irradiation of the laser beam.
[0057]
A plurality of evanescent excitation dichroic mirrors 33 are prepared in accordance with the wavelength of laser light for evanescent illumination and illumination of a scanning laser microscope, and the wavelength of fluorescence emitted from the sample 25. These evanescent excitation dichroic mirrors 33 are turrets. 331, and can be selectively switched on the optical path by an electric switching mechanism (not shown).
[0058]
In the optical path between the imaging lens 23 and the evanescent excitation dichroic mirror 33, a separation dichroic mirror 35 is disposed. The separation dichroic mirror 35 reflects the fluorescence emitted from the sample 25 by the evanescent illumination and transmits the wavelength of the other laser light for illumination of the scanning laser microscope and the wavelength of the fluorescence emitted from the sample 25 by the irradiation of the laser light. It has the following characteristics.
[0059]
The separation dichroic mirror 35 can be retracted from the optical path by an electric moving mechanism (not shown), and is retracted from the optical path when performing only illumination and observation by the scanning laser microscope.
[0060]
In the reflection optical path of the separation dichroic mirror 35, a barrier filter 36 as filter means, an imaging lens 37, and a CCD detector 38 as image pickup means are arranged.
[0061]
The barrier filter 36 has characteristics such that the respective wavelengths of the laser beams for evanescent illumination and scanning laser microscope illumination are cut, and the wavelength range of the fluorescence emitted from the sample 25 by the evanescent illumination is transmitted. The imaging lens 37 forms an image of the fluorescence from the sample 25 emitted by the evanescent illumination that has passed through the barrier filter 36 on the light receiving surface 38 a of the CCD detector 38. The CCD detector 38 captures the fluorescent light imaged on the light receiving surface 38a.
[0062]
Reference numeral 39 denotes a control unit. The control unit 39 electrically switches the evanescent excitation dichroic mirror 33, inserts and removes the separation dichroic mirror 35 from the optical path, captures an image with the CCD detector 38, and uses a galvanomirror in the scanning unit 10. The control of the unit 15, the wavefront conversion element 14, and the photodetector 20, the control of the scanning laser microscope in the laser light source unit 1, and the control of the AOTFs 8a and 8b for selecting each of the illumination laser wavelengths of evanescent are performed.
[0063]
Next, the operation of the first embodiment configured as described above will be described.
[0064]
In this embodiment, it is assumed that evanescent illumination is performed by 488 nm laser light of the Ar laser 2 and illumination of the scanning laser microscope is performed by 543 nm laser light of the green helium neon laser 3.
[0065]
In this case, the evanescent excitation dichroic mirror 33 has a characteristic of reflecting a wavelength of 488 nm and transmitting a wavelength range of 500 to 650 nm, and the separation dichroic mirror 35 reflects a wavelength range of 500 to 530 nm. Those having a characteristic of transmitting a wavelength range of 543 to 650 nm are used. The barrier filter 36 has a characteristic of transmitting fluorescence in a wavelength range of 500 to 530 nm and cutting a wavelength range of 488 nm and a wavelength range of 543 to 700 nm, and the barrier filter 19 in the scanning unit 10 has a wavelength of 488 nm. A material that cuts the wavelength range of ５４543 nm and transmits the wavelength range of 560 to 650 nm is used.
[0066]
First, fluorescence observation using evanescent illumination will be described.
[0067]
In this case, when a laser beam of 488 nm is oscillated by the Ar laser 2, the wavelength of the laser beam is selected by the AOTF 8b and guided to the epi-illumination tube 11 by the single mode fiber 9b for evanescent illumination.
[0068]
The laser light introduced into the epi-illumination light pipe 11 is collimated by a collimating lens 31 and is condensed by a condenser lens 32 at an end of a pupil position 24 a of the objective lens 24. In this case, as shown in FIG. 2, the tip lens 24b of the objective lens 24 is focused on a region of the sample 25 near the sample-side interface 28a of the cover glass 28. As a result, the laser beam 24c (hatched portion) condensed at the end of the pupil position 24a of the objective lens 24 becomes parallel light by the tip lens 24b, and irradiates the sample-side interface 28a of the cover glass 28 at an oblique direction at a certain angle. You. This angle is set to an angle at which the laser light 24c causes total reflection at the sample-side interface 28a, and the totally reflected laser light 24c passes through the direction 24c 'in the figure. At this time, a very small part of the laser light oozes from the sample-side interface 28a of the cover glass 28 to the sample 25 side, and the light that oozes out from the sample-side interface 28a in the depth direction of the sample 25 becomes evanescent light. The amount of the sample 25 oozing in the depth direction is about the wavelength of the light source (here, 500 nm).
[0069]
Therefore, the range of the fluorescence emitted from the sample 25 by such evanescent illumination becomes about the wavelength width in the depth direction, and the fluorescence image to be detected has a low background and can be observed with good S / N. A detailed description of this is described in Patent Document 2, for example.
[0070]
In addition, fluorescence (500 to 530 nm) emitted from the sample 25 by the laser beam for evanescent illumination of 488 nm passes through the center of the objective lens 24, passes through the evanescent excitation dichroic mirror 33, and is reflected by the separation dichroic mirror 35. . Then, only the fluorescence wavelength emitted from the sample 25 by the evanescent illumination is transmitted by the barrier filter 36 and captured by the CCD detector 38.
[0071]
Next, fluorescence observation using illumination by a scanning laser microscope will be described.
[0072]
In this case, when a laser beam of 543 nm is oscillated by the green helium neon laser 3, the wavelength of the laser beam is selected by the AOTF 8a and guided to the scanning unit 10 by the single mode fiber 9a for illumination of the scanning laser microscope.
[0073]
The 543 nm laser light introduced into the scanning unit 10 is collimated by the collimating lens 12 and reflected by the excitation dichroic mirror 13.
[0074]
The laser light reflected by the excitation dichroic mirror 13 is reflected by the wavefront conversion element 14 and enters the galvanomirror unit 15. Then, the light passes through the pupil projection lens 21a and the imaging lens 23, passes through the separation dichroic mirror 35, and the evanescent excitation dichroic mirror 33, enters the objective lens 24, and forms an image in the sample 25.
[0075]
When the sample 25 is irradiated with a laser beam of 543 nm, the fluorescence (560 to 650 nm) wavelength emitted from the sample 25 travels in the above-described path of the illumination light in the reverse direction and passes through the excitation dichroic mirror 13 in the scanning unit 10. Then, the light is condensed on a confocal pinhole 18 via a confocal lens 16 and a reflecting mirror 17. In this case, the confocal pinhole 18 is narrowed to the diffraction limit of the light to be collected. Thus, the fluorescent light emitted from the sample 25 can cut off light components other than the focal plane of the sample 25. Then, the fluorescent light (560 to 650 nm) transmitted through the confocal pinhole 18 transmits through the barrier filter 19 and is detected by the photodetector 20. In this case, the output detected by the photodetector 20 is used to acquire a slice image of the sample 25.
[0076]
Next, a case where the focusing position of the laser light for illumination of the scanning laser microscope in the sample 25 is variably controlled in the optical axis direction by the wavefront conversion element 14 will be described.
[0077]
In this case, when the reflection surface 14a of the wavefront conversion element 14 is flat as shown by the solid line in FIG. 3, there is no optical power, and the laser light reflected by the wavefront conversion element 14 As described above, the light enters the galvanomirror unit 15 as parallel light, and after the luminous flux diameter is enlarged by the pupil projection lens 21a and the imaging lens 23, the light enters the objective lens 24 as parallel light. The laser beam at this time is a solid line 24e shown in FIG. 2, and the position where the image is formed in the sample 25 coincides with the focal position of the objective lens 24, and is the same as the focus plane for observing the fluorescence by evanescent illumination.
[0078]
Next, when a voltage is applied to the wavefront conversion element 14, the reflecting surface 14a is slightly deformed (curved) as shown by the broken line in the figure, and the laser light reflected by the deformed reflecting surface 14b is slightly deformed as shown by the broken line 14d. The light is emitted as divergent light.
[0079]
When the diverging light 14d enters the objective lens 24, the diverging light 14d becomes like a diverging light 24f indicated by a dotted line shown in FIG. 2, and is collected in a position 25f inside the sample 25 inside the sample-side interface 28a of the cover glass 28. Light.
[0080]
Then, the light emitted from the position 25f in the sample 25 travels in the same direction as the above-mentioned illumination light in the opposite direction, returns to the wavefront conversion element 14, and is converted into parallel light by the deformed reflection surface 14b of the wavefront conversion element 14. The light is converted and returns to the excitation dichroic mirror 13 to be transmitted. Then, the light emitted from a position 25f in the sample 25 is detected by the photodetector 20 through the confocal lens 16, the reflection mirror 17, the confocal pinhole 18, and the barrier filter 19.
[0081]
Similarly, the voltage applied to the wavefront conversion element 14 is similarly varied, and the shape of the reflecting surface is arbitrarily changed, thereby condensing the laser light for scanning laser microscope illumination in the optical axis direction in the sample 25. The position can be arbitrarily changed, and light emitted from these focal positions can be detected.
[0082]
Therefore, this configuration includes variable control of the observation section in the optical axis direction with the mechanical positional relationship between the objective lens 24 and the sample 25 fixed so as to satisfy the condition for performing the evanescent illumination on the sample 25. Both confocal observation with a scanning laser microscope and fluorescence observation with evanescent illumination can be performed. This makes it possible to realize a microscope system that is easy to use for fluorescence observation by evanescent illumination even if the focal position in the optical axis direction on the scanning laser microscope side is positively changed. Further, since the reflection surface shape of the wavefront conversion element 14 can be switched at a high speed of several MHz as disclosed in Patent Document 3, for example, confocal observation of the inside of the sample 25 by a scanning laser microscope and evanescent illumination It is also possible to perform fluorescence observation of the cell surface by switching at high speed.
[0083]
At this time, the imaging timing of the CCD detector 38 for imaging the fluorescence by the evanescent illumination, the two-dimensional scanning of the laser light by the galvanomirror unit 15, the switching of the reflection surface shape by the wavefront conversion element 14, the wavelength selection of the AOTFs 8a and 8b Each control such as the above needs to be performed by the control unit 39 in synchronization. For example, when a laser beam for scanning laser microscope illumination is focused on a position 25f in the sample 25 and a partial area in the plane direction is observed at the position 25f, the wavefront conversion element 14 is used as shown in FIG. And the scanning angle of the galvanometer mirrors 15a and 15b of the galvanometer mirror unit 15 is changed in order to specify the scanning position of the sample 25 in a plane orthogonal to the optical axis. Further, the photodetector 20 controls to detect the intensity of light corresponding to the swing angle (scanning position) of the galvanometer mirror unit 15. The AOTF 8a selects light of 543 nm, and the AOTF 8b does not select light of 488 nm and 543 nm.
[0084]
Next, when switching to fluorescence observation by evanescent illumination, the AOTF 8a does not select the laser light of 488 nm and 543 nm, but selects the laser light of 488 nm by the AOTF 8b. Then, imaging is performed by the CCD detector 38. Here, not only the switching of the wavefront conversion element 14, but also the wavelength selection of the AOTFs 8a and 8b can be performed at a high speed of several MHz, so that both observation switching can be performed at a high speed.
[0085]
In this way, it is possible to realize a microscope system in which fluorescence observation by evanescent illumination and confocal observation by laser light for scanning laser microscope illumination can be synchronously switched at a high speed, and observation can be easily performed simultaneously.
[0086]
In the first embodiment, as a modification, when cells are used as the sample 25 instead of using laser light for scanning laser microscope illumination for confocal observation, light stimulation is applied to the cells. It is possible to use for. In this case, a light stimulus is applied to one point inside the sample 25, and the time-dependent change in the amount of fluorescence on the cell surface at that time is observed by fluorescence observation using evanescent illumination. At this time, the movement of the position of the light stimulus by the laser light for scanning laser microscope illumination can be performed at high speed by controlling the wavefront conversion element 14 and the galvanometer mirror unit 15. In this case, by changing the ratio of the laser light to the optical paths 6a and 6b separated by the beam splitter 7, the intensity of the laser light for scanning laser microscope illumination can be adjusted to an optimum intensity.
[0087]
In the first embodiment described above, the laser light source for scanning laser microscope illumination and the light source for laser light for evanescent illumination, that is, the Ar laser 2 and the green helium neon laser 3 are housed in the common laser light source unit 1. Then, the laser light emitted from the Ar laser 2 and the green helium neon laser 3 is split into two optical paths by the beam splitter 7 and introduced into two single-mode fibers 9a and 9b, which are respectively guided to the illumination optical paths. The Ar laser 2 and the green helium neon laser 3 may be provided separately. In this case, the light quantity is not reduced by the beam splitter 7.
[0088]
Further, the light source of the laser light for illumination of the scanning laser microscope and the light source of the laser light for evanescent illumination may be realized by one laser light source. In this case, since the laser light for scanning laser microscope illumination and the laser light for evanescent illumination have the same wavelength, confocal observation using a scanning laser microscope and fluorescence observation using evanescent illumination are alternately switched. do it.
[0089]
(Second embodiment)
Next, a second embodiment of the present invention will be described.
[0090]
FIG. 4 shows a schematic configuration of a second embodiment of the present invention, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0091]
In the second embodiment, an IR pulse laser capable of causing two-photon excitation to a sample is added as laser light for illumination of a scanning laser microscope.
[0092]
The two-photon excitation scanning laser microscope is disclosed in, for example, Japanese Patent Application Laid-Open No. H11-157, and is illuminated at an infrared wavelength, so that the illumination light has good permeability to the sample, and the illumination light extends to a deep part of the sample. It is suitable for thick samples because it reaches it. The location where two-photon excitation occurs is limited to the focus position where high instantaneous energy is collected, so fluorescence and photochemical reactions are limited to one point inside the thick sample. Can be generated. This photochemical reaction includes local release of a chemical substance in a sample, such as caged. Normally, ultraviolet light having a short wavelength and high energy is used to release the caged compound. In the case of two-photon excitation, an IR pulse laser having low energy can be used by collecting two photons.
[0093]
In the figure, reference numeral 41 denotes an IR pulse laser unit. The IR pulse laser unit 41 is an acousto-optic modulator (hereinafter referred to as AOM) which can switch between a pulse laser light source main body 42 for oscillating 720 nm pulse light and laser irradiation at high speed. 43. The AOM 43 is controlled by the control unit 39.
[0094]
A reflection mirror 44 is arranged on the optical path of the light emitted from the AOM 43.
[0095]
The second port 10b of the scanning unit 10 is disposed on the reflection optical path of the reflection mirror 44. At the second port 10b of the scanning unit 10, a beam expander unit 45 for converting the beam diameter of the laser beam from the reflection mirror 44 to an appropriate diameter is attached as a light-converging position control means.
[0096]
As shown in FIG. 5, the beam expander unit 45 has lenses 45a and 45b that constitute means for converting the laser beam diameter to an appropriate beam diameter on the optical axis. Of these lenses 45a and 45b, the position of the lens 45a can be moved in the optical axis direction, that is, in the direction of the arrow 45c by an electric moving mechanism (not shown), and the movement of the lens 45a causes the beam expander unit 45 to move. The degree of collimation of the emitted beam can be changed.
[0097]
A reflection mirror 46 is arranged on the optical path of the light emitted from the beam expander unit 45. In addition, an optical path combining dichroic mirror 47 is disposed on the reflection optical path of the reflection mirror 46.
[0098]
The optical path synthesizing dichroic mirror 47 is inserted in the optical path between the collimator lens 12 and the excitation dichroic mirror 13 and reflects the wavelength of 720 nm incident from the reflecting mirror 46 and transmits the wavelengths of 488 nm and 543 nm. The reflection optical path of the reflection mirror 46 is coupled to the optical path of the visible light laser (488 nm, 543 nm) from the visible laser light introduction port 10a.
[0099]
Note that when such an IR pulse laser is used, a fluorescence tomographic image can be obtained by two-photon excitation. In this case, the two-photon excitation phenomenon occurs only at the imaging position, so that the confocal pin Since a hole can be eliminated, fluorescence may be extracted from the optical path between the pupil projection lens 21a and the imaging lens 23 and detected by a photodetector such as a photomultiplier.
[0100]
Others are the same as FIG. 1 mentioned above.
[0101]
In the second embodiment, a pulse laser having a wavelength of 720 nm from the pulse laser light source main body 42 is used as the illumination laser light of the scanning laser microscope, and the three-dimensional thickness and surface directions of the sample 25 are used. By performing fluorescence observation by evanescent illumination using the wavelength (488 nm) of the Ar laser 2 mounted on the laser light source unit 1 while causing caged release at an arbitrary arbitrary position, the cell surface, which is the sample 25, can be measured over time. It is possible to observe target changes.
[0102]
Next, the operation of the second embodiment configured as described above will be described.
[0103]
In this case, the evanescent excitation dichroic mirror 33 has a characteristic of reflecting 488 nm and transmitting in a wavelength range of 500 to 900 nm, and can be selectively switched on the optical path by an electric switching mechanism (not shown). I have. The separation dichroic mirror 35 has a characteristic of reflecting a wavelength range of 500 to 650 nm and transmitting a wavelength of 720 nm. The barrier filter 36 has a characteristic of transmitting fluorescence in a wavelength range of 500 to 650 nm and cutting wavelengths of 488 nm and 720 nm. The excitation dichroic mirror 13b in the scanning unit 10 has a characteristic of reflecting a wavelength range of 720 to 900 nm and transmitting a wavelength range of 500 to 650 nm.
[0104]
Now, when a pulse laser of 720 nm oscillates from the pulse laser light source main body 42, the pulse laser is incident on the second port 10b of the scanning unit 10 via the AOM 43 and the reflection mirror 44.
[0105]
The pulse laser light introduced into the second port 10b passes through the beam expander unit 45, and is reflected by the excitation dichroic mirror 13 via the reflection mirror 46 and the optical path combining dichroic mirror 47.
[0106]
Further, the light is reflected by the wavefront conversion element 14, passes through the galvanomirror unit 15, the pupil projection lens 21 a, and the imaging lens 23, passes through the separation dichroic mirror 35, and the evanescent excitation dichroic mirror 33. Is collected.
[0107]
As a result, the sample 25 is excited by two photons at the focal point of the pulsed laser light in the sample 25, and the caged state is released. The position in the sample 25 to be caged can be arbitrarily changed in the optical axis direction, which is the thickness direction of the sample 25, by controlling the movement of the lens 45a of the beam expander unit 45 in the direction of the arrow 45c shown in FIG. By means of the 15 scanning controls, it can be arbitrarily changed in a plane perpendicular to the optical axis in the sample 25.
[0108]
Here, the operation of the beam expander unit 45 will be further described with reference to FIGS.
[0109]
Now, if the beam emitted from the lens 45a in the beam expander unit 45 is in a state of parallel light (solid line in the drawing), a laser beam shown by a solid line 24e in FIG. The light is focused near the interface 28a between the cover glass 28 and the sample 25 by the tip lens 24b. On the other hand, when the position of the lens 45a is moved to a position 45d indicated by a dotted line by an electric moving mechanism (not shown), the collimation state of the laser light emitted from the beam expander unit 45 changes, and a slight diverging light It becomes.
[0110]
When this diverging light is incident on the objective lens 24, it becomes like a diverging light 24f indicated by a dotted line in FIG. 2 and is condensed at a position 25f inside the sample 25 inside the sample-side interface 28a of the cover glass 28. I do. By controlling the beam expander unit 45 in this manner, the focusing position in the optical axis direction in the sample 25 can be changed.
[0111]
In this way, the beam expander unit 45 and the galvanomirror unit 15 control the condensing position in the sample 25, that is, the position at which the pulsed laser is irradiated and the caged position can be released by two-photon excitation. It can be freely selected dimensionally.
[0112]
Strictly speaking, when the light incident on the objective lens 24 becomes divergent light, a wavefront aberration occurs and the imaging performance deteriorates, but this is not a problematic level. In particular, in the second embodiment, since the illumination light from the scanning unit 10 is used to cause a photochemical reaction in the sample 25, it is not necessary to consider the performance degradation on the detection side.
[0113]
Next, the operation of fluorescence observation using evanescent illumination will be described.
[0114]
In this case, when a laser beam of 488 nm oscillates from the Ar laser 2 in the laser light source unit 1, the wavelength of the laser beam of 488 nm is selected by the AOTF 8b, and guided to the incident light projection tube 11 by the single mode fiber 9b. Then, the light is collimated by the collimator lens 31 and is condensed by the condenser lens 32 at the end of the pupil position 24a of the objective lens 24. At this time, the objective lens 24 is focused on a sample area close to the sample-side interface 28a of the cover glass 28. As a result, the evanescent light oozing from the interface of the cover glass 28 toward the sample emits fluorescence. As described in the first embodiment, the range in which the fluorescence is emitted is about the wavelength width in the depth direction, and observation with a low background and good S / N is possible.
[0115]
Fluorescence (500 to 530 nm) emitted from the sample 25 by the evanescent illumination of 488 nm passes through the center of the objective lens 24, passes through the evanescent excitation dichroic mirror 33, and is reflected by the separation dichroic mirror 35. Then, the fluorescence by the evanescent illumination is imaged by the CCD detector 38.
[0116]
Therefore, even in this case, the irradiation position of the sample 25 with the laser beam of 720 nm for the illumination of the scanning laser microscope can be changed with the galvanometer mirror unit 15 without changing the mechanical positional relationship between the objective lens 24 and the sample 25. Since the beam can be freely selected by the beam expander unit 45, a part of the deep part of the sample 25 is photostimulated by a laser beam of 720 nm for scanning laser microscope illumination, and evanescent illumination is performed while causing cage release by two-photon excitation. Can be observed. That is, at least one of the control of the condensing position of the laser light on the surface of the sample 25 by the beam expander unit 45 and the control of the two-dimensional scanning of the laser light on the sample 25 by the galvanometer mirror unit 15 and the operation of the CCD detector 38 By synchronizing the imaging of fluorescence, observation by evanescent illumination can be performed while cage release is caused by two-photon excitation.
[0117]
In this case, the timing of the irradiation of the 720 nm laser light can be controlled at a high speed by the AOM 43. For example, fluorescence observation by evanescent illumination is performed by the CCD detector before and after the cage release from the scanning unit 10 by the 720 nm laser light occurs. 38, the irradiation of 720 nm laser light from the scanning unit 10 is repeated at a predetermined cycle, and during this period, the fluorescence observation by the evanescent illumination is imaged plural times by the CCD detector 38. And so on. This makes it possible to observe, for example, a change with time of the surface of the sample 25 due to the release of the deep cage of the sample 25 with high S / N in real time. In such a synchronous operation, the irradiation timing of the 720 nm laser light from the scanning unit 10 and the imaging timing by the CCD detector 38 do not necessarily have to be the same.
[0118]
Further, in the above description, the number of points in the sample 25 where the cage release is to be performed is one, but a plurality of points may be used. In this case, the plurality of points are switched by the galvanometer mirror unit 15 in the plane of the sample 25, the beam expander unit 45 in the optical axis direction, and the AOM 43 in the irradiation timing control. , Respectively, in synchronization with the imaging by the CCD detector 38.
[0119]
In this manner, a scanning laser microscope system can be obtained in which a plurality of points in the sample 25 are sequentially irradiated with laser light, and the aging of the surface of the sample 25 can be observed with high S / N while canceling caged state. Further, the evanescent illumination and the imaging by the CCD detector 38 and the irradiation of the 720 nm laser light from the scanning unit 10 are not necessarily required to be simultaneous, and the timing of the irradiation time of the 720 nm laser light by the AOM 43 is controlled. It is also possible to aim at the time between each frame imaging of the CCD detector 38. In this case, as a characteristic of the barrier filter 36 in the optical path of the CCD detector 38, there is no need to cut off light having a wavelength of 720 nm, which not only facilitates manufacturing but also improves the transmittance in the fluorescent wavelength region to be captured. There are advantages such as.
[0120]
It should be noted that the second embodiment is not limited to the above-described contents, and various modifications are possible. For example, considering the characteristics of the separation dichroic mirror 35, the barrier filter 36, the excitation dichroic mirror 13 and the barrier filter 19 in the scanning unit 10, the laser light source unit 1 described in the first embodiment can be added to the system described above. It is also possible to incorporate confocal observation by using a laser beam for illumination of a scanning laser microscope of 543 nm oscillating from. In this case, the timing of irradiating the sample 25 with the 543 nm illumination laser light from the scanning unit 10 and the timing of irradiating the 488 nm evanescent illumination laser light onto the sample 25 are synchronized. Thereby, the confocal observation and the observation by the evanescent illumination are performed in synchronization, and the timing of the light detection by the photodetector 20 and the timing of the imaging of the fluorescence by the CCD detector 38 are also performed in synchronization.
[0121]
In this case, the separation dichroic mirror 35 has a characteristic of reflecting the wavelength range of 500 to 530 nm and transmitting the wavelength range of 543 to 720 nm, and the barrier filter 36 transmits the fluorescence of the wavelength range of 500 to 530 nm. In addition, those having characteristics of cutting a wavelength of 488 nm and a wavelength range of 543 to 720 nm are used. The excitation dichroic mirror 13 in the scanning unit 10 has a characteristic of reflecting the wavelengths of 543 nm and 720 nm and transmitting the wavelength range of 560 to 650 nm, and the barrier filter 19 has the wavelength of 488 to 543 nm and 720 nm. Is used, and a material having a characteristic of transmitting a wavelength range of 560 to 650 nm is used.
[0122]
When performing confocal observation of the inside of the sample 25 with the 543 nm laser light from the laser light source unit 1, the wavefront converting element 14 may change the focusing position in the sample 25 in the optical axis direction. , As described in the first embodiment.
[0123]
With the above configuration, cage release inside the sample by the pulse laser, confocal observation inside the sample 25 by the 543 nm laser light from the laser light source unit 1, and sample by evanescent illumination using the 488 nm laser light from the laser light source unit 1 A microscope system capable of performing fluorescence observation of the surface 25 simultaneously or with switching while the mechanical positional relationship between the objective lens 24 and the sample 25 is fixed.
[0124]
Further, the IR pulse laser was used to cause a photochemical reaction by two-photon excitation in the sample. For example, the wavelength of the IR pulse laser was set to about 800 nm, and the fluorescence tomography inside the sample 25 caused by the two-photon excitation was set. Obtaining an image and observing the cell surface by evanescent illumination may be combined. Although the irradiation position of the 720-nm pulse laser is varied in the optical axis direction by the beam expander unit 45, the irradiation position may be controlled by the wavefront conversion element 14. In this case, switching can be performed at a higher speed than that by the beam expander unit 45.
[0125]
Further, the imaging by the CCD detector 38 is limited to fluorescence observation generated by evanescent illumination, but this may be replaced by fluorescence observation by rotation of a disk such as a Nipkow disk or a stripe disk.
[0126]
FIG. 6 shows a schematic configuration of a main part of a microscope system which enables a fluorescence observation system using such a disk. In this case, a mercury lamp is separately prepared as an illumination light source. When the illumination light is emitted from this illumination light source, it enters the beam splitter 61. Then, the light transmitted through the beam splitter 61 is incident on a disk (Nipkow disk or stripe disk) 62 which is rotated at a constant speed by a motor. The light transmitted through the disk 62 enters the imaging lens 63. The light passing through the imaging lens 63 forms an image on the pupil of the objective lens 24 and irradiates the sample 25. The light returned from the sample 25 becomes parallel light through the objective lens 24 and forms an image on the disk 62 through the imaging lens 63. Then, the light emitted from the disk 62 is reflected by the beam splitter 61 and is captured as a confocal image by a CCD detector 64 as an imaging means. That is, the observation image obtained by rotating the Nipkow disk or the stripe disk is obtained by capturing the sliced surface of the cell with the CCD detector 64. Then, the focal position of the objective lens 24 is fixed to this slice plane, and the focal position of the laser light for illumination of the scanning laser microscope is selected using the wavefront conversion element 14 and the galvanomirror unit 15. For example, it is possible to perform imaging by the CCD detector 64 and scanning observation by the scanning laser microscope simultaneously or by switching.
[0127]
In the above description, a case in which a photochemical reaction is caused in the sample by two-photon excitation and a case in which confocal observation can be performed by switching between confocal observation using laser light for illumination with a scanning laser microscope have been described. If not, the wavefront conversion element 14 can be omitted.
[0128]
(Third embodiment)
Next, a third embodiment of the present invention will be described.
[0129]
FIG. 7 shows a schematic configuration of the third embodiment of the present invention, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0130]
In the first embodiment, the evanescent illumination is performed through the objective lens via the epi-illumination tube using the inverted microscope. However, in the third embodiment, the evanescent illumination is performed using the upright microscope. The difference is that the measurement is performed via a condenser unit on the opposite side of the objective lens with respect to the sample.
[0131]
In the figure, reference numeral 51 denotes an upright microscope main body, and a revo arm 52 to which the objective lens 24 is fixed is connected to the upright microscope main body 51 so as to be finely movable in the optical axis direction.
[0132]
A condenser lens 53a is arranged at a position facing the objective lens 24 on the optical axis. The condenser lens 53a is provided in the condenser lens unit 53. The condenser lens unit 53 is connected to the revo arm 52 by a guide mechanism (not shown), and is configured to be movable with respect to the revo arm 52 in the optical axis direction.
[0133]
A stage 54 supported by a support member (not shown) is disposed between the objective lens 24 and the condenser lens 53a. The sample 25 is mounted on the stage 54. In this case, the sample 25 is placed on a cover glass 28 fixed to a hole of a perforated petri dish 27 as shown in FIG. 8, and a petri dish 27 is provided between the tip lens 24b of the objective lens 24 and the sample 25. Is filled with water 27a contained therein. Further, between the back side of the cover glass 28 and the condenser lens 53a, immersion oil is filled to cause total reflection at the interface between the cover glass 28 and the sample 25 filled with water.
[0134]
An evanescent lighting unit 55 is attached to the condenser lens unit 53. The evanescent illumination unit 55 is connected to the emission end of the single mode fiber 9b for evanescent illumination, and is provided with a collimator lens 55a for collimating the light emitted from the single mode fiber 9b and a collimator light at the rear focal position of the condenser lens 53a. A condenser lens 55b for condensing light is provided.
[0135]
The condenser lens unit 53 is provided with a switching mirror 53c. The switching mirror 53c is inserted into the optical path when performing evanescent illumination, and is retracted from the optical path when performing transmission illumination by the transmission illumination lamp house 56 and observation (position 53c ').
[0136]
An intermediate lens barrel 57 is arranged above the objective lens 24. The intermediate lens barrel 57 performs coaxial epi-illumination of fluorescent light by a mercury lamp house 58, and includes a relay lens 57a and an excitation dichroic mirror 57b. In this case, the excitation dichroic mirror 57b is retracted from the optical path (position of 57b) when the observation by the evanescent illumination is performed by the CCD detector 38 and the illumination and scanning observation is performed by the scanning unit 10, and the mercury lamp lamp house 58 It is arranged in the optical path when performing fluorescent coaxial epi-illumination with a mercury lamp and observation (position 57b ').
[0137]
Above the intermediate lens barrel 57, a lens barrel 59 is arranged. The lens barrel 59 has an imaging lens 60, a separation dichroic mirror 35, and a barrier filter 36. The separation dichroic mirror 35 can be inserted into and removed from the optical path by a command from the control unit 39 by an electric mechanism (not shown). It has become. Further, a CCD detector 38 is arranged on the reflection optical path of the separation dichroic mirror 35. The timing of imaging of the CCD detector 38 is controlled by the control unit 39 as in the first embodiment.
[0138]
Reference numeral 21 denotes a pupil projection lens unit, which is disposed above the lens barrel 59, and further, the scanning unit 10 is disposed above.
[0139]
Other configurations such as the configuration inside the scanning unit 10 and the configuration of the laser unit 1 are the same as those in FIG.
[0140]
Next, the operation of the third embodiment configured as described above will be described.
[0141]
Here, as described in the first embodiment, the evanescent illumination is performed by the 488 nm laser light of the Ar laser 2, and the scanning laser microscope is illuminated by the 543 nm laser light of the green helium neon laser 3. I do. In this case, the separation dichroic mirror 35 has a characteristic of reflecting the wavelength range of 500 to 530 nm and transmitting the wavelength range of 543 to 650 nm, and the barrier filter 36 transmits the wavelength range of 500 to 530 nm. 488 nm and those having characteristics of cutting a wavelength range of 543 to 700 nm are used. The barrier filter 19 in the scanning unit 10 has a characteristic of cutting a wavelength range of 488 to 543 nm and transmitting a wavelength range of 560 to 650 nm. These characteristics are the same as in the first embodiment.
[0142]
In addition, the excitation dichroic mirror 57b in the intermediate lens barrel 57 that performs coaxial epi-illumination by the mercury lamp lamp house 58 is retracted to a position outside the optical path, and the switching mirror 53c and the separation dichroic mirror 35 in the condenser lens 53 are used to They are located on the street.
[0143]
First, the operation for fluorescence observation using evanescent illumination will be described.
[0144]
In this case, when a laser beam of 488 nm is oscillated by the Ar laser 2, the wavelength of the laser beam is selected by the AOTF 8b and guided to the evanescent illumination unit 55 by the single mode fiber 9b for evanescent illumination.
[0145]
The laser light introduced into the evanescent illumination unit 55 is collimated by a collimating lens 55a, and is condensed by a condenser lens 55b to an end of an aperture stop 53b which is a rear focal position of a condenser lens 53a. In this case, as shown in FIG. 8, the tip lens 24b of the objective lens 24 is focused on the sample 25 area close to the sample-side interface 28a of the cover glass 28, and the aperture stop of the condenser lens unit 53. 53 b is positioned so as to have a conjugate relationship with the pupil position of the objective lens 24.
[0146]
As a result, the laser beam 53d (hatched portion) condensed on the end of the aperture stop 53b of the condenser lens unit 53 becomes parallel light by the condenser lens 53a, and irradiates the sample-side interface 28a of the cover glass 28 from a diagonal direction at a certain angle. I do. This angle is set to an angle at which the laser light 53d causes total reflection at the sample-side interface 28a, and the totally reflected laser light 53d exits in the direction 53d 'shown in the figure. At this time, a very small part of the laser light oozes from the sample side interface 28a of the cover glass 28 to the sample 25 side, and the light oozing from the sample side interface 28a becomes evanescent light. The amount of the sample 25 oozing in the depth direction is about the wavelength of the light source (here, 500 nm).
[0147]
Accordingly, the range of the fluorescence emitted from the sample 25 by such evanescent illumination becomes about the wavelength width in the depth direction, and the fluorescence image to be detected can be observed with little background and high S / N. The fluorescence (500 to 530 nm) emitted from the sample by the evanescent illumination laser beam of 488 nm passes through the objective lens 24 and the imaging lens 60 and is reflected by the separation dichroic mirror 35. Then, only the fluorescence wavelength (500 to 530 nm) emitted from the sample by the evanescent illumination is extracted by the barrier filter 36, and the image is captured by the CCD detector 38.
[0148]
Next, fluorescence observation using illumination by a scanning laser microscope will be described.
[0149]
In this case, when a laser beam of 543 nm is oscillated by the green helium neon laser 3, the wavelength of the laser beam is selected by the AOTF 8a and guided to the scanning unit 10 by the single mode fiber 9a for illumination of the scanning laser microscope.
[0150]
The 543 nm laser light introduced into the scanning unit 10 is collimated by the collimating lens 12 and reflected by the excitation dichroic mirror 13.
[0151]
The laser light reflected by the excitation dichroic mirror 13 is reflected by the wavefront conversion element 14 and enters the galvanomirror unit 15. Then, the light passes through the pupil projection lens 21a and the imaging lens 23, passes through the separation dichroic mirror 35, and the evanescent excitation dichroic mirror 33, enters the objective lens 24, and forms an image in the sample 25.
[0152]
When the sample 25 is irradiated with a laser beam of 543 nm, the fluorescence (560 to 650 nm) wavelength emitted from the sample 25 travels in the above-described path of the illumination light in the reverse direction and passes through the excitation dichroic mirror 13 in the scanning unit 10. Then, the light is condensed on a confocal pinhole 18 via a confocal lens 16 and a reflecting mirror 17. The fluorescence (560 to 650 nm) transmitted through the confocal pinhole 18 passes through the barrier filter 19 and is detected by the photodetector 20 composed of a photomultiplier.
[0153]
In this state, the shape of the reflection surface of the wavefront conversion element 14 is changed by applying a voltage. For example, when the reflection surface of the wavefront conversion element 14 is changed and the laser beam is slightly converged as shown by a dotted line 24j in FIG. Is lighted. Then, the fluorescence emitted from this point 25f travels in the opposite direction along the same path as the illumination light, and returns to the wavefront conversion element 14. Further, the light is reflected by the wavefront conversion element 14, converted into parallel light, returned to the excitation dichroic mirror 13, and transmitted. Then, the light passes through the confocal lens 16, the reflecting mirror 17, the confocal pinhole 18, and the barrier filter 19, and is detected by the photodetector 20 as fluorescence emitted from the point 25 f of the sample 25. As described above, by arbitrarily changing the shape of the reflection surface of the wavefront conversion element 14 by applying a voltage, the direction of the optical axis in the sample 25 of the laser light for illumination of the scanning laser microscope from the scanning unit 10 in the sample 25 is obtained. Can be arbitrarily changed.
[0154]
Therefore, even in this case, similarly to the first embodiment, with the mechanical positional relationship between the objective lens 24 and the sample 25 fixed, the scanning laser microscope including the variable control of the observation section in the optical axis direction. , It is possible to perform both confocal observation by using and fluorescence observation by using evanescent illumination.
[0155]
As in the first embodiment, the high-speed switching between observation of the inside of the sample by the scanning laser microscope and observation of the sample surface by the evanescent illumination and use of illumination by the scanning laser microscope for optical stimulation of the sample are also similar to the first embodiment. It goes without saying that it is possible.
[0156]
Further, as an effect unique to the present embodiment that cannot be obtained in the first embodiment, the evanescent illumination is performed not from the objective lens 24 side but from the condenser lens 53a side. The high NA for generating the evanescent illumination and the oil immersion objective) are eliminated, and the degree of freedom of observation is increased. Further, since the evanescent excitation dichroic mirror (corresponding to 33 in the first embodiment) does not exist in the optical path for illuminating the scanning laser microscope, the observation optical path, and the optical path for fluorescence observation by evanescent illumination, bright observation is possible. It becomes. This further increases the degree of freedom in the combination of laser wavelength and fluorescence used in a scanning laser microscope and laser wavelength and fluorescence used in evanescent illumination.
[0157]
In addition, the present invention is not limited to the above-described embodiment, and can be variously modified in an implementation stage without departing from the spirit of the invention.
[0158]
Further, the embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent features. For example, even if some components are deleted from all the components shown in the embodiments, the problem described in the column of the problem to be solved by the invention can be solved, and the problem described in the column of the effect of the invention can be solved. In the case where the effect described above is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0159]
【The invention's effect】
As described above, according to the present invention, confocal observation by a scanning laser microscope and fluorescence observation by vanescent lighting can be performed simultaneously or by switching, and in particular, the focal position in the optical axis direction on the scanning laser microscope side can be set. Even if it is positively changed, it is possible to provide a microscope system that is easy to use with fluorescence observation using evanescent illumination.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.
FIG. 2 is a diagram illustrating a schematic configuration in which main parts of the first embodiment are enlarged.
FIG. 3 is a diagram for explaining a wavefront conversion element used in the first embodiment.
FIG. 4 is a diagram showing a schematic configuration of a second embodiment of the present invention.
FIG. 5 is a diagram for explaining a beam expander unit used in the second embodiment.
FIG. 6 is a diagram showing a schematic configuration of a modification of the second embodiment.
FIG. 7 is a diagram showing a schematic configuration of a third embodiment of the present invention.
FIG. 8 is a diagram illustrating a schematic configuration in which main parts of a third embodiment are enlarged.
[Explanation of symbols]
1. Laser light source unit 2. Ar laser
3 ... Green helium neon laser 4 ... Reflection mirror
5. Dichroic mirror, 6a. 6b: Optical path, 7: Beam splitter
8a. 8b ... AOTF, 9a. 9b… Single mode fiber
10: scanning unit, 10a: visible laser light introduction port
10b: second port, 11: reflected light projection tube, 11a: laser light introduction port
12: collimating lens, 13: excitation dichroic mirror
13a: Excitation dichroic mirror, 14: Wavefront conversion element
14a, 14b: reflective surface, 14d: divergent light
15 ... Galvano mirror unit, 15a. 15b: Galvanometer mirror
16: confocal lens, 17: reflection mirror, 18: confocal pinhole
19: barrier filter, 20: photodetector, 21: pupil projection lens unit
21a: pupil projection lens, 22: inverted microscope body, 22a: bottom port
23: imaging lens, 24: objective lens, 24a: pupil position
24b: tip lens, 24f: divergent light, 25: sample
26: Stage, 27: Petri dish, 27a: Water, 28: Cover glass
28a: sample side interface, 31: collimating lens, 32: condensing lens
33… Excitation dichroic mirror for evanescent
331: Turret, 35: Separable dichroic mirror
36: barrier filter, 37: imaging lens, 38: CCD detector
38a: light receiving surface, 39: control unit, 41: IR pulse laser unit
42: pulse laser light source body, 43: AOM, 44: reflection mirror
45 ... Beam expander unit, 45a. 45b ... Lens
46: Reflection mirror, 47: Optical path combining dichroic mirror
Reference numeral 50: Table, 50a: Hole, 51: Upright microscope body
52: Revo arm, 53: Condenser lens unit
53a: condenser lens, 53c: mirror
53d: laser beam, 54: stage, 55: evanescent lighting unit
55a: Collimating lens, 55b: Condensing lens
56: transmitted illumination lamp house, 57: middle lens barrel, 57a: relay lens
57b: Excitation dichroic mirror, 58: Mercury lamp house
59: lens barrel, 60: imaging lens, 61: beam splitter
62: disk, 63: imaging lens, 64: CCD detector

Claims (15)

A first light source for generating laser light,
An objective lens for converging laser light from the first light source on a sample,
Light scanning means for two-dimensionally scanning the sample with the laser light;
A second light source that generates light for performing evanescent illumination on the sample;
Focusing position control means provided in an optical path of laser light from the first light source and optically variably controlling a focusing position of the laser light on the sample in an optical axis direction of the objective lens;
Imaging means for imaging fluorescence generated by the evanescent illumination,
With the mechanical positional relationship between the objective lens and the sample fixed so as to satisfy the conditions for performing evanescent illumination on the sample, the focusing position of the laser light on the sample surface is controlled by the focusing position control means. A microscope system characterized in that the microscope can be controlled. 2. The microscope system according to claim 1, further comprising confocal observation means for confocal observation of light emitted from a condensing position on the sample on which the laser light from the first laser light source is condensed. . 2. The microscope system according to claim 1, wherein the laser light from the first laser light source causes a photochemical reaction at a focus position on the sample. The second light source is a laser light source that generates laser light, and constitutes a single laser light source unit together with the first light source,
The first and second light sources are respectively provided with first and second optical fibers for deriving laser light, respectively.
A light source for condensing the laser light passing through the first optical fiber on the sample by the objective lens and a light source for performing evanescent illumination on the sample with the laser light passing through the second optical fiber The microscope system according to any one of claims 1 to 3, wherein the microscope system is used. 5. The laser light source unit according to claim 4, wherein the wavelengths of the light generated by the first and second light sources are the same, and the first and second light sources are constituted by one light source. Microscope system. The microscope system according to claim 1, wherein the first and second light sources have different wavelengths of generated light. The microscope system according to any one of claims 1 to 6, wherein the focusing position control unit is a wavefront conversion element. The light condensing position controlling means has a beam diameter converting means for converting a beam diameter of the laser light condensed on the sample, and a means for changing a collimation degree of the laser light emitted from the beam diameter converting means. The microscope system according to claim 1, wherein: 9. The microscope system according to claim 1, wherein the first laser light source generates a two-photon excitation pulse laser. At least one of the control of the condensing position of the laser beam on the sample surface by the condensing position control unit and the control of the two-dimensional scanning of the laser beam on the sample by the optical scanning unit is performed by the imaging unit. The microscope system according to claim 1, wherein imaging of the fluorescence is controlled in synchronization. The confocal observation means has a light detection means for detecting light emitted from a condensing position on the sample,
The microscope system according to claim 10, wherein detection by the light detection unit and imaging of the fluorescence by the imaging unit are controlled in synchronization. 12. The apparatus according to claim 10, wherein timing of irradiating the sample with the laser light from the first laser light source and timing of irradiating the sample with the second light source are synchronized. Microscope system. A filter means for cutting a wavelength of laser light focused on the sample from the first light source is provided on an imaging optical path of an imaging means for imaging fluorescence generated by the evanescent illumination. Item 6. The microscope system according to Item 6, 10, or 11. The microscope system according to any one of claims 1 to 13, wherein the evanescent illumination is performed via a condenser lens disposed on a side opposite to the objective lens with the sample interposed therebetween. A first light source for generating laser light,
An objective lens that focuses laser light from the first laser light source on a sample, an optical scanning unit that two-dimensionally scans the laser light on the sample,
A second light source that generates light for performing illumination on the sample via the objective lens;
Imaging means for imaging light emitted from the sample by illumination from the second light source;
Optical path combining means for guiding the respective optical axes coaxially while the laser light from the light scanning means and the light emitted from the second light source are on their way to the objective lens;
Focusing position control means provided on an optical path of laser light from the first light source, and optically variably controlling a focusing position of the laser light on the sample in an optical axis direction of the objective lens; And
A microscope system, wherein a focusing position of the laser beam on the sample surface can be controlled by the focusing position control unit in a state where a mechanical positional relationship between the objective lens and the sample is fixed.

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