1. Field of the Invention
The present invention relates to a laser microscope, and more particularly to a laser microscope for detecting second harmonic light generated from a sample.
2. Description of Related Art
As a laser microscope utilizing a laser beam, a variety of microscopes have been developed for many purposes. The laser microscope focuses a laser beam emitted from a laser beam source onto a sample, and receives light reflected by or emitted from the sample to thereby observe and examine the sample.
As an example of the laser microscopes, there has been known a confocal microscope. The confocal microscope has attracted attentions in terms of a high resolution and an ability to acquire three-dimensional information on the sample. Known as an example of such a confocal microscope is a confocal microscope that scans the sample surface with irradiated light through the use of a rotating pinhole substrate (for example, see Japanese Unexamined Patent Application Publication No. 05-127090). The light from a light source enters the rotating pinhole substrate having plural pinholes. The split light from the pinholes moves on the sample. The pinholes are formed in the substrate in accordance with a spiral array at equal pitches in the radial and circumferential directions along spiral track so as to prevent the unevenness of brightness on the sample.
As another example of the confocal microscope, a confocal optical scanner having pinholes arranged at a constant density has been known (for example, see Japanese Unexamined Patent Application Publication No. 05-119262). The pinhole may be a microlens. However, the pinhole substrate having the thus-arranged pinholes is insufficient from the viewpoints of preventing the brightness unevenness and increasing the illumination on the sample in some cases. Further, an effective method of designing such an array substrate has not yet been well discussed.
To give another example of the laser microscope, a second harmonic microscope (SHG microscope) has been put into practical use, which measures various physical characteristics of a sample by use of second harmonic light generated by irradiating the sample with a laser beam (Nanophoton Corp., SHG-11 catalogue). The measurement of the characteristics with the SHG microscope is carried out by focusing a laser beam from a laser beam source onto a sample to scan the sample with the beam to detect second harmonic light emitted from the inside of the sample. The SHG microscope is regarded as being especially effective for detection of a structure or function of cells or protein levels in the medical field or in the field of biotechnology.
The SHG microscope detects second harmonic light emitted from the sample with an emission pattern of
However, the conventional SHG microscope detects light transmitted through the sample and thus cannot observe light from a sample if the sample is not transparent. Further, it is difficult to observe light from a sample if the sample is thick like a living body.
As described above, in the conventional SHG microscope, the second harmonic light is emitted in the same direction as the incident light-traveling direction, so only second harmonic light transmitted through a sample can be detected.
The present invention has been completed in view of the aforementioned problems, and it is accordingly an object of the invention to provide a laser microscope capable of detecting second harmonic light emitted in an opposite direction to a traveling direction of incident light.
A laser microscope according to an aspect of the invention includes: a laser beam source(for example, a laser beam source 10 of embodiments of the invention); a phase plate (for example, a phase plate 12 of embodiments of the invention) providing a phase difference for laser beam from the laser beam source in accordance with an incident position; an objective lens (for example, an objective lens 16 of embodiments of the invention) focusing light transmitted through the phase plate onto a sample; a first separating unit (for example, a fundamental light cut filter 22 of embodiments of the invention) separating second harmonic light emitted from the sample in a direction opposite to a traveling direction of the laser beam from a fundamental light reflected by the sample; and a photodetector (for example, a photodetector 19 of embodiments of the invention) detecting the second harmonic light separated from the fundamental light by the first separating unit. Hence, it is possible to detect second harmonic light emitted in the direction opposite to a laser beam traveling direction.
According to a second aspect of the invention, in the laser microscope of the first aspect, the phase plate preferably provides a phase difference of 180° for the laser beam in areas opposite to each other across an optical axis. Hence, an amount of second harmonic light emitted backward can be increased.
According to a third aspect of the invention, in the laser microscope of the first aspect, the phase plate includes ½ wavelength plates optical axes of which are shifted from each other by 90°, in areas opposite to each other across an optical axis. Hence, an intensity of second harmonic light emitted backward can be increased.
According to a fourth aspect of the invention, in the laser microscope of the second or third aspect, an oscillation direction of an electric vector of a laser beam in one of the areas opposite to each other across the optical axis is opposite to an oscillation direction in the other area. Hence, an amount of second harmonic light emitted backward can be increased.
According to a fifth aspect of the invention, the laser microscope of any one of the first to fourth aspects further includes: a second separating unit (for example, a fundamental light cut filter 21 of embodiments of the invention) for separating second harmonic light emitted from the sample in the same direction as the traveling direction of the laser beam from a fundamental light transmitted through the sample; and a photodetector (for example, a photodetector 18 of embodiments of the invention) detecting second harmonic light emitted from the light source in the same direction as a traveling direction of light, wherein the phase plate can be inserted/retracted to/from an optical path. Hence, it is possible to detect the second harmonic light emitted forward and backward.
According to the present invention, it is possible to provide a laser microscope capable of detecting second harmonic light emitted in an opposite direction to a traveling direction of incident light.
The above and other objects, advantages and features of the present invention will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:
The invention will be now described herein with reference to illustrative embodiments. Those skilled in the art will recognize that many alternative embodiments can be accomplished using the teachings of the present invention and that the invention is not limited to the embodiments illustrated for explanatory purposed.
Incidentally, the same components are denoted by identical reference numerals throughout the accompanying drawings, and description thereof is omitted if not necessary.
Referring to
In
In the SHG microscope structured as shown in
As the laser beam source 10 of this embodiment, a laser device capable of emitting second harmonic light and multiphoton-excited fluorescence is used. For example, at the time of observing living cells, an infrared light pulse laser device such a mode-locked titanium sapphire laser device may be used as the laser beam source 10, for example. Laser light characteristics such as a laser-light wavelength, a laser light intensity, an oscillation mode, a repetition frequency, and a pulse width are appropriately selected in accordance with a sample type or an observation method.
The beam expander 11 expands a beam diameter of light from the laser beam source 10 to emit the expanded beam. The expanded beam from the beam expander 11 enters the phase plate 12. In the case of detecting second harmonic light generated from the sample 30 and propagating backward, the phase plate 12 is placed in an optical path. In the case of detecting second harmonic light emitted from the sample 30 and propagating forward, the phase plate 12 is retracted from the optical path. The phase plate 12 is described below in detail. The light emitted from the phase plate 12 is refracted by the lenses 20a and 20b to enter the beam splitter 13. A part of the light incident on the beam splitter 13 passes through the beam splitter 13 and enters the galvanometer mirror 14.
The light reflected by the galvanometer mirror 14 is refracted by the lenses 20c and 20d to enter the galvanometer mirror 15. The galvanometer mirror 14 and the galvanometer mirror 15 scan a beam to enable imaging and observation of the entire surface of the sample. The light reflected by the galvanometer mirror 15 is refracted by the lenses 20e and 20f to enter the incidence-side objective lens 16. The objective lens 16 focuses the incident laser beam, and the focused beam enters the sample 30.
The sample 30 receives the incident light to emit multiphoton-excited fluorescence and second harmonic light having a frequency twice as high as the incident light. A typical multiphoton-excited fluorescence is a two-photon excited fluorescence. The second harmonic light and the multiphoton-excited fluorescence that are emitted from sample 30 and propagating forward partially transmit the sample 30. The second harmonic light and multiphoton-excited fluorescence transmitted the sample 30 are condensed by the transmission-side objective lens 17.
Further, the fundamental light transmitted through the sample 30 is also collected by the transmission-side objective lens 17. The transmission-side objective lens 17 and the incidence-side objective lens 16 are provided on opposite sides of the sample 30. The sample 30 is placed between the incidence-side objective lens 16 and the transmission-side objective lens 17. Preferably, the two objective lenses 16 and 17 can focus beams at substantially the same focal point on the sample.
The light from the objective lens 17 is refracted by the lenses 20g and 20h, and the refracted light enters the fundamental light cut filter 21. The fundamental light cut filter 21 separates the second harmonic light from the fundamental light and the multiphoton-excited fluorescence. That is, only the second harmonic light out of the light emitted from the objective lens 17 and incident to the fundamental light cut filter 21 passes through the fundamental light cut filter 21. The fundamental light cut filter 21 may be, for example, a band-pass filter or short wavelength pass filter that shields light having a wavelength region of output light from the laser beam source 10 and light having a wavelength region of multiphoton-excited fluorescence. Alternatively, the fundamental light cut filter 21 may be a dichroic mirror provided diagonally to an optical axis. If the fundamental light cut filter 21 is changed to a band-pass filter that shields light having a second harmonic light wavelength region and light having a wavelength region of the fundamental light, the multiphoton-excited fluorescence can be detected.
The second harmonic light transmitted through the fundamental light cut filter 21 enters the photodetector 18. The light incident on the photodetector 18 is focused by the lens 20h or the like to enter a light receiving surface of the photodetector 18. The photodetector 18 is, for example, a two-dimensional photosensor such as a CCD camera. It is preferred to provide the photodetector 18 with an image intensifier to effectively detect the second harmonic light as faint light. The photodetector 18 detects the incident light to convert the light into a video signal. The video signal is input to an image processor (not shown) for processing an image, Hand a taken image is displayed on a display.
Meanwhile, the second harmonic light emitted from the sample 30 toward a direction opposite to the propagating direction of incident light travels in the direction opposite to the propagating direction of incident light. That is, the second harmonic light emitted backward enters the objective lens 16 and travel towards the laser beam source 10. The second harmonic light is refracted by the objective lens 16, and the lenses 20f and 20e to enter the galvanometer mirror 15. The second harmonic light reflected by the galvanometer mirror 15 is refracted by the lenses 20d and 20c to enter the galvanometer mirror 14. The second harmonic light reflected by the galvanometer mirror 14 enters the beam splitter 13. The beam splitter 13 reflects a part of the incident light toward the photodetector 19. The light reflected by the beam splitter 13 is refracted by the lenses 20i and 20j to enter the fundamental light cut filter 22. The fundamental light cut filter 22 separates only the second harmonic light from the multiphoton-excited fluorescence and the fundamental light that propagates in the same direction as the second harmonic light. That is, only the second harmonic light out of the light incident from the objective lens 16 onto the fundamental light cut filter 22 passes through the fundamental light cut filter 22. The fundamental light cut filter 22 may be structured similar to the transmission-side fundamental light cut filter 21.
Then, the second harmonic light transmitted through the fundamental light cut filter 21 enters the photodetector 19. The light incident on the photodetector 19 is focused by the lens 20j or the like to enter the light receiving surface of the photodetector 19. The photodetector 19 is, for example, a two-dimensional sensor such as a CCD camera. It is preferred to provide the photodetector 19 with an image intensifier to effectively detect the second harmonic light as faint light. The photodetector 19 detects the incident light to convert the light into a video signal. The video signal is input to an image processor (not shown) for processing an image, and a taken image is displayed on a display.
Referring next to
The laser beam incident on the phase plate 12 is emitted with a phase difference in accordance with the thickness difference. In this example, a phase difference between the laser beam from the upper area 12a and that from the lower area 12b is 180°. That is, the thickness difference between the upper area 12a and the lower area 12b of the phase plate 12 is set to an optical distance that is half the wavelength of laser beam. Accordingly, the laser beam transmitted through the phase plate 12 involves a spatial phase difference; the phase difference between the upper half and the lower half is 180°. That is, the phase plate 12 provides a phase difference for the laser beam in accordance with incident positions. Incidentally, in the above description, the thickness of the phase plate 12 is non-uniform, but a transmissive film may be formed on a flat transparent plate.
If the phase plate 12 is placed in the optical path, the second harmonic light from the sample 30 is emitted backward. A mechanism of emitting the second harmonic light backward is described below. First, an effect of arranging the phase plate 12 in the optical path is described with reference to
If the phase plate 12 as shown in
The arrow of
Description is made of an example where the light oscillating in such a direction is focused by the objective lens 16. The light transmitted through the upper area 12a is refracted downwardly by the objective lens 16. Accordingly, the oscillation direction of the electric vector of the light extends diagonally upward to the right as shown in
Next, description is given of a state in which the light transmitted through the phase plate 12 is focused on the sample 30 by the objective lens 16. In this example, the oscillation direction of the electric vector of the light is described with regard to two component types: components vertical to the light traveling direction and components parallel to the light traveling direction. Incidentally, in
After the transmission through the objective lens 16, the oscillation direction of the electric vector extends diagonally upward right in the upper area 12a, and the oscillation direction extends diagonally downward right in the lower area 12b. Hence, the components in the vertical direction are opposite to each other. Thus, in such a state that the light is focused on the sample 30, the vertical components in the oscillation direction of the electric vector cancel each other. Accordingly, components of the electric vector vertical to the light traveling direction are reduced to substantially 0. That is, on the sample, the electric vector of the light does not oscillate in the direction vertical to the traveling direction.
The oscillation direction of the electric vector extends diagonally upward right in the upper area 12a and extends diagonally downward right in the lower area 12b. Thus, the horizontal components extend to the right. As a result, the horizontal components of the electric vector in the upper area 12a and the lower area 12b are reinforced with each other. Accordingly, components of the electric vector parallel to the light traveling direction are enhanced to the right. That is, the electric vector of the light oscillates in the direction parallel to the light traveling direction. The objective lens 16 focuses the laser beam that has the phase different after transmitted through the phase plate 12, so the light is irradiated to the sample 30 in such a state that the electric vector oscillates in the direction parallel to the traveling direction.
Next, the light is irradiated to the sample 30 in such a state that the electric vector oscillates in the direction parallel to the traveling direction. The principle for emitting the second harmonic light backward is described with reference to
First, referring to
Meanwhile, description is given of the emission pattern of the second harmonic light in the SHG microscope having the phase plate 12 according to this embodiment. In this embodiment, since the phase plate 12 is provided, the electric vector of the light irradiated to the sample 30 oscillates in parallel to the traveling direction. When the light oscillating in parallel to the traveling direction is irradiated to the sample 30, the polarized molecules oscillate laterally. That is, the oscillation direction of the polarized molecules is changed in accordance with the oscillation direction of the electric vector. An emission pattern obtained at this time is inclined with respect to the emission pattern 40 of
Here, from the viewpoint of increasing a light intensity of the second harmonic light emitted backward, it is preferable to use the objective lens 16 having the large numerical aperture. Thus, an angle at which the light is refracted by the objective lens 16 increases, making it possible to increase components oscillating in the direction parallel to the traveling direction. For example, as shown in
In this way, the second harmonic light emitted in the direction opposite to the traveling direction of the incident light enters the photodetector 19 by way of the objective lens 16 or the like. Then, an image of the second harmonic light emitted backward is taken by the photodetector 19. Hence, an second harmonic light image can be easily taken in the case of using a non-transparent sample such as a semiconductor or a thick sample such as a living body, and an application range of the SHG microscope can be widened.
Incidentally, the phase plate 12 is structured as shown in
The phase plate 12 structured as shown in
As described above, if the phase plate 12 is structured as shown in
The phase plate 12 of
If the thus-structured phase plate 12 is used, the oscillation direction of the electric vector is shifted by 180° between the opposite areas. That is, the oscillation direction of the electric vector of the light transmitted through the upper area 12a is opposite to that of the light transmitted through the lower area 12b. Further, the oscillation direction of the electric vector of the light transmitted through the left area 12c is opposite to that of the light transmitted through the right area 12d. The objective lens focuses the light transmitted through the phase plate 12, and thus the electric vector of the incident light oscillates in parallel to the traveling direction on the sample. Hence, the second harmonic light is emitted in the direction opposite to the traveling direction. Then, an image of the second harmonic light emitted backward is taken by the photodetector 19. As a result, an the second harmonic light image can be easily taken in the case of using a non-transparent sample such as a semiconductor or a thick sample such as a living body, and an application range of the SHG microscope can be widened. As described above, the phase plate 12 divided into four areas is used, making it possible to increase components oscillating in parallel to the direction of the electric vector as compared with the phase plate 12 divided into two areas as shown in
Incidentally, in the illustrated examples of
Needless to say, the phase plate 12 is not limited to the above structure, and may be structured insofar as a phase difference corresponding to an incident position can be set to the laser beam to shift the phase of the electric vector. For example, if a liquid crystal optical element is used, the phase of the electric vector can be shifted in accordance with an incident position. Further, the objective lens focuses the beam having the electric vectors out of phase, and thus the electric vector of the incident light has components oscillating in parallel to the traveling direction. Hence, the second harmonic light can be emitted in the direction opposite to the traveling direction of the incident light.
For detecting the second harmonic light emitted forward, the phase plate 12 may be retracted from the optical path. That is, second harmonic light emitted forward or backward can be detected by inserting or retracting the phase plate 12 to/from the optical path.
Referring to
In this embodiment, a sample 30 is put on the XY stage (not shown). Then, the XY stage is scanned to observe and image the entire surface of the sample. In this SHG microscope as well, if the phase plate 12 of the first embodiment is used, the second harmonic light emitted backward can be detected as in the first embodiment. Thus, similar effects to the first embodiment can be obtained. Further, in this embodiment, the sample is scanned using the XY stage, so an optical system can be simplified.
Referring to
In this embodiment, the microlens array disk 25 is interposed between the beam expander 11 and the lens 20a. Further, the phase plate 12 is provided between the lens 20a and the lens 20b. The laser light incident on the microlens array disk 25 is split into plural beams and then enters the lens 20a. The objective lens 16 focuses the incident multi-beam onto the sample. The multi-beam split by the microlens array disk 25 is concentrated to form multi-focal points on the sample 30 due to an image formation function of the objective lens 16. The laser beams move on the sample 30 by the microlens array disk 25 rotating. In this SHG microscope as well, if the phase plate 12 of the first embodiment is used, the second harmonic light emitted backward can be detected as in the first embodiment. Thus, similar effects to the first embodiment can be obtained.
It is apparent that the present invention is not limited to the above embodiment that may be modified and changed without departing from the scope and spirit of the invention.
Number | Date | Country | Kind |
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2005-249191 | Aug 2005 | JP | national |