The present invention relates to a beam splitter apparatus, a scanning observation apparatus, a laser-scanning microscope, and a laser-scanning endoscope.
In the related art, there are known scanning microscopes that acquire an image of a specimen by two-dimensionally scanning a beam over the specimen (for example, see Patent Literature 1). According to Patent Literature 1, it is possible to change an image-acquisition region in the depth direction of the specimen by moving the focal point of the beam also in the optical-axis direction by using a wavefront converting device.
The lifetime of fluorescence generated by molecular reactions in a biological subject is assumed to be about three nanoseconds. Therefore, in order to observe reactions occurring at different depths in the biological subject at substantially same timescale as the fluorescence lifetime, the beam needs to be modulated at a high speed of several hundreds of megahertz. Because the wavefront converting device moves the position of the focal point of the beam by mechanically changing the shape of a reflecting surface thereof, there is a limit in principle to enhancing the speed at which the focal point is moved.
A first aspect of the present invention is a beam splitter apparatus that is applied to an observation apparatus that irradiates a specimen with pulsed light beams to induce a molecular reaction in the specimen and that observes signal lights generated by this reaction, the beam splitter apparatus including a demultiplexing portion that splits an input pulsed light beam into a plurality of optical paths; relay optical systems that are provided in the plurality of optical paths and that relay the pulsed light beams guided through the individual optical paths; a multiplexing portion that multiplexes the plurality of the pulsed light beams that have been relayed through the individual optical paths by the relay optical systems; delaying portions that are provided in the plurality of optical paths and that give the pulsed light beams, which are guided through the individual optical paths, relative time delays that are large enough to separate the plurality of the signal lights from each other; and divergence-angle setting portions that are provided in the plurality of optical paths and that give the pulsed light beams that are guided through the individual optical paths divergence angles that are different from each other.
A second aspect of the present invention is a scanning observation apparatus including any one of the beam splitter apparatuses described above; a scanning portion that scans a plurality of pulsed light beams emitted from the beam splitter apparatus in a direction that intersects the optical axes; an observation optical system that irradiates the specimen with the pulsed light beams scanned by the scanning portion; and a detection system that detects the signal lights coming from the specimen.
A third aspect of the present invention is a laser-scanning microscope including any one of the scanning observation apparatuses described above; and a laser light source that supplies the beam splitter apparatus with pulsed laser beams that serve as the pulsed light beams.
A fourth aspect of the present invention is a laser-scanning endoscope including any one of the scanning observation apparatuses described above.
A beam splitter apparatus 1 according to a first embodiment of the present invention and a laser-scanning microscope 100 provided with the same will be described below with reference to
First, the overall configuration of the laser-scanning microscope 100 will be described.
As shown in
The laser light source 2 emits the pulsed laser beam L0 that induces a reaction, for example, photoemission, of specific molecules contained in the specimen A.
As will be described later in detail, the beam splitter apparatus 1 generates the four pulsed laser beams L1 to L4 by dividing the ray bundle of the single pulsed laser beam L0, which has entered the beam splitter apparatus 1 by coming from the laser light source 2, into a plurality of beams, and individually applies different delay times and divergence angles to the four generated pulsed laser beams L1 to L4. By doing so, as shown in
The scanning portion 3 is provided with two mirrors 3a and 3b that are rotatable about two mutually perpendicular axes. The scanning portion 3 is configured so as to perform raster scanning of the pulsed laser beams L1 to L4 in a plane that intersects the optical axis by appropriately changing the direction in which the pulsed laser beams L1 to L4 are reflected by controlling the rotational angles of the two mirrors 3a and 3b. A plane that is optically conjugate with the pupil of the objective lens 5b is positioned on the reflecting surfaces of the mirrors 3a and 3b or between the mirrors 3a and 3b. A lens pair that relays the pupil may be disposed between the beam splitter apparatus 1 and the mirror 3a as needed.
The observation optical system 5 is provided with an imaging lens 5a that forms an image by using the pulsed laser beams L1 to L4 that have passed through the pupil projection lens 10 and an objective lens 5b that makes the pulsed laser beams L1 to L4 with which an image is formed by the imaging lens 5a converge on the specimen A.
The detection system 6 is provided with a dichroic mirror 6a that reflects, among the beams that have been made to converge by the observation optical system 5, only the signal light LS, a detecting portion 6b that detects the signal light LS reflected by the dichroic mirror 6a, and a detection lens 6c that focuses the signal light LS at an photoreceiver of the detecting portion 6b.
The control portion 7 synchronizes the timing at which the signal light LS is detected by the detecting portion 6b with the timing at which the pulsed laser beam L0 is emitted from the laser light source 2.
The restoring portion 8 restores two-dimensional information or three-dimensional information by associating the signal light LS detected by the detecting portion 6b with the scanning positions of the pulsed laser beams L1 to L4, and outputs the restored two-dimensional information or three-dimensional information to the display portion 9.
Next, the operation of the thus-configured laser-scanning microscope 100 will be described.
The pulsed laser beam L0 emitted from the laser light source 2 is converted to the four pulsed laser beams L1 to L4 at the beam splitter apparatus 1, and these are subsequently made to enter the observation optical system 5 via the scanning portion 3, are radiated onto the specimen A from the observation optical system 5, and are used to perform raster scanning over the specimen A.
Regarding the signal light LS, such as fluorescence, generated at the specimen A due to the irradiation with the pulsed laser beams L1 to L4, the light-detecting portion 6b detects the signal light LS and converts it to an electrical signal corresponding to the intensity thereof, the restoring portion 8 associates the signal with the position on the specimen A, thus generating an image, and the generated two-dimensional image or three-dimensional image is displayed on the display portion 9. At this time, as shown in
Next, the beam splitter apparatus 1 according to this embodiment will be described.
As shown in
In the main optical path 11a and 11b, a first beam splitter (demultiplexing portion) 13a, a second beam splitter (demultiplexing portion) 13b, and a third beam splitter (multiplexing portion) 13c are provided in series in this order from the entrance side. The single pulsed laser beam L0 is divided twice by the first beam splitter 13a and the second beam splitter 13b, and thus, the four pulsed laser beams L1 to L4 that have travelled along the different optical paths 11a, 11b, 12a, and 12b join at the third beam splitter 13c so as to be emitted therefrom along an exit optical axis Oout, which is an extension of the entrance optical axis Oin. In the following, of the main optical path, the portion between the first beam splitter 13a and the second beam splitter 13b will be referred to as a first main optical path 11a, and a portion between the second beam splitter 13b and the third beam splitter 13c will be referred to as a second main optical path 11b.
Specifically, the first beam splitter 13a divides the pulsed laser beam L0 into two beams, one of which is reflected into the first delay optical path 12a, and the other beam passes straight through into the first main optical path 11a. The second beam splitter 13b divides the pulsed laser beam that comes thereinto via the first main optical path 11a into two beams, one of which is reflected into the second delay optical path 12b, and the other beam passes straight through into the third beam splitter 13c. Furthermore, the second beam splitter 13b divides the pulsed laser beam that comes thereinto via the first delay optical path 12a into two beams, one of which is reflected into the second delay optical path 12b, and the other beam passes straight through into the third beam splitter 13c.
By doing so, the pulsed laser beam L1 that has passed through the first main optical path 11a and the second main optical path 11b, the pulsed laser beam L2 that has passed through the first main optical path 11a and the second delay optical path 12b, the pulsed laser beam L3 that has passed through the first delay optical path 12a and the second main optical path 11b, and the pulsed laser beam L4 that has passed through the first delay optical path 12a and the second delay optical path 12b join at the third beam splitter 13c. The third beam splitter allows the pulsed laser beams that have travelled along the second main optical path 11b to pass therethrough and reflects the pulsed laser beams that have travelled along the second delay optical path 12b, thus emitting the four pulsed laser beams L1 to L4 along the single exit optical axis Oout.
Note that, in the optical-path configuration shown in
In the individual optical paths 11a, 11b, 12a, and 12b, pairs of lenses 141a and 142b, 142a and 142b, 143a and 143b, and 144a and 144b are provided as relay optical systems that form conjugate surfaces S that are conjugate with the image planes.
In addition, first mirror pair (divergence-angle setting portions) 151a and 151b are provided between the pair of lenses 143a and 143b in the first delay optical path 12a, and second mirror pair (divergence-angle setting portions) 152a and 152b are provided between the pair of lenses 144a and 144b in the second delay optical path 12b. The first mirror pair 151a and 151b fold back the pulsed laser beam that has been reflected by the first beam splitter 13a toward the second beam splitter 13b so as to reach the main optical path 11a by tracing out a rectangular shape. The second mirror pair 152a and 152b fold back the pulsed laser beam that has been reflected by the second beam splitter 13b toward the third beam splitter 13c so as to reach the main optical path 11b by tracing out a rectangular shape.
Here, divergence angles that the individual mirror pair 151a and 151b and mirror pair 152a and 152b give to the pulsed laser beams are determined in accordance with the positions of the individual mirror pair 151a and 151b and mirror pair 152a and 152b in the direction perpendicular to the main optical path 11a and 11b (hereinafter, referred to as Z′-direction), and focal points of the individual pulsed laser beams are formed at different positions in the optical-axis direction depending on the differences among the divergence angles. Specifically, the individual mirror pair 151a and 151b and mirror pair 152a and 152b are disposed at positions that are shifted, in directions perpendicular to the main optical path 11a and 11b, from the reference positions indicated by two-dot chain lines in
By doing so, when the four pulsed laser beams L1 to L4 emitted from the beam splitter apparatus 1 are focused by the objective lens 5b, focal points F1 to F4 are formed at different depths in the specimen A, as shown in
In this case, with the beam splitter apparatus 1 according to this embodiment, the four pulsed laser beams L1 to L4 that are emitted from the third beam splitter 13c and that are finally radiated onto the specimen A have relative time delays due to the fact that the optical path lengths of the individual optical paths 11a, 11b, 12a, and 12b are different from each other, and are sequentially emitted from the beam splitter apparatus 1 with time intervals that correspond to the optical-path-length differences d therebetween. Specifically, when assuming that the frequency of the pulsed laser beam L0 is Q Hz, the speed of light is C m/s, a delay level d of the second delay optical path 12b is c/4Q m, the frequency of the pulsed laser beams L1 to L4 emitted from the beam splitter apparatus 1 is 4Q Hz, and thus, the repetition frequency of the pulsed laser beams L0 appears to be multiplied.
Here, the optical-path-length differences d are set so that the relative time delays possessed by the pulsed laser beams L1 to L4 become greater than the lifetime of the signal light LS. For example, in the case of observing fluorescence from GFP, which is a typical fluorescent protein, because the lifetime of this fluorescence is about three nanoseconds, the pulsed laser beams L1 to L4 possess relative time delays with respect to each other that are equal to or greater than three nanoseconds.
By shifting the times at which the specimen A is irradiated with the pulsed laser beams L1 to L4 by the amount of time attributed to the relative time delays, it is possible to make the time intervals among the four pulsed laser beams L1 to L4 sufficiently short so that the four pulsed laser beams L1 to L4 can be assumed to be radiated onto the specimen A essentially at the same time while allowing signal lights LS generated at the observation planes P1 to P4 to be detected as signals that are distinct from each other. By doing so, there is an advantage in that it is possible to observe molecular reactions occurring at the four observation planes P1 to P4 having different depths at the same point in time.
Note that, in this embodiment, the individual mirror pair 151a and 151b and mirror pair 152a and 152b may be provided in such a way that they can be moved together in the Z′-direction.
By doing so, it is possible to change the intervals among the focal points F1 to F4 of the pulsed laser beams L1 to L4 in the optical-axis direction, that is, the intervals in the Z-direction among the observation planes P1 to P4. Specifically, it is possible to change the positions of the third observation plane P3 and the fourth observation plane P4 in the Z-direction together by moving the first mirror pair 151a and 151b, and it is possible to change the positions of the second observation plane P2 and the fourth observation plane P4 in the Z-direction together by moving the second mirror pair 152a and 152b.
In addition, although the beam splitter 13c possessing no polarizing property is employed as a multiplexing portion in this embodiment, alternatively, a half-wave plate may be disposed in any of the main optical paths 11a and 11b and the second delay optical path 12b in addition to employing a polarizing beam splitter.
By doing so, it is possible to decrease the amount of light loss in the multiplexing portion.
Next, modifications of the beam splitter apparatus 1 according to this embodiment will be described.
As shown in
The first delay optical path 12a has a rectangular optical path formed via mirrors M1 and M2 and the other mirror pair 153a and 153b. By moving the other mirror pair 153a and 153b together in the direction parallel to the main optical path 11a and 11b, the positions of the focal points F1 to F4 of the individual pulsed laser beams L1 to L4 are changed. The relationship between the amount of movement XM of the mirror pair 153a and 153b and the amount of movement XE of the focal points F1 to F4 in the specimen A at this time is expressed by the expression below. Therefore, by employing lenses 145a and 145b having short focal distances, it is possible to decrease the amount of movement of the mirror pair 153a and 153b that is required to change the positions of the focal points F1 to F4.
2XM=XE(mfR/fp)2
Assuming that m is the magnification of the objective lens 5b, d is the working distance of the objective lens 5b, fPB is the back focus of the pupil projection lens 10, fp is the focal distance of the pupil projection lens 10, and fR is the focal distances of the lenses 145a and 145b in front of and behind the mirror pair 153a and 153b, the relationship below is satisfied:
|2XM|<fR,fPB(fR/fp)2,d(mfR/fp)2
As shown in
In the case in which the specimen A is a scatterer, such as biological tissue, the signal lights LS are subjected to increasing influences of scattering and aberration due to the specimen A with increasing depths of the positions of the observation planes P1 to P4, and thus, the intensities of the signal lights LS detected by the detecting portion 6b decrease. Therefore, by adjusting the intensities of the individual pulsed laser beams L1 to L4 as in this modification, it is possible to compensate for the variability in the detected intensities of the signal lights LS caused by the fact that irradiation positions of the individual pulsed laser beams L1 to L4 differ in the depth direction.
In addition, in the case in which the mirror pair 151a and 151b and the mirror pair 152a and 152b are provided in a movable manner, the intensities of the pulsed laser beams L1 to L4 may be automatically adjusted by moving the ND filters 16 together with the movement of the mirror pair 151a and 151b and that of the mirror pair 152a and 152b by using the ND filters 16 whose transmittances change in a continuous manner. In this case, the relationship between the amounts of movement of the mirror pair 151a and 151b and that of the mirror pair 152a and 152b and the amounts of change in the detected intensities of the signal lights LS should be ascertained by measuring or calculating them in advance.
As shown in
Of the pulsed laser beams that have entered them, the individual polarizing beam splitters 13a and 13c allow P-polarization components to pass therethrough and reflect S-polarization components. Therefore, by adjusting the polarization state of the pulsed laser beam L0 by using the half-wave plate 17a in front of the first polarizing beam splitter 13a, it is possible to change the splitting ratio of the pulsed laser beam at the polarizing beam splitter 13a. The half-wave plate 17c converts the polarization states of the pulsed laser beams that pass through the first delay optical path 12a to P-polarization from S-polarization, thus converting the pulsed light beams that have passed through the second beam splitter 13b so as to be uniformly P-polarized. The half-wave plates 17b and 17d can change transmittances/reflectances of the pulsed laser beams at the polarizing beam splitter 13c by adjusting the polarization states of the pulsed laser beams in the individual optical paths 11b and 12b, which are P-polarized. As a result, it is possible to adjust the intensities of the individual pulsed laser beams L1 to L4.
By doing so also, as with the second modification, it is possible to compensate for the variability in the detected intensities of the signal lights LS caused by the fact that the irradiation positions of the individual pulsed laser beams L1 to L4 differ in the depth direction.
As shown in
By doing so, as shown in
Furthermore, by horizontally moving the optical axes of the pulsed laser beams between the mirror pairs in the direction perpendicular to the plane of the figure by changing the angles of the mirror pair 151a and 151b and that of the mirror pair 152a and 152b, it is possible to make the positions of the focal points F1 to F4 of the pulsed laser beams L1 to L4 also differ in the X-direction.
As shown in
By doing so, as shown in
In this modification, the first mirror pair 151a and 151b may be shifted from the reference positions in the Y′-direction, and the second mirror pair 152a and 152b may be shifted from the reference positions in the Z′-direction. In this case, in
As shown in
By doing so, as compared with the positions of the focal points F1 to F4 shown in
In this modification, the first mirror pair 151a and 151b may be shifted from the reference positions in the Z′-direction, and the second mirror pair 152a and 152b may be shifted from the reference positions in the Y′-direction and the Z′-direction. In this case, in
Next, a beam splitter apparatus 1′ according to a second embodiment of the present invention and a laser-scanning microscope provided with the same will be described below with reference to the drawings.
In this embodiment, configurations differing from those of the above-described first embodiment will mainly be described, and configurations common with those of the first embodiment will be given the same reference signs and descriptions thereof will be omitted.
A laser-scanning microscope according to this embodiment is configured in the same manner as the laser-scanning microscope 100 according to the first embodiment.
As shown in
Here, the individual focusing lenses 18a to 18c are disposed at positions that are shifted, in the respective optical-axis directions, from the reference positions at which focal points of all of the pulsed laser beams L1 to L4 are formed at the same position. By doing so, as with the first embodiment, when the four pulsed laser beams L1 to L4 emitted from the beam splitter apparatus 1′ are focused by the objective lens 5b, focal points are formed at different depths in the specimen A, as shown in
Note that, in this embodiment, the individual focusing lenses 18a to 18c may be provided so as to be movable in the optical-axis directions of the individual optical paths 11a, 12a, and 12b. By doing so, it is possible to change the intervals in the Z-direction among the individual observation planes P1 to P4 by changing the intervals in the Z-direction among the focal points F1 to F4 of the individual pulsed laser beams L1 to L4.
Specifically, it is possible to change the positions of the first observation plane P1 and the second observation plane P2 in the Z-direction together by moving the focusing lens 18a, it is possible to change the positions of the third observation plane P3 and the fourth observation plane P4 in the Z-direction together by moving the focusing lens 18b, and it is possible to change the positions of the second observation plane P2 and the fourth observation plane P4 in the Z-direction together by moving the focusing lens 18c. At this time, because the delay times possessed by the individual pulsed laser beams L1 to L4 do not fluctuate in association with the movement of the focusing lenses 18a to 18c, it is possible to shift the positions of the focal points F1 to F4 in the optical-axis direction without changing the respective time-delay levels of the four of pulsed light beams L1 to L4. Therefore, there is an advantage in that time control can easily be performed.
In addition, in this embodiment, although
In addition, as the third beam splitter 13c of the multiplexing portion in this embodiment, a half-wave plate may be disposed in the second delay optical path 12b in addition to employing a polarizing beam splitter. By doing so, it is possible to decrease the amount of light loss in the multiplexing portion.
In addition, this embodiment may be combined with the configuration described in the first embodiment in which divergence angles are given to the individual pulsed laser beams L1 to L4 by adjusting the positions of the individual mirror pair 151a and 151b and mirror pair 152a and 152b.
In addition, in this embodiment, as in the second modification and the third modification of the first embodiment, the intensities of the individual pulsed laser beams L1 to L4 may be adjusted by using the ND filters disposed in the individual optical paths 11a, 11b, 12a, and 12b or by utilizing a combination of the polarizing beam splitters and the half-wave plates.
Next, a modification of the beam splitter apparatus 1′ according to this embodiment will be described.
A beam splitter apparatus according to a modification of this embodiment differs from the beam splitter apparatus 1′ in that the focusing lens 18a and the focusing lens 18b are provided so as to be movable along the optical axes in synchronization with each other by means of motors (not shown).
The first observation plane P1 and the second observation plane P2 are moved in the Z-direction by moving the focusing lens 18a, and the third observation plane P3 and the fourth observation plane P4 are moved in the Z-direction by moving the focusing lens 18b. Therefore, by moving these two focusing lenses 18a and 18b in a synchronized manner, it is possible to move all of the observation planes P1 to P4 together in the Z-direction. By doing so, it is possible to acquire a three-dimensional image of the specimen A at high speed by scanning the four pulsed laser beams L1 to L4 at high speed not only in the X-direction and the Y-direction but also in the Z-direction.
Note that in the first and second embodiments, another set of the first to third beam splitters 13a to 13c may be connected in series behind the first to third beam splitters 13a to 13c, and another set of the main optical path 11a and 11b and the delay optical paths 12a and 12b described above may be formed.
By doing so, the number of pulsed laser beams to be generated from the single pulsed laser beam L0 by using the beam splitter apparatus 1 or 1′ can be increased from four to 16, 64, and so on.
Note that, although the first and second embodiments have been described by using a laser-scanning microscope as an example, the beam splitter apparatuses and the scanning observation apparatuses of the present invention can also be applied to a laser-scanning endoscope. Specifically, by providing an observation optical system 5 and a wave guiding path (for example, an optical fiber) that receives the signal lights LS and transmits them to the detection system 6 at the distal-end portion of an inserted portion provided in the laser-scanning endoscope, it suffices to supply the pulsed laser beams L1 to L4 to the observation optical system 5, via an optical fiber or the like, from the beam splitter apparatus 1 or 1′ disposed at the basal end of the inserted portion.
Number | Date | Country | Kind |
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2013-034592 | Feb 2013 | JP | national |
This is a continuation of International Application PCT/JP2014/054513, with an international filing date of Feb. 25, 2014, which is hereby incorporated by reference herein in its entirety. This application claims the benefit of Japanese Patent Application No. 2013-034592, filed on Feb. 25, 2013, the content of which is incorporated herein by reference.
Number | Date | Country | |
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Parent | PCT/JP2014/054513 | Feb 2014 | US |
Child | 14833252 | US |