JP4912738B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope, and more particularly to a laser scanning microscope that combines observation laser light and operation laser light and irradiates a specimen with the same objective lens.

2. Description of the Related Art Conventionally, there is known a laser scanning microscope that includes an observation laser light source and an operation laser light source and two-dimensionally scans a specimen with observation laser light and operation laser light by separate scanning means (for example, , See Patent Document 1).
In this laser scanning microscope, the observation laser beam and the operation laser beam are combined and then focused on the specimen by the same objective lens.
JP 2002-82287 A

  However, since the fluorescence generated in the specimen is branched from the observation laser light after returning the optical path of the observation laser light via the scanning means, the multiplexing means for combining the operation laser light with the observation laser light Fluorescence is lost when passing through. In particular, when the number of operating laser beams increases, the loss of fluorescence increases, and there is a disadvantage that a bright fluorescent image cannot be obtained.

  The present invention has been made in view of the above-described circumstances, and even if the number of operation laser beams combined with the observation laser beam is increased, a fluorescence loss is reduced and a bright fluorescence image is acquired. It is an object of the present invention to provide a laser scanning microscope that can be used.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to an observation laser light source that emits an observation laser light, an operation laser light source that emits a plurality of operation laser lights for operating a sample, and a plurality of operation lasers emitted from the operation laser light source. An operation laser beam combining means for combining the light, a scanning means for two-dimensionally scanning the observation laser light, and a plurality of the operation laser lights. to a plurality of laser beams position adjusting means for adjusting the irradiation position, respectively two-dimensionally, and the observation laser beam scanned by the scanning means and the position adjusted manipulation laser beam by the laser beam positioning means multiplexes An objective lens for condensing the fluorescence generated in the specimen while condensing the observation laser light and the operation laser light multiplexed by the multiplexing means and irradiating the specimen; and the objective lens By A light detector for detecting the emitted fluorescence, and the operation laser light combining means combines the plurality of operation laser lights before combining with the observation laser light by the combining means. A laser scanning microscope is provided that is positioned.

According to the present invention, the observation laser light emitted from the observation laser light source is two-dimensionally scanned by the scanning unit and condensed on the sample by the objective lens. On the other hand, the plurality of operation laser beams emitted from the operation laser light source are adjusted to the two-dimensional arrangement by the operation of the laser beam position adjusting unit, and then combined with the observation laser beam by the operation of the combining unit. Waves are collected on the specimen by the objective lens.

  The observation laser light is condensed on the specimen, thereby exciting the fluorescent substance in the specimen and generating fluorescence. The generated fluorescence returns through the objective lens, the multiplexing unit, and the scanning unit, and is detected by the photodetector. A fluorescence image is constructed based on the operation position information on the specimen by the scanning means and the light quantity information of the fluorescence detected by the photodetector.

On the other hand, the operation laser light is condensed on the specimen by the objective lens, and the specimen is operated for optical stimulation, laser trap, and the like. According to the present invention, since the irradiation positions of the plurality of operation laser beams are adjusted and incident on the specimen, a plurality of light stimulations and laser traps can be performed on the specimen.

  In this case, the plurality of operation laser lights emitted from the operation laser light source are combined by the operation laser light combining means and then combined with the observation laser light by the combining means. Therefore, no multiplexing means for multiplexing a plurality of operation laser lights is arranged on the optical path of the observation laser light that the fluorescence follows, and even if the number of operation laser lights increases, It is possible to obtain a bright fluorescent image with a low loss of the image.

In the above invention, it is preferable that the plurality of operation laser beams have polarization directions orthogonal to each other, and the operation laser beam multiplexing means is constituted by a polarization beam splitter.
By comprising in this way, the several laser beam for operation can be combined efficiently.

In the above invention, the branching means for branching the operation laser light from the operation laser light source into a plurality of parts, and the λ / 2 plate for making the polarization directions of the plurality of branched operation laser lights orthogonal to each other It is preferable to provide.
By doing in this way, a plurality of operation laser beams can be obtained from a single operation laser light source, and a plurality of operation laser beams can be efficiently combined. Therefore, the apparatus can be configured simply and inexpensively.
Further, in the above invention, an operation laser beam condensing position adjusting means for changing a condensing position in the specimen in the optical axis direction may be provided in each optical path of the plurality of operation laser beams. .

  According to the present invention, even if the number of operation laser beams combined with the observation laser beam is increased, it is possible to obtain a bright fluorescence image by reducing the loss of fluorescence.

Hereinafter, a laser scanning microscope 1 according to an embodiment of the present invention will be described with reference to FIGS.
The laser scanning microscope 1 according to the present embodiment is a laser scanning confocal microscope. In FIG. 1, optical components such as various lenses and pinholes are omitted for simplification of description.

As shown in FIG. 1, the laser scanning microscope 1 according to the present embodiment generates an observation laser light generation unit 2 that generates an observation laser light L ₁ and operation laser lights L ₂ and L ₃ . a manipulation laser beam generator 3, the first and multiplexing means 4, which have been multiplexed observation laser beam L ₁ and the operation of multiplexing the observation laser beam L ₁ and the manipulation laser beam L _2, L ₃ While the sample laser beams L ₂ and L ₃ are condensed and irradiated on the sample P, the sample P is irradiated with the observation laser beam L ₁ to excite the fluorescence F generated by exciting the fluorescent substance in the sample P. The objective lens 5 which condenses, and the photodetector 6 which detects the fluorescence F condensed with this objective lens 5 are provided.

Observation laser light generator 2, 2 observation laser beam L ₁ and the observation laser light source 7 that emits the observation laser beam L ₁ emitted from the observation laser light source 7 in a direction intersecting the optical axis And a first scanner (scanning means) 8 that scans dimensionally. Reference numeral 9 denotes a mirror.
Further, between the observation laser light source 7 of the observation laser light generation unit 2 and the first scanner 8, the light is generated in the sample P and is collected by the objective lens 5, and is combined with the multiplexing means 4, the mirror 9, and the first scanner 8. There is provided a dichroic mirror 10 that divides the fluorescence F returning through the first scanner 8 from the observation laser light L ₁ and directs it to the photodetector 6.

Manipulation laser beam generator 3, branches manipulation laser light source 11 for emitting a manipulation laser beam L _2, L _3, the manipulation laser beam L _2, L ₃ emitted from該操effect laser light source 11 A second mirror (laser beam position adjusting unit) that adjusts the position of the half mirror (branching unit) 12 and the two branched operation laser beams L ₂ and L ₃ in a direction crossing the optical axis. 13 and 14, and second combining means (operation laser light combining means) 15 for combining the two operation laser lights L ₂ and L ₃ whose positions are adjusted by the second scanners 13 and 14, respectively. I have. Reference numeral 16 denotes a mirror.

The operation laser light generator 3 includes a λ / 2 plate 17 that rotates the polarization direction of one operation laser light L ₃ branched by the half mirror 12 by 90 °, and the operation laser light on the specimen P. In-focus position adjusting means (operating laser beam focusing position adjusting means) 18 and 19 for adjusting the in-focus positions of L ₂ and L ₃ are provided. The focusing position adjusting means 18 and 19 are composed of a wavefront conversion element or a plurality of lens groups (not shown) at least partially supported so as to be movable in the optical axis direction.

The second multiplexing means 15 is constituted by a polarization beam splitter. In addition, shutters 21 and 22 that are controlled to be opened and closed by a control device 20 to be described later are provided on the optical paths of the _two operation laser beams L ₂ and L ₃ branched by the half mirror 12, respectively.
Instead of the shutters 21 and 22, an acoustooptic device such as AOTF or AOM may be used.
The operating laser beams L ₂ and L ₃ are used as optical tweezers for holding the specimen P by being focused on the position of actin or myosin bound to the specimen P.

The objective lens 5 is supported by a focusing mechanism 23 that moves the objective lens 5 along the optical axis direction.
The focusing mechanism 23 and the focusing position adjusting means 18 and 19 are connected to the control device 20. The control device 20 stores the relationship between the amount of movement of the objective lens 5 by the focusing mechanism 23 and the amount of movement of the focusing position of the two operation laser beams L ₂ and L _{3 by} the focusing position adjusting means 18 and 19. ing.

In the observation mode in which both the observation laser beam L ₁ and the operation laser beams L ₂ and L ₃ are irradiated, the control device 20 interlocks the focusing mechanism 23 and the focusing position adjusting means 18 and 19. The in-focus positions of the _two operation laser beams L ₂ and L ₃ are moved by the same distance in the opposite direction to the moving direction of the objective lens 5.

Further, in the preparation mode in which only the observation laser beam L ₁ is irradiated and the operation laser beams L ₂ and L ₃ are stopped, the control device 20 stops the focusing position of the observation laser beam L _1. The focusing mechanism 23 and the in-focus position in a state where the in-focus positions of the operating laser beams L ₂ and L ₃ (the in-focus position achieved when the operating laser beams are emitted) are matched. The interlocking with the adjusting means 18, 19 is stopped.
Thus, in the preparation mode, when the focusing mechanism 23 is operated to move the objective lens 5 in the optical axis direction, not only the observation laser beam L ₁ but also the operation laser beams L ₂ and L ₃ that are stopped. The in-focus position can be moved together with the objective lens 5 in the optical axis direction.

The control device 20 is connected to the photodetector 6, first and second scanners 8, 13, a monitor 24 and a mouse 25.
Controller 20, the scan position information at the first scanner 8 observation laser beam L ₁ on the specimen P by, on the basis of the light intensity information of the fluorescence F detected by the photodetector 6, a two-dimensional A fluorescent image is constructed and displayed on the monitor 24.

The operation of the laser scanning microscope 1 according to the present embodiment configured as described above will be described below with reference to FIGS.
When observing a specimen P, for example, a cell floating in the culture medium 27 stored in the petri dish 26, using the laser scanning microscope 1 according to this embodiment, as shown in FIG. Actin or myosin is bound to the end points A and B (step S1 in FIG. 8), and the control device 20 is switched to the preparation mode (step S2 in FIG. 8).

As a result, as shown in FIG. 2, only the observation laser light L ₁ from the observation laser light generator 2 is irradiated onto the specimen P, and the two operation laser lights L ₂ and L ₃ are stopped. The operation laser lights L ₂ and L ₃ are stopped by turning off the operation laser light source 11, closing the shutters 21 and 22, or turning off the acousto-optic elements. Here, description will be made on the assumption that the shutters 21 and 22 are closed.

The operator operates the focusing mechanism 23 to move the objective lens 5 so that the focal plane FP of the observation laser light L ₁ is arranged in the vicinity of the uppermost position of the sample P, for example, and the first scanner 8. Is activated. Thus, the fluorescence image of the specimen P in the focal plane FP of the observation laser beam L ₁ is acquired (step S3 in FIG. 8). In the example shown in FIG. 2, since the end point A of the sample P is the highest position, a fluorescence image of a cross section at the highest position including the end point A is acquired.

While confirming the acquired fluorescent image on the monitor 24, the operator instructs the end point A to the fluorescent image on the monitor 24 using the input means such as the mouse 25 (step S4 in FIG. 8). Thus, since the gripping position of the optical tweezers according to one of the manipulation laser beam L ₂ is specified, the controller 20 actuates the second scanner 13, one of the irradiation position of the manipulation laser beam L ₂ after positioning two-dimensionally so as to match the specified end point a (step S5 in FIG. 8), it opens the shutter 21 of the optical path of the one of the operating laser beam L _2, the manipulation laser beam L ₂ irradiates the specimen P.

In the preparation mode, since the in-focus position of the stopped operation laser light L ₂ is maintained in a state coincident with the focal plane FP of the observation laser light L ₁ , the operation irradiated by opening the shutter 21 is performed. As shown in FIG. 3, the laser beam L ₂ for use is focused on the end point A, and the specimen P can be held at the end point A.
At the same time, controller 20 releases the preparation mode for the shutter 21 manipulation laser beam L ₂ the open said one of the switches to the observation mode (step S6 in FIG. 8).

The operator then actuates the focusing mechanism 23, as shown in FIG. 4, gradually lowers the focal plane FP of the observation laser beam L _1. At this time, since the manipulation laser beam L ₃ in which the shutter 22 is closed is maintained in the preparation mode, the focus position is focused on the state that matches the focal plane FP of the observation laser beam L ₁ It is lowered according to the operation of the mechanism 23.

On the other hand, with respect to the operation laser light L ₂ that has been released from the preparation mode and switched to the observation mode, the focusing mechanism 23 and the focusing position adjusting means 18 are interlocked by the operation of the control device 20. Thus, the manipulation laser beam L _2, while being moved together with the focal plane FP of the observation laser beam L ₁ by the focusing mechanism 23, the amount of movement manipulation laser beam in the opposite direction at the same movement and L _The focus position adjusting means 18 is operated so that the _second focus position moves. As a result, the manipulation laser beam L _2, as shown in FIG. 4, is maintained in a stationary state without moving the focus position holding the end point A of the specimen P.

Then, the operator operates the focusing mechanism 23 so that, for example, as shown in FIG. 5, the objective lens is arranged so that the focal plane FP of the observation laser light L ₁ is arranged near the lowest position of the sample P. 5 is moved to activate the first scanner 8. In the example shown in FIG. 5, since the end point B of the sample P is the lowest position, a fluorescence image of a cross section at the lowest position including the end point B is acquired (step S7 in FIG. 8).

The operator designates the end point B with the input means such as the mouse 25 while confirming the acquired fluorescent image on the monitor 24 (step S8 in FIG. 8). Thus, since the gripping position of the optical tweezers by the other manipulation laser beam L ₃ is designated, the controller 20 actuates the second scanner 14, and the other of the irradiation position of the manipulation laser beam L ₃ after positioning two-dimensionally so as to match the specified end point B (step S9 in FIG. 8), opens the shutter 22 of the optical path of the other manipulation laser beam L _3, as shown in FIG. 6 as, illuminating the manipulation laser beam L ₃ in the specimen P.

The other operation laser light L ₃ irradiated by opening the shutter 22 is focused on the end point B, and the specimen P can be held at the end point B.
At the same time, the controller 20 also cancels the preparation mode for the laser beam L ₃ for the other operation which opens the shutter 22 is switched to the observation mode (step S10 in FIG. 8). Accordingly, as shown in FIG. 7, even if the focusing mechanism 23 is operated to move the objective lens 5 in the optical axis direction, the other operation laser light L ₃ also holds the end point B of the sample P. The in-focus position is maintained without moving.

In the subsequent observation mode, even if the objective lens 5 is moved by operating the focusing mechanism 23 and the focal plane FP of the observation laser light L ₁ is moved, the two end points A and B are held. The in-focus positions of the operation laser beams L ₂ and L ₃ are kept stationary. Therefore, the manipulation laser beam L _2, L ₃ samples P to end point A by optical tweezers using while kept stationary at two locations B, by sequentially moving the focal plane FP of the observation laser beam L ₁ Thus, the acquisition position of the fluorescence image can be displaced in the optical axis direction of the objective lens 5. Then, by acquiring a plurality of fluorescence images shifted in the optical axis direction of the objective lens 5, it is possible to acquire a three-dimensional fluorescence distribution of the specimen P using these fluorescence images (FIG. 8). Step S11).

In this case, according to the laser scanning microscope 1 according to the present embodiment, the focus positions L ₂ and L _{3 of} the operation laser beam branched into two are respectively brought into focus positions adjusting means 18 and 19. It is adjusted in the optical axis direction of the objective lens 5, and is adjusted by the second scanners 13 and 14 in a two-dimensional direction intersecting the optical axis of the objective lens 5. Then, after the two operating laser beams L ₂ and L ₃ whose focusing positions are adjusted are combined by the second combining unit 15, the first combining unit 4 performs the observation laser beam L _1. Therefore, the fluorescence F collected by the objective lens 5 does not have to pass through the second multiplexing means 15. As a result, even when two operation laser beams L ₂ and L ₃ are irradiated, the loss of fluorescence F at the combined position of the operation laser beams L ₂ and L ₃ and the observation laser beam L ₁ increases. There is an advantage that a bright fluorescent image can be obtained.

Further, since the two operation laser beams L ₂ and L ₃ are combined by the λ / 2 plate 17 and the second combining means 15 comprising a polarization beam splitter, the operation laser light source 11 is 1 Therefore, the apparatus can be reduced in size. Further, by reducing the loss of the operating laser beams L ₂ and L ₃ , waste can be eliminated and power consumption can be reduced.

Further, according to the laser scanning microscope 1 according to the present embodiment, the specimen is connected via the single objective lens 5 by linking the focusing mechanism 23 and the focusing position adjusting means 18 and 19 in the observation mode. only the focal plane FP of the observation laser beam L ₁ that is emitted to P is moved according to the movement of the objective lens 5 can be allowed to quiesce the manipulation laser beam L _2, L _3. As a result, it is possible to move the focal plane FP of the observation laser light L _{1 in} the direction of the optical axis of the objective lens 5 while keeping the specimen P stationary, and without changing the state of the specimen P. A three-dimensional fluorescence distribution can be observed without applying an external force to the specimen P.

In the present embodiment, the operation laser beams L ₂ and L ₃ emitted from the single operation laser light source 11 are branched by the half mirror 12. An operation laser light source 11 may be disposed for each of the lights L ₂ and L ₃ . In this case, by changing the installation angle of the operation laser light source 11 by 90 °, two operation laser lights L ₂ and L ₃ whose polarization directions are orthogonal to each other can be used.

Moreover, although the case where the two operation laser beams L ₂ and L ₃ are used has been described, three or more operation laser beams may be used instead. In this case, the operation laser light combined after combining the two operation laser lights in the same manner as described above may be combined with another operation laser light.

Moreover, although the case where the operation laser beams L ₂ and L ₃ are used as optical tweezers has been described, they may be used for light stimulation. By doing in this way, the three-dimensional fluorescence distribution of the sample P can be acquired while giving a light stimulus to two or more fixed positions.
Further, as the focusing mechanism 23, the one that moves the objective lens 5 in the optical axis direction is illustrated, but instead, the stage 28 that supports the specimen P is moved in the optical axis direction while the objective lens 5 remains fixed. It may be moved.

Further, in the present embodiment, the two operating laser beams L ₂ and L ₃ are used as optical tweezers, and the focusing mechanism 23 is operated while the different end points A and B of the sample P are held stationary. by moving the objective lens 5 in the optical axis direction by, but the focal plane FP of the observation laser beam L ₁ is moved in the optical axis direction, instead of this, the end point by the optical tweezers a, after gripping the B The specimen P may be moved in the optical axis direction of the objective lens 5 by the operation of the focusing position adjusting means 18 and 19.

  In this way, the three-dimensional fluorescence distribution of the specimen P can be acquired without moving the objective lens 5 and the stage 28 by the focusing mechanism 23. As a result, a motor that generates a large driving force for moving the objective lens 5 and the stage 28 becomes unnecessary, and the apparatus can be downsized.

1 is an overall configuration diagram showing a laser scanning microscope according to a first embodiment of the present invention. It is explanatory drawing which shows the preparation mode which irradiated the sample only with the laser beam for observation from the laser scanning microscope of FIG. It is explanatory drawing which shows the state which irradiated the sample laser beam for one operation in the origin position in which the focal plane of the laser beam for observation from the laser scanning microscope of FIG. 1 corresponded to the highest position of the sample. It is explanatory drawing which shows the state which lowered the focal plane of the laser beam for observation from the origin position of FIG. It is explanatory drawing which shows the state in which the focal plane of the laser beam for observation from the laser scanning microscope of FIG. 1 corresponded to the lowest position of the sample. It is explanatory drawing which shows the state which irradiated the sample with the other observation laser beam in the state of FIG. FIG. 6 is an explanatory diagram for explaining an observation mode in which two operation laser beams are fixed in the state of FIG. 5 and only the observation laser beams are moved. It is a flowchart which shows the observation procedure of the sample using the laser scanning microscope of FIG.

Explanation of symbols

F fluorescence L ₁ laser beam for observation L ₂ , L ₃ laser beam for operation P specimen 1 laser scanning microscope 4 first multiplexing means (multiplexing means)
5 Objective Lens 6 Photodetector 7 Laser Light Source for Observation 8 First Scanner (Scanning Means)
11 Laser light source for operation 12 Half mirror (branching means)
13, 14 Second scanner (laser beam position adjusting means)
15 Second multiplexing means (operational laser beam multiplexing means)
17 λ / 2 plate 18, 19 Focus position adjusting means (operation laser beam condensing position adjusting means)

Claims (4)

An observation laser light source that emits an observation laser beam;
An operation laser light source that emits a plurality of operation laser lights for operating the specimen;
An operation laser beam combining means for combining a plurality of operation laser beams emitted from the operation laser light source;
Scanning means for two-dimensionally scanning the observation laser beam;
A plurality of laser beam position adjusting means which are provided corresponding to the plurality of operation laser beams and adjust the irradiation positions of the plurality of operation laser beams two-dimensionally;
A combining unit that combines the observation laser beam scanned by the scanning unit and the operation laser beam adjusted in position by the laser beam position adjusting unit;
An objective lens for condensing the fluorescence generated in the specimen, while condensing and irradiating the specimen with the observation laser light and the operation laser light multiplexed by the multiplexing means;
A photodetector for detecting the fluorescence collected by the objective lens,
The laser scanning microscope in which the operation laser beam combining unit is arranged at a position where the plurality of operation laser beams are combined before being combined with the observation laser beam by the combining unit. The plurality of operation laser beams have polarization directions orthogonal to each other,
The laser scanning microscope according to claim 1, wherein the operation laser beam multiplexing means is constituted by a polarization beam splitter. Branching means for branching the operation laser light from the operation laser light source into a plurality of parts;
The laser scanning microscope according to claim 2, further comprising a λ / 2 plate that makes the polarization directions of the plurality of branched operation laser beams orthogonal to each other.   4. The operation laser light condensing position adjusting means for changing the condensing position of the specimen in the optical axis direction is provided in each optical path of the plurality of operation laser lights. 5. The laser scanning microscope described.

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