JP5591073B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus.

2. Description of the Related Art Conventionally, there is known a scanning confocal microscope apparatus in which laser light whose wavefront is deformed by a deformable mirror is incident on an objective lens via a galvanometer mirror unit (see, for example, Patent Document 1). This apparatus is configured such that the condensing point of the laser light changes in the depth direction by changing the reflecting surface of the deformable mirror.
In addition, when the galvano mirror is oscillated, the laser beam is shifted from the center of the pupil of the objective lens and is kicked by the lens frame, etc. There is known a scanner device that translates a galvanometer mirror in the optical axis direction in synchronization with movement (see, for example, Patent Document 2).

  When the light incident on the galvanometer mirror is a plane wave, the scanner device disclosed in Patent Document 2 can continue to cause the laser beam to be incident on the center of the pupil of the objective lens. The performance can be maintained.

JP 2005-165212 A Japanese Patent Laid-Open No. 10-68901

  However, when the light incident on the entrance pupil position of the objective lens is not a plane wave but a wavefront modulated light as disclosed in Patent Document 1, the method disclosed in Patent Document 2 Since the optical path length of the laser light is changed by translating the galvanometer mirror, there is a problem that the position optically conjugate with the pupil position of the objective lens is changed, and the condensing performance of the laser light is changed. This problem can occur strictly when the light incident on the entrance pupil position of the objective lens is a plane wave, but in the case of light subjected to wavefront modulation, the influence of deteriorating the light collecting performance is relatively large.

  The present invention has been made in view of the above-described circumstances, and two-dimensionally scans illumination light on a specimen without changing the light condensing performance of the illumination light guided from the light source onto the specimen. It aims at providing the microscope device which can do.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a spatial light modulation element disposed at a position conjugate to the pupil position of an objective optical system, which receives a substantially plane wave of illumination light from a light source and modulates the wavefront thereof , and around two non-parallel axes. has two mirrors that swings each relay a scanner that scans the illumination light wavefront is modulated two-dimensionally by the spatial light modulator, an image in the scanner pupil position of the objective optical system A relay optical system; a modulation area adjustment unit that moves a modulation area of the wavefront that forms an image on the spatial light modulation element according to the swing of the mirror; and the movement of the modulation area by the modulation area adjustment unit And a light beam shift mechanism that moves the light beam of the illumination light incident on the spatial light modulation element from the light source in a direction intersecting the light beam, and the modulation area adjustment unit includes the spatial light modulation element. Fixing the image above In the case where it is assumed that the mirror is stopped in the direction opposite to the moving direction of the image relayed to the pupil position of the objective optical system by the relay optical system when it is assumed that the mirror is swung. The modulation area is moved so that the image of the pupil position of the objective optical system is moved, and the light beam shift mechanism is moved by the modulation area adjustment unit. The modulation area of the spatial light modulation element and the spatial light modulation element There is provided a microscope apparatus for moving the light beam of the illumination light so that an illumination area illuminated by the illumination light above matches.

  According to the present invention, the illumination light whose wavefront is modulated by the spatial light modulation element is guided to the scanner, and the illumination light emitted from the scanner is guided to the objective optical system via the relay optical system. Focused on the specimen. At this time, the image in the scanner is relayed to the pupil position of the objective optical system by the relay optical system. Since the two mirrors constituting the scanner are swung around two axes that are not parallel, the illumination light is scanned two-dimensionally on the specimen.

  In this case, if the image on the spatial light modulator is fixed, of the two mirrors constituting the scanner, the mirror which is not arranged at a position optically conjugate with the pupil position of the objective optical system is swung. As a result, the image relayed to the pupil position of the objective optical system is moved in the direction intersecting the optical axis. According to the present invention, an image relayed to the pupil position of the objective optical system by the modulation area adjustment unit moving the modulation area of the wavefront forming the image on the spatial light modulation element according to the swing of the mirror. Can be canceled and stopped.

  In this case, since there is no fluctuation in the optical path length, the relay relationship from the image position in the scanner to the pupil position of the objective optical system does not change. Therefore, even when the wavefront of the illumination light to be relayed is not necessarily a plane wave, the wavefront formed in the scanner is accurately relayed to the pupil position of the objective optical system by modulating with the spatial light modulation element, and the light collection performance is improved. A decrease can be prevented.

  Furthermore, the light shift mechanism moves the irradiation position of the illumination light that is irradiated onto the spatial light modulation element following the movement of the wavefront modulation area, so that the spatial light modulation includes the entire area where the wavefront modulation area moves. There is no need to illuminate a wide area on the device. That is, the light density of the illumination light can be increased and the illumination efficiency can be improved by narrowing the luminous flux diameter of the illumination light to the size of the wavefront modulation region.

In the above invention, the light beam shift mechanism is provided on a light path between the light source and the spatial light modulation element, and includes a movable mirror that deflects the illumination light from the light source to the spatial light modulation element. The movable mirror may be moved in a direction parallel to the incident light beam.
Further, in the above invention, a parallel plate disposed on an optical path between the light source and the spatial light modulator and having a refractive index for refracting the light beam, the light beam shift mechanism includes You may rock | fluctuate the rocking | fluctuation axis line which cross | intersects a light beam.
By doing in this way, the light beam of illumination light can be moved with the simple structure which only moves or rocks a movable mirror or a parallel plate.

In the above invention, the relay optical system may relay an image on the swing axis of the mirror that is swung at a higher speed among the two mirrors.
By doing so, it is sufficient for the modulation area adjusting unit and the light beam shift mechanism to move the wavefront modulation area and the light beam in accordance with the oscillation of the mirror having the slower oscillation speed. Thereby, the movement of the wavefront modulation area and the illumination area on the spatial light modulation element can be matched with the movement of the image at the pupil position of the objective optical system more easily.

  According to the present invention, the illumination light can be two-dimensionally scanned on the specimen without changing the light collection performance of the illumination light guided from the light source onto the specimen.

1 is an overall configuration diagram showing a microscope apparatus according to an embodiment of the present invention. It is the figure which expanded a part of microscope apparatus of FIG. 1, and is a figure explaining the movement of a movable mirror. FIG. 2 is a perspective view showing an example in which the scanner of the microscope apparatus of FIG. 1 is shown and a position optically conjugate with the pupil position of the objective optical system is arranged on a high-speed mirror. FIG. 4 is a diagram for explaining the movement of the modulation region in the spatial light modulation element of the microscope apparatus of FIG. 1 in the case of FIG. 3. FIG. 6 is a modification of FIG. 4 and is a diagram for explaining the movement of the modulation region when the spatial light modulation element is irradiated with laser light over the entire region where the modulation region is moved. FIG. 3 is a modification of FIG. 3, showing the scanner of the microscope apparatus of FIG. 1, showing an example in which a position optically conjugate with the pupil position of the objective optical system is arranged between the high-speed side mirror and the low-speed side mirror. It is a perspective view. FIG. 7 is a diagram for explaining the movement of the modulation region in the spatial light modulation element of the microscope apparatus of FIG. 1 in the case of FIG. 6. FIG. 5 is a diagram illustrating an example of a configuration in which laser light is moved by swinging a parallel plate, which is a modification of FIG. 2.

A microscope apparatus 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the microscope apparatus 1 according to the present embodiment includes a light source 2 that generates laser light (illumination light) L, and a collimator that converts a wavefront of the laser light L emitted from the light source 2 into a substantially plane wave. The wavefront is modulated by the lens 3, the wavefront modulation unit 4 that modulates the wavefront of the laser light L converted into a substantially plane wave, the movable mirror 5 disposed between the collimator lens and the wavefront modulation unit, and the wavefront modulation unit 4. The first relay optical system 6 that relays the laser light L, the scanner 7 that scans the laser light L relayed by the first relay optical system 6 two-dimensionally, and the scanner 7 The second relay optical system 8 that relays the laser light L, the objective optical system 9 that condenses the laser light L relayed by the second relay optical system 8, the wavefront modulation unit 4, the movable mirror 5, and the scanner Control 7 A control unit 10, and a photodetector 11 for detecting the fluorescence from the specimen A, collected by the objective optical system 9. In the figure, reference numeral 12 denotes a stage on which a specimen A placed on a slide glass is mounted.

The laser light L emitted from the light source 2 is converted into a substantially plane wave by the collimator lens 3 and then reflected toward the wavefront modulation unit 4 by the movable mirror 5.
As shown in FIG. 2, the movable mirror 5 is provided so as to be movable back and forth in a direction parallel to the incident axis of the laser light L from the light source 2 by a motor 5 a. As the movable mirror 5 moves, the reflected laser beam (light beam) L is translated in a direction intersecting the optical axis.

  The wavefront modulation unit 4 reflects the laser beam L reflected by the movable mirror 5 and the laser beam L reflected by the prism 13, and in this case, the laser beam L has a form according to its surface shape. And a reflective spatial light modulator 14 that modulates the wavefront of the light and returns it to the prism 13.

  The optical path of the laser light L reflected by the prism 13 is folded back by the spatial light modulator 14 so as to return to the same prism 13, and the optical path of the optical axis parallel to the laser light L from the light source 2 is returned by the prism 13. It has become. Here, when the movable lens 5 is moved, the position of the laser light L incident on the prism 13 moves, and the position of the laser light L incident on the spatial light modulator 14 and the prism 13 are emitted. The position of the laser beam L is also translated in the direction intersecting the optical axis.

  The spatial light modulator 14 is configured by a segment type MEMS that can arbitrarily change its surface shape by a shape command signal from the control unit 10. The spatial light modulator 14 and the entrance pupil position of the objective optical system 9 are arranged in an optically conjugate positional relationship.

  As shown in FIG. 3, the scanner 7 includes two mirrors 7a and 7b provided so as to be swingable around two swing axes S1 and S2 arranged at twisted positions. One swing axis S1 is arranged in a plane orthogonal to the other swing axis S2. Thus, when viewed from one direction along the plane, as shown in FIG. 1, the two swing axes S1 and S2 are arranged orthogonal to each other.

The swing speed of one mirror 7a is set sufficiently higher than the swing speed of the other mirror 7b. The high speed side mirror 7a is used for scanning the sample A of the laser light L, and the low speed side mirror 7b is used for sending the scanning position of the laser light L on the sample A. In the figure, reference numerals 7c and 7d are motors for swinging the mirrors 7a and 7b, and reference numeral 15 is a mirror.
As shown in FIG. 3, the high-speed side mirror 7 a is disposed at a position optically conjugate with the entrance pupil position of the objective optical system 9. The hatching in FIG. 3 indicates that the high-speed mirror 7 a is disposed at a position optically conjugate with the entrance pupil position of the objective optical system 9.

  The first relay optical system 6 and the second relay optical system 8 are composed of a plurality of lenses. The first relay optical system 6 is configured to relay an image formed on the surface of the spatial light modulator 14 to the surface of the high-speed side mirror 7a. The second relay optical system 8 is configured to relay an image formed on the surface of the high-speed side mirror 7 a to the entrance pupil position of the objective optical system 9.

  In FIG. 1, reference numeral 16 denotes a dichroic mirror that reflects the laser light L and transmits fluorescence, and reference numeral 17 denotes a condenser lens. The fluorescence transmitted through the dichroic mirror 16 is collected by the condenser lens 17 and detected by the photodetector 11. By storing the intensity of the fluorescence detected by the photodetector 11 and the scanning position information of the laser beam L by the scanner 7 at the time of detection in association with each other, a two-dimensional fluorescence image can be acquired. It can be done.

  The control unit 10 outputs a shape command signal to the spatial light modulation element 14 so that the surface shape of the spatial light modulation element 14 in the modulation region B becomes a preset shape. The surface shape of the modulation region B of the spatial light modulation element 14 is such that the wavefront of the plane wave incident on the modulation region B can be modulated and condensed at one point at the focal position of the objective optical system 9. It is a shape, and can be calculated or measured in advance in consideration of aberrations of various optical systems, refractive index distribution in the specimen A, and the like.

  Further, the control unit 10 outputs an angle command signal for instructing the swing angle to the motors 7c and 7d that swing the mirrors 7a and 7b of the scanner 7. Then, the control unit 10 outputs a movement command signal for moving the modulation region B realizing the surface shape as described above as indicated by an arrow D in FIG. 4 in synchronization with the angle command signal. It has become.

  Specifically, when it is assumed that the modulation region B of the spatial light modulation element 14 is fixed and the low-speed mirror 7b is swung, the moving direction of the image of the laser light L at the entrance pupil position of the objective optical system 9 Moves the wavefront modulation region B in the spatial light modulator 14 in accordance with the oscillation of the mirror 7b so that the image of the laser beam L is moved in the opposite direction, assuming that the low-speed mirror 7b is fixed. It is supposed to let you.

  Further, the control unit 10 outputs a movement command signal for moving the movable mirror 5 in parallel to the motor 5a in synchronization with the movement command signal of the modulation region B to the spatial light modulation element 14.

  Specifically, the control unit 10 causes the illumination area C of the laser light L on the spatial light modulation element 14 to follow the movement of the modulation area B shown in FIG. Thus, a movement command signal for translating the laser beam L reflected by the movable mirror 5 in the direction intersecting the optical axis is output. Here, the position where the movable mirror 5 should be moved by the motor 5a may be calculated in advance based on the design value or by measurement. The beam diameter of the laser light L is reduced by a beam expander (not shown) or the like so that the illumination area C covers the entire modulation area B and has the smallest possible diameter.

The operation of the microscope apparatus 1 according to the present embodiment configured as described above will be described below.
In order to perform fluorescence observation of the specimen A using the microscope apparatus 1 according to the present embodiment, a shape command signal for instructing the surface shape in the modulation region B is output from the control unit 10 to the spatial light modulation element 14. The laser light L generated in the light source 2 is converted into a substantially plane wave through the collimator lens 3 and reflected by the movable mirror 5 to be incident on the wavefront modulation unit 4.

  The laser beam L incident on the wavefront modulation unit 4 is reflected by the prism 13 and incident on the spatial light modulator 14. The spatial light modulator 14 is irradiated with the laser light L on the illumination region C including the modulation region B, and only the portion of the laser light L incident on the modulation region B is modulated in wavefront, reflected by the prism 13 and reflected by the first light. 1 is incident on the relay optical system 6.

  The first relay optical system 6 relays the image of the modulation region B of the spatial light modulator 14 on the surface of the mirror 7a on the high speed side of the scanner 7 arranged at a position optically conjugate with this. In the scanner 7, the reflected laser beam L is oscillated in the scanning direction by oscillating the high speed side mirror 7a, and the reflected laser beam is oscillated by oscillating the low speed side mirror 7b. The light L is swung in the feeding direction. Thereby, the laser beam L is scanned two-dimensionally.

The laser beam L scanned by the scanner 7 is incident on the second relay optical system 8.
The second relay optical system 8 relays the image of the laser light L formed on the surface of the mirror 7a on the high speed side to the entrance pupil position of the objective optical system 9 arranged at a position optically conjugate with this. To do.
By doing so, the image of the modulation region B of the spatial light modulator 14 is relayed to the entrance pupil position of the objective optical system 9.

  In this case, if the scanner 7 is operated with the modulation region B in the spatial light modulation element 14 fixed, the relay is moved to the entrance pupil position of the objective optical system 9 according to the oscillation of the low-speed mirror 7b. The image of the laser beam L thus moved linearly moves in the direction intersecting the optical axis. This moving direction is defined as P direction and the moving amount is defined as ΔP. Conversely, even if the modulation area B in the spatial light modulation element 14 is moved as indicated by the arrow D while the low-speed mirror 7b of the scanner 7 is stopped, it is relayed to the entrance pupil position of the objective optical system 9. The image of the laser beam L moves linearly in the direction intersecting the optical axis. This movement direction is defined as Q direction and the movement amount is defined as ΔQ.

  In the present embodiment, the control unit 10 moves the modulation region B of the spatial light modulation element 14 so that the P direction and the Q direction are opposite and ΔP = ΔQ. Regardless of the oscillation of 7b, the image of the laser beam L relayed to the entrance pupil position of the objective optical system 9 can be kept stationary. Since the laser light L incident on the entrance pupil position does not change in the direction crossing the optical axis, the laser light L can be incident so as to cover the entire entrance pupil, and illumination with the maximum brightness can be performed.

  Further, in this case, in order to prevent the image at the entrance pupil position of the objective optical system 9 from moving due to the swing of the mirrors 7a and 7b of the scanner 7, the modulation region B in the spatial light modulation element 14 is laser-beamed. Since the optical path length from the spatial light modulator 14 to the entrance pupil position of the objective optical system 9 does not change, the optical path length from the spatial light modulator 14 to the entrance pupil position of the objective optical system 9 does not change. There is an advantage that it is not necessary to change the relay relationship. Therefore, even when the wavefront of the laser beam L to be relayed is not necessarily a plane wave, the wavefront modulated by the spatial light modulator 14 is accurately relayed to the entrance pupil position of the objective optical system 9 to prevent degradation of the light collecting performance. can do.

  As a result, aberrations caused by various optical systems, refractive index distribution in the specimen A, and the like are compensated, and the laser light L is accurately focused on a desired point in the specimen A by the objective optical system 9. There is an advantage that can be. If the laser light L generated from the light source 2 is an ultrashort pulse laser light, fluorescence can be generated by the multiphoton excitation effect only at the focal position of the objective optical system 9 and a clear fluorescent image can be obtained. .

  Further, as shown in FIG. 5, on the spatial light modulation element 14, the laser light L may be irradiated to a wide area including the modulation area B and covering the entire area where the modulation area B moves. The image can be stopped at the entrance pupil position of the objective optical system 9. On the other hand, according to this embodiment, the beam diameter of the laser light L can be reduced to the diameter of the modulation area B by causing the illumination area C to follow the movement of the wavefront modulation area B. That is, the sample A can be illuminated more brightly by increasing the light density of the laser light L.

  In the present embodiment, the oscillation axis S1 of the high-speed mirror 7a constituting the scanner 7 is positioned optically conjugate with the surface of the spatial light modulator 14 and the entrance pupil position of the objective optical system 9. Since it is set, it is not necessary to move the modulation area B according to the swing of the mirror 7a on the high speed side.

Therefore, if the spatial light modulator 14 and the motor 5a move the modulation area B or the movable mirror 5 in accordance with the oscillation of the low-speed mirror 7b, which is sufficiently slower than the high-speed mirror 7a, respectively. Sufficient and responsiveness may be low. That is, the displacement of the image of the laser beam L incident on the entrance pupil position of the objective optical system 9 due to the swinging of the mirrors 7a and 7b can be prevented more reliably, and the laser is prevented from moving in the modulation region B The illumination area C of the light L can be followed more accurately.
Further, since the spatial light modulation element 14 is not moved, but the modulation region B on the spatial light modulation element 14 is moved, the spatial light modulation element 14 can be moved at high speed without vibration.

  In this embodiment, the position optically conjugate with the entrance pupil position of the objective optical system 9 is arranged on the swing axis S1 of the mirror 7a on the high speed side of the scanner 7, but this is replaced. As shown in FIG. 6, it may be arranged between the two mirrors 7a and 7b. In the figure, hatching indicates a position optically conjugate with the entrance pupil position of the objective optical system 9.

  By doing in this way, the image of the laser beam L relayed to the entrance pupil position of the objective optical system 9 is affected by the swinging of the two mirrors 7a and 7b. Therefore, in order to keep the image stationary, another movable mirror (not shown) is provided, and as shown in FIG. 7, not only in the direction of the arrow D but also in the direction of the arrow E perpendicular thereto, the modulation region B Need to be moved.

  On the other hand, by placing a position optically conjugate with the entrance pupil position of the objective optical system 9 between the two mirrors 7a and 7b, the incidence of the objective optical system 9 due to the swinging of the mirrors 7a and 7b. The amount of movement in one direction of the image relayed to the pupil position is small. Therefore, compared with the above embodiment, the movement range of the modulation area B becomes smaller, and the movable mirror 5 can be moved within a narrower range. In particular, when the position optically conjugate with the entrance pupil position of the objective optical system 9 is arranged in the middle of the two mirrors 7a and 7b, it is arranged in one of the two mirrors 7a and 7b. In comparison, the amount of movement in one direction of the image relayed to the entrance pupil position of the objective optical system 9 can be halved.

  In the present embodiment, the laser beam L incident on the wavefront modulation unit 4 is translated by the movable mirror 5, but instead of this, as shown in FIG. A parallel plate 18 is disposed in the middle of the optical path to the modulation element 14, and the parallel plate 18 is swung around a swing axis S3 perpendicular to the optical axis of the laser light L, whereby the laser light L is parallelized. It is good also as moving.

The parallel plate 18 is made of a material having a refractive index that refracts the incident laser beam L. Reference numeral 18 a denotes a motor that swings the parallel plate 18 in accordance with a swing command signal from the control unit 10.
Even in this case, the illumination area C on the spatial light modulator 14 can be moved with a simple configuration.

  In the present embodiment, a segment type MEMS mirror that changes the surface shape is exemplified as the spatial light modulator 14, but instead, any other spatial light modulator 14, for example, a liquid crystal element A deformable mirror or the like may be used.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Light source 3 Collimator lens 4 Wavefront modulation part 5 Movable mirror (light beam shift mechanism)
5a motor 6 first relay optical system 7 scanner 7a, 7b mirror 7c, 7d motor 8 second relay optical system (relay optical system)
9 Objective optical system 10 Control unit (modulation area adjustment unit, light beam shift mechanism)
11 Photodetector 12 Stage 13 Prism 14 Spatial Light Modulation Element 15 Mirror 16 Dichroic Mirror 17 Condensing Lens 18 Parallel Plate A Sample B Modulation Area C Illumination Area L Laser Light (Illumination Light, Light)
S1 to S3 Oscillation axis (axis)

Claims (4)

A spatial light modulation element disposed at a position conjugate with the pupil position of the objective optical system, in which a substantially plane wave of illumination light from the light source is incident and modulates the wavefront;
A scanner that has two mirrors each swinging about two non-parallel axes, and that scans illumination light whose wavefront is modulated by the spatial light modulator in a two-dimensional manner;
A relay optical system for relaying an image in the scanner pupil position of the objective optical system,
A modulation region adjustment unit that moves a modulation region of the wavefront that forms an image on the spatial light modulation element in accordance with the swing of the mirror;
A light beam shift mechanism that moves the light beam of the illumination light incident on the spatial light modulation element from the light source in a direction intersecting the light beam in accordance with the movement of the modulation region by the modulation region adjustment unit;
The moving direction of the image relayed to the pupil position of the objective optical system by the relay optical system when it is assumed that the modulation area adjusting unit fixes the image on the spatial light modulation element and swings the mirror In the opposite direction, the modulation region is moved so that the image of the pupil position of the objective optical system moves when the state where the mirror is stopped is assumed,
The illumination light is adjusted so that the modulation region of the spatial light modulation element moved by the modulation region adjustment unit matches the illumination region illuminated by the illumination light on the spatial light modulation element. A microscope apparatus for moving the light beam. A movable mirror disposed on an optical path between the light source and the spatial light modulator, and deflecting the illumination light from the light source to the spatial light modulator;
The microscope apparatus according to claim 1, wherein the light beam shift mechanism moves the movable mirror in a direction parallel to a light beam of the incident illumination light. A parallel plate disposed on an optical path between the light source and the spatial light modulator and having a refractive index that refracts the light beam;
The beam shifting mechanism, the microscope apparatus according to the parallel plate in claim 1, Ru is swung to swing axis line crossing the beams.   The microscope apparatus according to claim 1, wherein the relay optical system relays an image on a swing axis of a mirror that is swung at a higher speed among the two mirrors.

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