JP2012150238A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To two-dimensionally scan illumination light without changing the capability of collecting the illumination light on a specimen.SOLUTION: A microscope device 1 includes: a spatial optical modulation element 14 for modulating wavefront of illumination light from a light source 2; a scanner 5 including a first mirror 5a and a second mirror 5b each rotating about two non-parallel shaft lines and for two-dimensionally scanning the illumination light with the wavefront modulated by the spatial optical modulation element 14; a first relay optical system 9 for relaying an image from the first mirror 5a to the second mirror 5b of the scanner 5; and a second relay optical system 10 for relaying the image from the second mirror 5b to a pupil position of an objective optical system 6.

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.

JP 2005-165212 A

  However, in the scanning confocal microscope apparatus disclosed in Patent Document 1, the modulated wavefront relayed to the pupil plane of the objective lens by the lens and the imaging lens is shifted along the optical axis on the pupil plane by the oscillation of the galvanometer mirror. Will be shifted in the direction orthogonal to When such a shift occurs, there is an inconvenience that coma similar to decentration increases. Such a shift is small at the center of the imaging region, but increases as it goes to the periphery, resulting in a significant decrease in optical performance.

  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 that modulates a wavefront of illumination light from a light source, and a first mirror and a second mirror that respectively swing about two non-parallel axes, and the spatial light modulation element A scanner for two-dimensionally scanning the illumination light whose wavefront is modulated by the first relay optical system for relaying an image from the first mirror of the scanner to the second mirror, and the second And a second relay optical system for relaying an image from the mirror to the pupil position of the objective optical system.

  According to the present invention, the illumination light from the light source whose wavefront is modulated by the spatial light modulator is incident on the first mirror and deflected in a direction according to the swing angle of the first mirror, It is relayed by the first relay optical system, is incident on the second mirror, and is deflected in a direction corresponding to the swing angle of the second mirror. Since the first mirror and the second mirror are swung around non-parallel axes, respectively, the illumination light can be scanned two-dimensionally by changing the swing angles of the two mirrors. The illumination light scanned two-dimensionally is relayed by the second relay optical system, is incident on the objective optical system, and is irradiated on the specimen.

  In this case, by arranging the first relay optical system between the two mirrors constituting the scanner, the two mirrors are both arranged at a position optically conjugate with the pupil position of the objective optical system. Can do. As a result, even if each of the two mirrors is swung, the image relayed to the pupil position of the objective optical system does not move in the direction intersecting the optical axis, so the performance of condensing the illumination light on the specimen changes. Without illumination, the illumination light can be scanned two-dimensionally on the specimen.

In the above-described invention, a third relay optical system that relays an image on the spatial light modulation element onto the first mirror of the scanner may be provided.
By doing so, the position between the position on the first mirror and the position on the spatial light modulator that is optically conjugate with the pupil position of the objective optical system by the first and second relay optical systems. Can be prevented by relaying with the third relay optical system, and the wavefront correction at the pupil position of the objective optical system can be performed more accurately.

  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 a perspective view which shows the scanner and 1st relay optical system of the microscope apparatus of FIG.

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, a microscope apparatus 1 according to this embodiment includes a light source 2 that generates laser light (illumination light), and a collimator lens 3 that converts a wavefront of the laser light emitted from the light source 2 into a substantially plane wave. A wavefront modulation unit 4 that modulates the wavefront of the laser light converted into a substantially plane wave, a scanner 5 that scans the laser light whose wavefront is modulated by the wavefront modulation unit 4, and scanning by the scanner 5 Objective optical system 6 for condensing the laser beam thus collected on the specimen A, a control section 7 for controlling the scanner 5 and the wavefront modulation section 4, and light for detecting fluorescence from the specimen A collected by the objective optical system 6 And a detector 8.

In addition, the microscope apparatus 1 according to the present embodiment includes a first relay optical system 9 provided in the scanner 5 and a second relay optical system 10 disposed between the scanner 5 and the objective optical system 6. And a third relay optical system 11 disposed between the wavefront modulation unit 4 and the scanner 5.
In the figure, reference numeral 12 denotes a stage on which a specimen A placed on a slide glass is mounted.

  The wavefront modulation unit 4 reflects the laser beam reflected by the prism 13 that reflects the laser beam converted into a substantially plane wave by the collimator lens 3, and reflects the laser beam reflected by the prism 13, and the laser beam is shaped in accordance with the 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 reflected by the prism 13 is folded back by the spatial light modulation element 14 so as to return to the same prism 13, and the prism 13 returns the optical path coaxial with the laser light from the light source 2.
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 7. The spatial light modulator 14 and the entrance pupil position of the objective optical system 6 are arranged in an optically conjugate positional relationship.

As shown in FIG. 2, the scanner 5 includes a first mirror 5a and a second mirror 5b which are provided so as to be able to swing around two swing axes S ₁ and S ₂ arranged at twisted positions. And. One swing axis lines S ₁ is disposed in a plane perpendicular to the other of the swing axis S _2. Thereby, when viewed from one direction along the plane, the two swing axes S ₁ and S ₂ are arranged orthogonal to each other.

In FIG. 1, for ease of illustration, the two swing axes S ₁ and S ₂ are shown in parallel with each other for convenience, but actually, as shown in FIG. Are arranged in a non-parallel positional relationship.

  The swing speed of the first mirror 5a is set sufficiently higher than the swing speed of the second mirror 5b. The first mirror 5a on the high speed side is used for scanning on the sample A of laser light, and the second mirror 5b on the low speed side is used for sending the scanning position on the sample A of laser light. In the figure, reference numerals 5c and 5d denote motors for swinging the mirrors 5a and 5b.

Between the 1st mirror 5a and the 2nd mirror 5b, the 1st relay optical system 9 which relays the image on the 1st mirror 5a on the 2nd mirror 5b is arrange | positioned.
The second relay optical system 10 is arranged between the scanner 5 and the objective optical system 6 and relays the image on the second mirror 5b of the scanner to the pupil position of the objective optical system 6. Yes.

Further, the third relay optical system 11 is disposed between the wavefront modulation unit 4 and the scanner 5, and relays an image on the spatial light modulation element 14 of the wavefront modulation unit 4 onto the first mirror 5 a of the scanner 5. It is supposed to be.
Each of the first relay optical system 9, the second relay optical system 10, and the third relay optical system 11 includes a plurality of lenses.

  As a result, the surface position of the spatial light modulator 14, the surface position of the first mirror 5a of the scanner 5, and the surface position of the second mirror 5b are all optically conjugate with the entrance pupil position of the objective optical system. Placed in position. The hatching in FIG. 2 indicates that the surfaces of the first mirror 5 a and the second mirror 5 b are both arranged at positions optically conjugate with the entrance pupil position of the objective optical system 6.

  In FIG. 1, reference numeral 15 denotes a dichroic mirror that reflects laser light and transmits fluorescence, and reference numeral 16 denotes a condenser lens. The fluorescence transmitted through the dichroic mirror 15 is condensed by the condenser lens 16 and detected by the photodetector 8. By storing the fluorescence intensity detected by the photodetector 8 and the scanning position information of the laser beam by the scanner 5 at the time of detection in association with each other, a two-dimensional fluorescence image can be acquired. It is like that.

The control unit 7 outputs a shape command signal to the spatial light modulation element 14 so that the surface shape in the modulation region of the spatial light modulation element 14 becomes a preset shape. The surface shape of the modulation region of the spatial light modulation element 14 is such that the wavefront of the plane wave incident on the modulation region can be modulated and condensed at one point at the focal position of the objective optical system 6. Yes, it 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 7 outputs an angle command signal for instructing the swing angle to the motors 5c and 5d that swing the mirrors 5a and 5b of the scanner 5.

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, the light source 2 outputs the shape command signal for instructing the surface shape to the spatial light modulator 14 from the control unit 7. The generated laser light is incident on the wavefront modulation unit 4 as a substantially plane wave through the collimator lens 3.

  The laser light incident on the wavefront modulation unit 4 is reflected by the prism 13 and incident on the spatial light modulator 14. In the spatial light modulation element 14, the laser light incident on the modulation region is reflected after the wavefront is modulated, reflected by the prism 13, and incident on the third relay optical system 11.

  The third relay optical system 11 relays the image of the modulation area of the spatial light modulation element 14 onto the surface of the first mirror 5a of the scanner 5 disposed at a position optically conjugate with this. In the scanner 5, the first mirror 5a on the high speed side is swung, so that the reflected laser light is swung in the scanning direction, and the second mirror 5b on the low speed side is swung. The reflected laser beam is swung in the feeding direction. Thereby, the laser beam is scanned two-dimensionally.

  The laser beam scanned in one direction by the first mirror 5a of the scanner 5 passes through the first relay optical system 9, and then is scanned in the other direction by the second mirror 5b. Thereby, the image on the surface of the first mirror 5a is relayed on the surface of the second mirror 5b by the first relay optical system 9.

  The laser beam scanned two-dimensionally by the scanner 5 is incident on the second relay optical system 10. The second relay optical system 10 relays the image of the laser beam formed on the surface of the second mirror 5b to the entrance pupil position of the objective optical system 6 arranged at a position optically conjugate with this. To do.

By doing so, the image of the modulation region of the spatial light modulator 14 is relayed to the entrance pupil position of the objective optical system 6.
That is, according to the microscope apparatus 1 according to the present embodiment, the image of the laser beam relayed to the entrance pupil position of the objective optical system 6 is made stationary regardless of the swinging of the two mirrors 5a and 5b of the scanner 5. Can be maintained in a state.

Since the laser beam incident on the entrance pupil position does not fluctuate in the direction crossing the optical axis, the laser beam can be incident over the entire entrance pupil, and the illumination that is maximally bright can be performed.
In this case, the image at the entrance pupil position of the objective optical system 6 does not move even when the mirrors 5a and 5b of the scanner 5 are swung. Therefore, the wavefront modulated by the spatial light modulator 14 is incident on the objective optical system 6. It is possible to accurately relay to the pupil position and prevent the light collecting performance from deteriorating.

  As a result, aberrations caused by various optical systems, refractive index distribution in the specimen A, and the like are compensated, and the objective optical system 6 can focus the laser beam on a desired point in the specimen A with high accuracy. There is an advantage that you can. If the laser beam generated from the light source 2 is an ultrashort pulse laser beam, fluorescence can be generated by the multiphoton excitation effect only at the focal position of the objective optical system 6 and a clear fluorescence image can be acquired.

  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.

1 microscope apparatus 2 light sources 5 scanner 5a first mirror 5b the second mirror 6 objective optical system 9 first relay optical system 10 and the second relay optical system 11 third relay optical system 14 spatial light modulator S _1, S ₂ swing axis (axis)

Claims (2)

A spatial light modulator for modulating the wavefront of the illumination light from the light source;
A scanner having a first mirror and a second mirror that respectively swing about two non-parallel axes, and two-dimensionally scanning illumination light whose wavefront is modulated by the spatial light modulator;
A first relay optical system for relaying an image from the first mirror of the scanner to the second mirror;
A microscope apparatus comprising: a second relay optical system that relays an image from the second mirror to a pupil position of the objective optical system.   The microscope apparatus according to claim 1, further comprising a third relay optical system that relays an image on the spatial light modulation element onto the first mirror of the scanner.

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