JP2011133580A - Method and device for projecting hologram image - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simultaneously condense laser beams onto a plurality of desired condensing points in a sample even when aberration exists in an objective lens. <P>SOLUTION: A hologram image projection method includes: a step S1 of setting positional information on a focal plane of the objective lens in the sample about the plurality of condensing points; a step S2 of calculating the wave front at an entrance pupil position of the laser beams from each condensing point by performing inverse light tracing from each condensing point to the entrance pupil position of the objective lens using the positional information of each condensing point, refractive index of the sample, and lens data of the objective lens; a step S3 of calculating the synthetic wave front by synthesizing a plurality of calculated wave fronts; a step S4 of setting a phase pattern applied to a wave front modulation element based on the synthetic wave front; and a step S5 of applying the set phase pattern to the wave front modulation element to make laser beams enter and condensing the laser beams whose wave front is modulated with the phase pattern to the sample via the objective lens. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a hologram image projection method and a hologram image projection apparatus.

Conventionally, it is known to use a hologram in order to simultaneously irradiate a plurality of locations on a specimen with light (see, for example, Non-Patent Document 1).
In this method, a fluorescence image of a specimen is acquired by a fluorescence microscope or the like, and a plurality of regions of interest that should be subjected to light stimulation or the like are specified in the acquired fluorescence image, thereby generating a stimulation light irradiation pattern. Is subjected to Fourier transform to create a hologram phase pattern. Then, the created phase pattern is applied to the wavefront modulation element, and laser light composed of substantially parallel light guided from the light source is incident on the wavefront modulation element, thereby modulating the laser light, and the modulated laser light. Is condensed by an objective lens. In this way, the hologram image is projected onto the specimen, and the stimulation light is condensed simultaneously at a plurality of locations.

Volodymyr Nikolenko et al, "SLM microscopy: scanless two-photon imaging andphotostimulation with spatial light modulators", Frontiers in Neural Circuits, Vol 2, Article 5, 19 December 2008, p1-15

  However, in the conventional method, when there is aberration in the objective lens, the laser beam can be accurately focused on a small spot at the center of the optical axis of the objective lens, but it is separated from the center of the optical axis in the radial direction. Accordingly, there is a disadvantage that the laser beam is not condensed at one point and the spot is greatly blurred.

  The present invention has been made in view of the above-described circumstances, and can condense laser light simultaneously on a plurality of desired condensing points in a specimen even when there is aberration in the objective lens. An object of the present invention is to provide a holographic image projection method and a holographic image projection apparatus.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a position setting step for setting position information on a focal plane of the objective lens in a sample for a plurality of condensing points on which laser light is to be condensed via the objective lens, and each set condensing point The position of the entrance pupil of the objective lens from each condensing point assumed to be a set position using the position information in the specimen, the refractive index of the specimen, and lens data of each lens constituting the objective lens A wavefront calculation step of calculating a wavefront at the entrance pupil position of the laser light from each of the condensing points, and the plurality of wavefronts calculated corresponding to each of the condensing points A wavefront combining step for combining and calculating a combined wavefront; a phase pattern setting step for setting a phase pattern to be applied to the wavefront modulation element based on the calculated combined wavefront; and A condensing step of applying the phase pattern to the wavefront modulation element to make the laser light incident, and condensing the laser light, the wavefront of which is modulated by the phase pattern, onto the sample through the objective lens; A holographic image projection method is provided.

  According to the present invention, when the position information of the respective condensing points arranged at different positions in the sample is set in the position setting step, the entrance pupil position of the objective lens from each condensing point is set in the wavefront calculating step. The reverse ray tracing is performed, and a plurality of wavefronts at the entrance pupil position of the laser light from each condensing point are calculated. The calculated plurality of wavefronts are combined in the wavefront combining step so that the wavefronts that need to be incident on the entrance pupil position of the objective lens in order to simultaneously focus the laser light at different positions on the focal plane A synthetic wavefront is obtained by computation. Then, a phase pattern is given to the wavefront modulation element based on the synthesized wavefront so that the wavefront of the laser light incident on the wavefront modulation element is modulated to the same wavefront as the obtained synthesized wavefront.

  In this case, since the backward ray tracing is performed using the lens data of each lens constituting the objective lens in addition to the position information of each condensing point and the refractive index of the sample, the calculated wavefront is the objective wavefront. This includes the influence of aberration present in the lens. Therefore, laser light having this wavefront is incident on the entrance pupil position of the objective lens from the opposite side of the sample, so that the laser light is accurately focused on the focal plane of the objective lens. Therefore, by making the laser beam incident on the wavefront modulation element to which such a wavefront is obtained, the laser beam is simultaneously focused on a plurality of focusing points on the focal plane of the objective lens. A simple hologram image can be projected into the specimen.

In the above invention, the wavefront combining step may be a step of calculating the combined wavefront by calculating a linear sum of the plurality of wavefronts.
In the above invention, the wavefront synthesis step may be a step of calculating the synthesized wavefront by arranging the plurality of wavefronts divided into regions at a ratio of the reciprocal of the number of the condensing points. Good.

  Further, the present invention provides a position setting step for setting position information on the focal plane of the objective lens in the sample for a plurality of condensing points on which the laser light is to be collected via the objective lens, and each set collection point. The forward ray tracing from the entrance pupil position of the objective lens to the focal plane is performed using the position information of the light spot in the specimen, the refractive index of the specimen, and the lens data of each lens constituting the objective lens. A virtual image generation step of generating a virtual image having a plurality of spots formed at each of the condensing points, and the generated virtual image by deforming the wavefront of the laser light at the entrance pupil position of the objective lens A wavefront calculating step for calculating the wavefront that minimizes the diameter of each spot in the image, and a phase pattern to be applied to the wavefront modulation element based on the calculated wavefront A phase pattern setting step to be set; and the laser beam is incident on the wavefront modulation element by applying the set phase pattern to the wavefront modulation element, and the laser beam whose wavefront is modulated by the phase pattern is passed through the objective lens. And a condensing step for condensing the sample.

  According to the present invention, when the position information of the respective condensing points arranged at different positions in the sample is set in the position setting step, from the entrance pupil position of the objective lens to the focal plane in the virtual image generation step. , A virtual image having a plurality of spots formed at each condensing point is generated. Next, in the wavefront calculating step, the wavefront of the laser beam at the entrance pupil position of the objective lens is calculated such that the diameter of each spot in the generated virtual image is minimized. A phase pattern is applied to the wavefront modulation element based on the wavefront calculated in the wavefront calculation step so that the wavefront of the laser light incident on the wavefront modulation element is modulated to the same wavefront as the obtained wavefront. The

  In this case, the forward ray tracing is performed by using the lens data of each lens constituting the objective lens in addition to the position information of each condensing point and the refractive index of the sample. Each spot includes an influence of aberration existing in the objective lens. For this reason, laser light having a wavefront that minimizes the diameter of each spot is incident on the entrance pupil position of the objective lens from the opposite side of the sample, so that the laser light is accurately focused on the focal plane of the objective lens. Is done. Therefore, by making the laser beam incident on the wavefront modulation element to which such a wavefront is obtained, the laser beam is simultaneously focused on a plurality of focusing points on the focal plane of the objective lens. A simple hologram image can be projected into the specimen.

  In the invention described above, the position information is a condensing point map having a plurality of condensing points assumed on a focal plane of the objective lens in the specimen, and the virtual image generating step includes the concentrating point generation step. The step of calculating the wavefront of the laser light at the entrance pupil position of the objective lens by Fourier transforming the light spot map, and using the refractive index of the sample and the lens data of each lens constituting the objective lens The laser beam having the wavefront may be subjected to forward ray tracing from the entrance pupil position of the objective lens to the focal plane to generate the virtual image.

  The present invention also provides a wavefront modulation element that modulates a wavefront of a laser beam with a phase pattern, an objective lens that focuses the laser beam modulated by the wavefront modulation element on a sample, and a laser beam that passes through the objective lens. A position information setting unit for setting position information on the focal plane of the objective lens in a sample of a plurality of condensing points on the focal plane on which the light is to be collected, and each condensing point set by the position information setting unit Using the position information of the sample, the refractive index of the specimen, and the lens data of each lens constituting the objective lens, the back ray tracing from each condensing point assumed at the set position to the entrance pupil position of the objective lens And combining a plurality of wavefronts calculated corresponding to the respective condensing points with a wavefront calculating unit that calculates a wavefront at the entrance pupil position of the laser light from each of the condensing points. Calculate wavefront Providing a surface combining portion, and the phase pattern setting unit for setting the phase pattern imparted to the wavefront modulation element based on the calculated combined wavefront, the hologram image projection apparatus comprising a.

  According to the present invention, when the position information setting unit sets the position information of the respective condensing points arranged at different positions in the sample, the wavefront calculation unit sets the entrance pupil position of the objective lens from each condensing point. The reverse ray tracing is performed, and a plurality of wavefronts at the entrance pupil position of the laser light from each condensing point are calculated. The calculated wavefronts are synthesized by the wavefront synthesis unit, so that the wavefronts that need to be incident on the entrance pupil position of the objective lens in order to simultaneously focus the laser light at different positions on the focal plane A synthetic wavefront is obtained by computation. Then, based on the composite wavefront obtained in this way, the phase pattern setting unit sets the phase pattern.

  In this case, since the backward ray tracing is performed using the lens data of each lens constituting the objective lens in addition to the position information of each condensing point and the refractive index of the sample, the calculated wavefront is the objective wavefront. This includes the influence of aberration present in the lens. Therefore, laser light having this wavefront is incident on the entrance pupil position of the objective lens from the opposite side of the sample, so that the laser light is accurately collected on the focal plane of the objective lens. Therefore, by applying a laser beam to the wavefront modulation element in a state where a phase pattern capable of obtaining such a wavefront is applied to the wavefront modulation element, the laser is simultaneously applied to a plurality of condensing points on the focal plane of the objective lens. A hologram image that condenses light can be projected into the specimen.

  According to the present invention, even when there is aberration in the objective lens, there is an effect that laser light can be simultaneously focused on a plurality of desired condensing points in the sample.

1 is an overall configuration diagram schematically showing a hologram image projection apparatus according to a first embodiment of the present invention. It is a flowchart explaining the hologram image projection method which concerns on the 1st Embodiment of this invention using the hologram image projector of FIG. It is a whole block diagram which shows typically the hologram image projector which concerns on the 2nd Embodiment of this invention. It is a flowchart explaining the hologram image projection method which concerns on the 2nd Embodiment of this invention using the hologram image projector of FIG. FIG. 5A is a diagram illustrating a condensing point map when it is assumed that the objective lens has no aberration in the hologram image projecting method of FIG. 4, and FIG. 5B is a diagram illustrating a virtual image on the focal plane.

A hologram image projection apparatus and a hologram image projection method according to a first embodiment of the present invention will be described below with reference to FIGS.
The hologram image projector 1 according to the present embodiment is a microscope system, and as shown in FIG. 1, a light source device 2 that generates laser light and laser light incident from the light source device 2 are applied to a specimen A. A microscope apparatus 3 for irradiating and an adjusting apparatus 4 for adjusting laser light incident on the microscope apparatus 3 from the light source device 2 are provided.

  The light source device 2 includes a laser light source 5 that generates laser light, a collimator lens 6 that converts the laser light emitted from the laser light source 5 into collimated light, and a wavefront modulation unit that modulates the wavefront of the laser light composed of the collimated light. 7, relay lenses 8 and 10, and a scanner 9 that scans the laser beam.

  The wavefront modulation unit 7 reflects the laser beam reflected by the prism 11 and the laser beam reflected by the prism 11. At this time, the wavefront modulation unit 7 modulates the wavefront of the laser beam by the phase pattern and returns it to the prism 11. And a wavefront modulation element 12.

The laser beam reflected by the prism 11 is folded back so as to return to the same prism 11 by the wavefront modulation element 12, and returned to the optical path coaxial with the laser beam from the laser light source 5.
The wavefront modulation element 12 is configured by a segment type MEMS mirror whose surface shape can be arbitrarily changed by the adjusting device 4 described later. In this case, the surface shape formed by the unevenness of each segment of the MEMS mirror becomes a phase pattern for modulating the wavefront of the laser light. The wavefront modulation element 12 and the entrance pupil position of the objective lens 13 are arranged in an optically conjugate positional relationship.

  The scanner 9 is a so-called proximity galvanometer mirror in which two galvanometer mirrors 9a and 9b that can be swung around an axis arranged in a direction intersecting each other are arranged close to each other. Can be scanned automatically.

  The microscope apparatus 3 focuses the laser beam on the specimen A placed on the stage 14, while receiving the objective lens 13 that receives the light from the specimen A, and photoelectrons that detect the fluorescence received by the objective lens 13. A photo detector 15 composed of a multiplier tube, a camera 16 such as a CCD for taking a fluorescent image in the specimen A, and a mirror 17 inserted into and removed from the optical path so as to switch the optical path to the photo detector 15 or the camera 16. I have. Reference numerals 18 to 20 are condenser lenses, and reference numeral 21 is a dichroic mirror. The objective lens 13 has known lens data for each lens constituting the objective optical system, and is provided so that the distance in the optical axis direction from the stage 14 can be changed.

  By setting the surface shape of the wavefront modulation element 12 to a flat reflecting surface shape, laser light having a wavefront consisting of plane waves can be incident on the entrance pupil position of the objective lens 13. As a result, the laser beam can be condensed on the focal plane of the objective lens 13.

  In a state where the mirror 17 is switched to the light detector 15 side (position removed from the optical path indicated by the broken line), the laser light is emitted from the laser light source 5 and the scanner 9 is driven to collect on the focal plane in the sample A. The two-dimensional scanning of the specimen A spreading along the focal plane of the objective lens 13 is performed by detecting the fluorescence generated at each condensing position by the photodetector 15 while scanning the radiated laser beam two-dimensionally. A simple fluorescence image can be acquired.

  As shown in FIG. 1, the adjusting device 4 includes a storage unit 22 that stores lens data of the objective lens 13, and an input unit (position information setting unit) that sets position information of a laser beam condensing point in the sample A. 23, the position information of each condensing point set by the input unit 23, the lens data of each lens constituting the objective lens 13 stored in the storage unit 22, and the refractive index of the specimen A. Corresponding to the wavefront calculating unit 24 for calculating the wavefront at the entrance pupil position of the objective lens 13, the wavefront combining unit 25 for combining a plurality of wavefronts calculated for all the condensing points, and the wavefront combining unit 25. And a phase pattern setting unit 26 for setting a phase pattern to be applied to the wavefront modulation element 12 based on the synthesized wavefront.

  The input unit 23 sets the two-dimensional position information of each condensing point by designating the position where the user wants to condense the laser beam, that is, the condensing point on a monitor (not shown). ing. Here, the condensing point in the sample A may be set at an arbitrary position or may be set at a position responding to the light stimulus. When it is desired to set the focal point to a position that responds to light stimulation, a monitor (not shown) displays a two-dimensional image acquired by the microscope device 3 so that the user can easily specify the focal point. It is preferable.

For each set condensing point, the wavefront calculation unit 24 uses the position information set from the input unit 23 and the lens data of the objective lens 13 stored in the storage unit 22 for each condensing point. Calculate the corresponding wavefront.
Specifically, a point light source is assumed at a position specified by position information set for a condensing point, and laser light is used from the point light source to the entrance pupil position of the objective lens 13 using lens data of the objective lens 13. The wavefront is calculated by performing reverse ray tracing of light.
The wavefront synthesis unit 25 synthesizes the wavefronts calculated for all the condensing points. In the present embodiment, the wavefront synthesis unit 25 calculates a linear sum of wavefronts calculated for all the condensing points.

  The phase pattern setting unit 26 sets a phase pattern to be applied to the wavefront modulation element 12 based on the combined wavefront obtained at the entrance pupil position of the objective lens 13 and outputs the phase pattern to the wavefront modulation element 12. . Here, since the wavefront modulation element 12 is in a conjugate relationship with the entrance pupil of the objective lens 13, the phase pattern applied to the wavefront modulation element 12 is the same as or the phase of the combined wavefront at the entrance pupil position of the objective lens 13. Wrapping process. In the phase wrapping process, when the phase modulation range in the wavefront modulation element 12 is set to 2nπ (n is an integer), the portion having a phase difference exceeding 2nπ in the combined wavefront at the entrance pupil position of the objective lens 13 , By subtracting 2nπ from the phase difference. In the segment type MEMS mirror in the present embodiment, since the range of phase modulation is normally set to a range of 2nπ, phase wrapping processing is performed as necessary.

  In this manner, the wavefront modulation element 12 is adjusted to have a surface shape that matches the input phase pattern. As a result, the laser beam reflected by the wavefront modulation element 12 is modulated so as to have the same wavefront as the combined wavefront obtained at the entrance pupil position of the objective lens 13 on the surface of the wavefront modulation element 12. Therefore, such laser light is condensed by the objective lens 13 and is simultaneously condensed on each set condensing point while the objective lens 13 is fixed.

  A hologram image projecting method for projecting a hologram image for condensing laser light at a plurality of different positions in the specimen A using the hologram image projecting apparatus 1 according to the present embodiment configured as described above will be described.

First, as shown in FIG. 2, in the input unit 23, position information of each condensing point is set (step S1).
Here, when the condensing point is set at a position that responds to the light stimulus in the sample A, the wavefront modulation element 12 is set to a phase pattern having a flat reflecting surface shape by the output from the adjusting device 4. In the microscope apparatus 3, the mirror 17 is retracted from the optical path. Then, laser light is generated from the laser light source 5 and the laser light is scanned two-dimensionally by the scanner 9.

  The laser light emitted from the laser light source 5 is propagated through the optical path without changing the wavefront, scanned two-dimensionally by the scanner 9, reflected by the dichroic mirror 21, and incident on the objective lens 13 to enter the sample A. Focused on the focal plane. Fluorescence is generated at the condensing position of the laser beam, and the generated fluorescence is received by the objective lens 13, passes through the dichroic mirror 21, is collected by the collecting lenses 18 and 19, and is detected by the photodetector 15. .

  Since fluorescence is generated only in a very thin region near the focal plane of the objective lens 13, the intensity of the fluorescence detected by the photodetector 15 and the scanning position of the laser beam by the scanner 9 are stored in association with each other. Thus, a fluorescence image (slice image) of the specimen A extending along the focal plane can be acquired.

The observer designates the position of the condensing point where the laser beam is to be condensed in a two-dimensional fluorescent image displayed on a monitor (not shown). For example, in the case where the specimen A is a nerve cell and a behavior is observed by applying a stimulus by laser light, the condensing points to which the stimulus is desired are distributed. In this case, the observer sets the two-dimensional position information of the condensing point by designating all the condensing points.
On the other hand, when the condensing point is set at an arbitrary position in the specimen A, it is not necessary to acquire such a two-dimensional fluorescence image.

  In the wavefront calculation unit 24, reverse ray tracing is performed using the position information set by the input unit 23 and the lens data of each lens constituting the objective lens 13 stored in the storage unit 22 and the refractive index of the sample A. Then, the wavefront at the entrance pupil position of the objective lens 13 of the laser light generated from the point light source assumed at each condensing point is calculated (step S2).

When the wavefronts at the entrance pupil positions of the objective lens 13 are calculated for all the condensing points, the wavefront synthesizing unit 25 calculates the linear sum of the wavefronts and generates a synthesized wavefront (step S3). The generated combined wavefront is input to the phase pattern setting unit 26 so that the phase pattern applied to the wavefront modulation element 12 is the same as the combined wavefront at the entrance pupil position of the objective lens 13 or a phase-wrapped pattern. It is set (step S4). The phase pattern set by the phase pattern setting unit 26 is output to the wavefront modulation element 12.
Thereby, the preparation for observing the specimen A while performing light stimulation is completed.

  In this state, in the microscope apparatus 3, the mirror 17 is inserted on the optical path (positioned at a position indicated by a solid line in the drawing), and the fluorescence condensed by the objective lens 13 is collected by the condenser lens 20. The camera 16 is set to be photographed. Then, when the laser light emitted from the laser light source 5 is incident on the wavefront modulation unit 7 with the scanner 9 stopped at the origin position, the wavefront of the laser light is modulated according to the phase pattern displayed on the wavelength modulation element 12. Is done. The modulated laser light passes through the relay lens 8, the scanner 9, and the relay lens 10, is reflected by the dichroic mirror 21, and enters the entrance pupil of the objective lens 13 (step S5).

Since the entrance pupil position of the objective lens 13 is disposed at a position optically conjugate with the wavefront modulation element 12, the laser light incident on the entrance pupil position is the same as the wavefront when modulated by the wavefront modulation element 12. Has a wavefront.
Then, by condensing such laser light by the objective lens 13, it is possible to project a hologram image in the sample A so as to be simultaneously condensed at a plurality of condensing points arranged at different positions. .

  By irradiating laser light to a plurality of designated condensing points at the same time, the fluorescence emitted from the specimen A is received by the objective lens 13 while the specimen A is stimulated, and passes through the dichroic mirror 21. Then, it is reflected by the mirror 17, condensed by the condenser lens 20, and photographed by the camera 16. Thereby, the fluorescence image of the sample A when a light stimulus is given can be acquired.

  As described above, according to the hologram image projection apparatus 1 and the hologram image projection method according to the present embodiment, it is possible to simultaneously focus laser beams on a plurality of regions of interest arranged at different positions in the specimen A. As a result, when the condensing point is set in the specimen A at a position that responds to the light stimulus, there is an advantage that the behavior of the specimen A generated immediately after the light stimulus can be accurately observed without a time difference.

  In the present embodiment, the wavefront synthesis in the wavefront synthesis unit 25 is performed by calculating the linear sum of the wavefronts. Instead, a plurality of wavefronts calculated for each condensing point are used. It is good also as performing by dividing | segmenting the area | region into the ratio of the reciprocal number of the total number of condensing points.

  Specifically, when the total number of the condensing points is n, the area on the wavelength modulation element 12 is divided without calculating the linear sum of the wavefronts calculated for each condensing point, and the divided areas are divided. One of a plurality of condensing points is associated with each other. At this time, it is preferable to perform the association so that the corresponding region of each condensing point is periodically distributed at a ratio of the reciprocal of the total number n of condensing points. Further, the distribution of the corresponding region of each condensing point may be a mosaic shape or a concentric shape. After making such association, a wavefront element of a portion overlapping with a corresponding region on the wavelength modulation element 12 is extracted from the wavefronts calculated from the respective condensing points, and all the extracted wavefront elements are extracted. Arrange over the area to synthesize the wavefront. A phase pattern to be applied to the wavelength modulation element 12 is set based on the composite wavefront obtained in this way.

A hologram image projection apparatus and hologram image projection method according to a second embodiment of the present invention will be described below with reference to FIGS.
In the first embodiment, the wavefront is generated by performing the backward ray tracing from each set condensing point to the entrance pupil position of the objective lens 13, but in this embodiment, instead of this, A spot image is obtained by tracing forward rays from the entrance pupil position of the objective lens to each condensing point, and a wavefront that minimizes the diameter of the spot is generated.

  As shown in FIG. 3, the hologram image projector 100 according to the present embodiment is different from the first embodiment in the configuration of the adjusting device 4. That is, the adjusting device 4 stores the lens data of each lens constituting the objective lens 13 and the refractive index of the sample A, and the input unit (sets the position information of the laser beam condensing point in the sample A). (Position information setting unit) 23, position information of each condensing point set by the input unit 23, lens data of each component lens of the objective lens 13 stored in the storage unit 22, and the refractive index of the specimen A A virtual image generation unit 27 that performs forward ray tracing from the entrance pupil position of the objective lens to the focal plane of the objective lens and generates a virtual image having a plurality of spots formed at each condensing point; A wavefront calculator 28 for calculating the wavefront of the laser beam at the entrance pupil position of the objective lens so that the spot diameter in the virtual image is minimized, and the wave based on the wavefront calculated by the wavefront calculator 28 And a phase pattern setting unit 26 for setting the phase pattern imparted to the modulation element 12.

Here, the forward ray tracing in the virtual image generation unit 27 is performed using the position information of each condensing point, the lens data of each component lens of the objective lens 13, and the refractive index of the specimen A.
The wavefront calculation unit 28 performs an operation for deforming the wavefront of the laser light at the entrance pupil position of the objective lens, and calculates the spot diameter in the virtual image by forward ray tracing. Then, the wavefront calculation unit 28 repeats this operation to calculate the wavefront of the laser light that minimizes the spot diameter in the generated virtual image.

  A holographic image projection method for projecting a holographic image that simultaneously condenses laser light at a plurality of different positions in the specimen A using the holographic image projection apparatus 100 according to the present embodiment configured as described above will be described.

First, as shown in FIG. 4, in the input unit 23, position information of each condensing point on the focal plane of the objective lens is set (step S11). Here, when the condensing point is set at a position that responds to the light stimulus in the specimen A, each condensing point in the slice image acquired on the focal plane of the objective lens 13 is designated by the input unit 23. Do.
Next, in the input unit 23, when it is assumed that the objective lens 13 has no aberration, all the condensing points are condensed at different positions on the focal plane of the objective lens 13 to form the spot R. A condensing point map is generated (step S12).

  However, since there is aberration in the actual objective lens 13, even if a laser beam for obtaining a hologram image as shown in FIG. As shown in FIG. 5B, some of the spots R1 are distorted by aberrations and become large. Therefore, the virtual image generation unit 27 performs Fourier transform on the condensing point map to calculate the wavefront of the laser light at the entrance pupil position of the objective lens 13 (step S13). Then, in the virtual image generation unit 27, the entrance pupil position of the objective lens 13 for the laser light having the wavefront at the calculated entrance pupil position using the lens data of each lens constituting the objective lens 13 and the refractive index of the specimen A. A normal ray is traced from the focal plane to the focal plane to generate a virtual image (step S14). At this time, in the generated virtual image, the influence of the actual aberration is reflected, and a part of the spot R1 is distorted and enlarged by the aberration.

  For each condensing point forming such a spot R1, the wavefront of the laser light incident on the objective lens 13 at the entrance pupil position of the objective lens 13 is repeatedly deformed so that the spot diameter of the spot R1 is minimized. A simple wavefront is calculated (step S15). Specifically, the coefficient of the Zernike polynomial is repeatedly changed using the spot diameter of each spot R1 as an evaluation value, and the coefficient of the Zernike polynomial that minimizes the spot diameter as the evaluation value is specified.

Here, the cylindrical function w (r, θ) that gives the shape of the wavefront that is a Zernike polynomial is expressed by the following equation. That is,
w (r, θ) = Z1
+ Z2 ・ ρcosθ
+ Z3 · ρsinθ
+ Z4 · (2ρ ² -1)
+ Z5 · ρ ² cos 2θ
+ Z6 · ρ ² sin2θ
+ Z7 · (3ρ ² -2) ρcosθ
+ Z8 · (3ρ ² -2) ρsinθ
...
It is. Here, ρ is the distance from the pupil center when normalized by the radius r from the pupil center of the entrance pupil in the objective lens 13, and θ is on the surface of the wavefront modulation element 12 or the entrance pupil of the objective lens 13. An angle with respect to a predetermined reference line by polar coordinates on the surface is shown. Zn is a Zernike coefficient.

  The wavefront calculated in this way is input to the phase pattern setting unit 26, and the phase pattern applied to the wavefront modulation element 12 is the same as the wavefront at the entrance pupil position of the objective lens 13, or a phase-wrapped pattern. (Step S16). Then, the phase pattern set by the phase pattern setting unit 26 is output to the wavefront modulation element 12. In this state, when laser light is generated from the laser light source 5 and incident on the wavefront modulation unit 7, the wavefront of the laser light is modulated according to the phase pattern of the wavelength modulation element 12. The modulated laser light is condensed and irradiated onto the sample by the objective lens 13 via the relay lens 8, the scanner 9, the relay lens 10 and the dichroic mirror 21 (step S17).

  Thus, the forward ray tracing in the virtual image generation unit 27 is performed using the lens data of each lens constituting the objective lens 13 in addition to the position information of each condensing point and the refractive index of the specimen A. Therefore, each spot of the generated virtual image includes the influence of the aberration existing in the objective lens 13. Therefore, by changing the coefficient Zn of the Zernike polynomial so that the spot diameter of the spot R1 distorted by aberration is minimized, the wavefront at the entrance pupil position of the objective lens 13 is modulated into a wavefront that anticipates aberration in advance. The Rukoto.

  Therefore, by making a laser beam having such a wavefront incident on the entrance pupil position of the objective lens 13 from the opposite side of the sample A, the wavefront is restored while being focused by the objective lens 13, and on the focal plane. Light can be collected with high accuracy. Therefore, the laser light is simultaneously focused on a plurality of condensing points on the focal plane of the objective lens 13 by making the laser light incident on the wavefront modulation element 12 to which such a wavefront is provided. Such a hologram image can be projected into the specimen.

  Further, in the present embodiment, the segment type MEMS that changes the surface shape is exemplified as the wavefront modulation element 12, but instead of this, any other wavefront modulation element 12, such as a liquid crystal element, deformable A mirror or the like may be used. In the case of a liquid crystal element, the refractive index distribution due to the orientation of liquid crystal molecules is a phase pattern, and in the case of a deformable mirror, the surface shape is a phase pattern.

R, R1 Spots S1, S11 Steps for setting position information S2, S15 Steps for calculating a wavefront at the entrance pupil position S3 Steps for calculating a composite wavefront S4, S16 Steps for setting a phase pattern S5, S17 Step of condensing on specimen S12 Step of generating condensing point map S14 Step of generating virtual image 1 Hologram image projection device 12 Wavefront modulation element 13 Objective lens 23 Input unit (position information setting unit)
24 Wavefront Calculation Unit 25 Wavefront Synthesis Unit 26 Phase Pattern Setting Unit

Claims (6)

A position setting step for setting position information on a focal plane of the objective lens in the sample for a plurality of condensing points on which the laser light is to be collected via the objective lens;
From each condensing point assumed at a set position by using position information of each set condensing point in the sample, a refractive index of the sample, and lens data of each lens constituting the objective lens A wavefront calculating step of performing a ray tracing to the entrance pupil position of the objective lens and calculating a wavefront at the entrance pupil position of the laser light from each of the condensing points;
A wavefront synthesizing step for calculating a combined wavefront by combining the plurality of wavefronts calculated corresponding to the respective condensing points;
A phase pattern setting step for setting a phase pattern to be applied to the wavefront modulation element based on the calculated composite wavefront;
A condensing step of applying the set phase pattern to the wavefront modulation element to make the laser light incident, and condensing the laser light, the wavefront of which is modulated by the phase pattern, onto the sample through the objective lens And a holographic image projection method.   The hologram image projection method according to claim 1, wherein the wavefront synthesis step is a step of calculating the synthesized wavefront by calculating a linear sum of the plurality of wavefronts.   2. The hologram image projection according to claim 1, wherein the wavefront synthesizing step is a step of calculating the synthesized wavefront by arranging the wavefronts obtained by dividing the plurality of wavefronts at a ratio that is a reciprocal of the number of the condensing points. Method. A position setting step for setting position information on a focal plane of the objective lens in the sample for a plurality of condensing points on which the laser light is to be collected via the objective lens;
Using the position information of each set condensing point in the sample, the refractive index of the sample, and lens data of each lens constituting the objective lens, from the entrance pupil position of the objective lens to the focal plane A virtual image generation step of performing forward ray tracing to generate a virtual image having a plurality of spots formed at each of the condensing points;
A wavefront calculating step of calculating the wavefront such that the diameter of each spot in the generated virtual image is minimized by deforming the wavefront of the laser light at the entrance pupil position of the objective lens;
A phase pattern setting step for setting a phase pattern to be applied to the wavefront modulation element based on the calculated wavefront;
A condensing step of applying the set phase pattern to the wavefront modulation element to make the laser light incident, and condensing the laser light, the wavefront of which is modulated by the phase pattern, onto the sample through the objective lens And a holographic image projection method. The position information is a condensing point map having a plurality of the condensing points assumed on a focal plane of the objective lens in the specimen;
The virtual image generating step includes a step of calculating a wavefront of the laser light at an entrance pupil position of the objective lens by performing Fourier transform on the condensing point map, a refractive index of the sample, and each lens constituting the objective lens Generating a virtual image by performing forward ray tracing from the entrance pupil position of the objective lens to the focal plane for the laser light having the calculated wavefront using the lens data of Item 5. The hologram image projecting method according to Item 4. A wavefront modulation element that modulates the wavefront of the laser beam with a phase pattern;
An objective lens that focuses the laser light modulated by the wavefront modulation element on a sample;
A position information setting unit for setting position information on the focal plane of the objective lens in a sample of a plurality of focal points on the focal plane on which the laser light is to be collected via the objective lens;
Each condensing assumed at a set position using the position information of each condensing point set by the position information setting unit, the refractive index of the sample, and lens data of each lens constituting the objective lens A wavefront calculation unit that performs reverse ray tracing from a point to the entrance pupil position of the objective lens and calculates a wavefront at the entrance pupil position of the laser beam from each of the condensing points;
A wavefront synthesizing unit that calculates a combined wavefront by combining the plurality of wavefronts calculated corresponding to each of the condensing points;
A hologram image projection apparatus comprising: a phase pattern setting unit that sets the phase pattern to be applied to the wavefront modulation element based on the calculated composite wavefront.

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