JP2011128572A - Hologram image projection method and hologram image projector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simultaneously focus stimulus light on stimulus positions arranged in different positions in a depth direction in a sample. <P>SOLUTION: A hologram image projection method includes: a step S1 of setting the three dimensional positional information of a plurality of focal points on which a laser beam is focused through an objective lens in a sample; a step S2 of performing reverse ray tracking from each of focal points to an entrance pupil of the objective lens using positional information on each of focal points in the sample, the refractive index of the sample and characteristic data of the whole system of the objective lens to calculate a wave front at the entrance pupil of the laser light from each of the focal points; a step S3 of calculating a combination wave front by combining a plurality of the calculated wave fronts; a step S4 of setting a phase pattern added to a wave front modulating element based on the calculated combination wave front; and a step S5 of adding the set phase pattern to the wave front modulating element and making the laser light enter the element to focus the laser light of which the wave front is modulated by the phase pattern on the sample through 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. The phase pattern of the hologram is created by performing Fourier transform on. 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 and photostimulation with spatial light modulators", Frontiers in Neural Circuits, Vol 2, Article 5, 19 December 2008, p1-15

  However, in the conventional method, although light can be condensed simultaneously on a plurality of target portions existing on the focal plane of the objective lens, they are arranged at positions shifted in the optical axis direction from the focal plane. There is an inconvenience that light cannot be focused on the site of interest.

  The present invention has been made in view of the above-described circumstances, and a hologram image projection method and hologram image projection capable of simultaneously condensing light on regions of interest arranged at different positions in the depth direction within a specimen The object is to provide a device.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a position setting step for setting three-dimensional position information in a sample of a plurality of condensing points on which laser light is to be condensed via an objective lens, and the inside of the sample at each set condensing point. Using the position information, the refractive index of the sample, and the characteristic data of the entire objective lens system, the ray tracing from each condensing point assumed at the set position to the entrance pupil position of the objective lens is performed. Performing a wavefront calculating step for calculating a wavefront at the entrance pupil position of the laser light from each condensing point, and combining a plurality of wavefronts calculated corresponding to each condensing point to obtain a combined wavefront A wavefront synthesis step for calculating, a phase pattern setting step for setting a phase pattern to be applied to the wavefront modulation element based on the calculated composite wavefront, and applying the set phase pattern to the wavefront modulation element Is incident the laser beam Te, the laser light wavefront modulated by the phase pattern, to provide a hologram image projection method comprising a condensing step of condensing the specimen via the objective lens.

  According to the present invention, the three-dimensional position information of the respective condensing points that are three-dimensionally arranged at different positions in the sample is set by the position setting step, and in the wavefront calculating step, the objective is determined from each condensing point. Back ray tracing to the entrance pupil position of the lens is performed. The plurality of wavefronts calculated in the wavefront calculation step need to be incident on the entrance pupil position of the objective lens in order to simultaneously collect the laser light at three-dimensionally different positions by being synthesized in the wavefront synthesis step. A synthetic wavefront as a simple wavefront is obtained by calculation. 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. Thereby, it is possible to project a hologram image into the sample such that the laser beam is simultaneously focused on the condensing points arranged three-dimensionally at different depth positions.

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 three-dimensional position information in a sample of a plurality of condensing points on which laser light is to be condensed via the objective lens, and the above-described condensing points of the set condensing points. Using the position information in the sample, a virtual image generating step for generating a virtual image obtained by projecting the respective condensing points on a predetermined reference plane in the sample, and the generated virtual image is used as a refraction of the sample. A wavefront calculating step for calculating a wavefront of the laser light at the entrance pupil position of the objective lens by performing Fourier transform using characteristic data of the entire system of the objective lens, and wavefront modulation based on the calculated wavefront A phase pattern setting step for setting a phase pattern to be applied to the element; and applying the laser beam by applying the set phase pattern to the wavefront modulation element, and changing the wavefront by the phase pattern. Said laser light to provide a hologram image projection method comprising a condensing step of condensing the specimen via the objective lens.

  According to the present invention, the three-dimensional position information of the respective condensing points arranged three-dimensionally at different positions in the sample is set by the position setting step, and the virtual image generation step is performed via the objective lens. A virtual image is generated by projecting a plurality of condensing points on which laser light is to be condensed onto a predetermined reference plane. In the wavefront calculating step, the wavefront at the entrance pupil position of the objective lens is calculated by Fourier transforming the virtual image. Then, a phase pattern to be applied to the wavefront modulation element is set in the phase pattern setting step, and in the condensing step, the set phase pattern is applied to the wavefront modulation element, and laser light is incident on the wavefront modulation element. Laser light whose wavefront is modulated by the phase pattern is condensed on the sample via the objective lens. Thereby, it is possible to project a hologram image in which a laser beam is simultaneously focused on a plurality of focusing points arranged three-dimensionally in the sample.

  In the above invention, the position setting step sets a planar position setting step for setting planar position information in the sample of the plurality of focusing points, and sets depth information for each focusing point with respect to the reference plane. Depth information setting step, wherein the virtual image generation step uses planar position information of each of the condensing points in the sample, so that each of the condensing points is on a predetermined reference plane in the sample. A first virtual image generating step for generating a first virtual image when it is assumed that a plurality of spots are formed by condensing the light, and the depth information of the set condensing points A second virtual image generating step of generating a second virtual image by correcting a corresponding spot in the first virtual image.

  By doing in this way, the set depth information is set for the spot corresponding to the condensing point that is not condensed on the reference plane among the spots in the first virtual image where all the spots are condensed on the predetermined reference plane. Since the phase pattern is generated from the second virtual image generated by correcting the spot based on the wavefront modulation element, the wavefront modulation element can be obtained simply by irradiating the wavefront modulation element provided with the phase pattern with a laser beam composed of parallel light. The laser beam modulated by can be condensed at a depth position different from the reference plane in the sample. Thereby, a laser beam can be simultaneously condensed on the several condensing point which differs in a depth direction.

Further, in the above invention, the second virtual image generation step is obtained by ray tracing the laser beam that focuses the spot in the first virtual image at each condensing point to the reference plane. It may be a step of correcting the corresponding spot in the first virtual image by replacing it with a spot of laser light.
By doing in this way, the spot in the first virtual image is replaced with a spot obtained by performing forward ray tracing or backward ray tracing to the reference plane with the laser beam condensed at each condensing point. It is possible to easily generate a second virtual image that forms spots that converge at different positions in the depth direction by calculation.

  In the above invention, the second virtual image generating step is performed on the reference plane in a state where the objective lens is arranged so that the focal position of the objective lens is shifted from the reference plane by a predetermined distance in the optical axis direction. Dividing the vector information change amount when the vector information representing the aberration at the focal plane of the objective lens is changed so as to illuminate, and calculating a proportionality coefficient by the predetermined distance; and And correcting the spot using vector information obtained by multiplying the depth information.

  In this way, by multiplying the calculated proportionality coefficient by depth information indicating the distance between the actual condensing point and the reference surface, the objective lens for condensing the laser light on the reference surface can be obtained. In the position, vector information representing the aberration in the focal plane of the objective lens necessary for condensing the laser beam at a depth position different from the reference plane can be obtained, and the second corrected spot based on this can be obtained. By generating the virtual image, it is possible to simultaneously focus the laser light on a plurality of condensing points with different depth directions while fixing the objective lens.

  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 the laser through the objective lens. A position information setting unit for setting three-dimensional position information in a sample of a plurality of condensing points to collect light, and the position in the sample of each condensing point set by the position information setting unit Using the information, the refractive index of the sample, and the characteristic data of the entire system of 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 is performed, A wavefront calculator that calculates a wavefront at the entrance pupil position of the laser beam from each condensing point, and a plurality of wavefronts calculated by the wavefront calculator corresponding to each condensing point are combined and combined Wave to calculate wavefront Providing a synthesis section, a hologram image projection apparatus and a phase pattern setting unit for setting the phase pattern imparted to the wavefront modulation element based on the calculated combined wavefront.

  According to the present invention, when the three-dimensional position information of each condensing point is set by the position information setting unit, the set position information, the refractive index of the sample, and the entire objective lens system are set by the wavefront calculation unit. Using the characteristic data, reverse ray tracing from the focal point to the entrance pupil position of the objective lens is performed. Thereby, the wavefront at the entrance pupil position of the objective lens of the laser light from each condensing point is calculated. Then, the plurality of wavefronts obtained for each condensing point are synthesized in the wavefront synthesis unit to obtain a synthesized wavefront. A phase pattern is set by the phase pattern setting unit based on the composite wavefront obtained in this way. As a result, the laser beam condensed by the objective lens can be simultaneously applied to a plurality of condensing points arranged at three-dimensionally different positions by simply making the laser beam incident on the wavefront modulation element provided with the set phase pattern. The focused hologram image can be projected into the specimen.

  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 the laser through the objective lens. A position information setting unit for setting three-dimensional position information in a sample of a plurality of condensing points to collect light, and the position in the sample of each condensing point set by the position information setting unit Using the information, a virtual image generation unit that generates a virtual image obtained by projecting each of the focal points onto a predetermined reference plane in the sample, and the virtual image generated by the virtual image generation unit Based on the calculated wavefront, a wavefront calculating unit that calculates the wavefront of the laser light at the entrance pupil position of the objective lens by performing Fourier transform using the refractive index and characteristic data of the entire system of the objective lens Wavefront modulation Providing a hologram image projection apparatus and a phase pattern setting unit for setting the phase pattern imparted to the child.

  According to the present invention, when the three-dimensional position information of each condensing point is set by the position information setting unit, each condensing point is set in the sample using the set position information by the virtual image generating unit. A virtual image projected on a predetermined reference plane is generated, and the wavefront calculation unit Fourier transforms the virtual image using the refractive index of the sample and the characteristic data of the entire system of the objective lens, and the wavefront at the entrance pupil position of the objective lens Is calculated. Then, the phase pattern setting unit sets a phase pattern to be applied to the wavefront tuning element based on the calculated wavefront. As a result, the laser beam condensed by the objective lens can be simultaneously applied to a plurality of condensing points arranged at three-dimensionally different positions by simply making the laser beam incident on the wavefront modulation element provided with the set phase pattern. The focused hologram image can be projected into the specimen.

  According to the present invention, there is an effect that it is possible to simultaneously collect light on a site of interest arranged at a different position in the depth direction in the specimen.

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. It is a figure explaining the production | generation method by (a) forward ray tracing and (b) back ray tracing of the 2nd virtual image in the hologram image projection method of FIG. It is a figure which shows the 2nd virtual image produced | generated by the method of FIG. 5 by (a) forward ray tracing and (b) reverse ray tracing. FIG. 5 is a diagram for explaining a modification of the hologram image projection method of FIG. 4 (a) a state in which the reference plane is made coincident with the focal plane of the objective lens, and (b) the focal plane of the objective lens is shifted from the reference plane in the depth direction. (C) is a diagram schematically showing a state in which the focal position of the objective lens is made to coincide with the reference plane by adjusting the Zernike coefficient.

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 the wavefront of 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 light 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 characteristic data relating to the entire known system, and is provided so that the distance in the optical axis direction from the stage 14 can be changed. Here, the characteristic data of the entire system of the objective lens 13 includes the focal length, the numerical aperture, and the diameter of the entrance pupil of the entire system of the objective lens 13.

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. Thereby, 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.

  Then, by relatively moving the distance between the objective lens 13 and the stage 14 and changing the position of the focal plane of the objective lens 13, a plurality of two-dimensional fluorescence images (slice images) are acquired. A three-dimensional fluorescence image of the specimen A can be acquired.

  As shown in FIG. 1, the adjusting device 4 includes a storage unit 22 that stores characteristic data of the entire system of the objective lens 13 and a refractive index of the sample A, and three-dimensional position information of a laser beam condensing point in the sample A. An input unit (position information setting unit) 23 for setting the position information of each condensing point set by the input unit 23, the characteristic data of the entire system of the objective lens 13 stored in the storage unit 22, and the sample A A wavefront calculating unit 24 for calculating the wavefront at the entrance pupil position of the objective lens 13 corresponding to each condensing point based on the refractive index, and a wavefront combining unit 25 for combining the wavefronts calculated for all the condensing points; A phase pattern setting unit for setting a phase pattern to be applied to the wavefront modulation element 12 based on the combined wavefront synthesized by the wavefront synthesis unit 25;

The input unit 23 sets the three-dimensional position information of each condensing point by designating the position where the observer wants to condense the laser beam, that is, the condensing point on a monitor (not shown). It has become. Here, the position information in the depth direction of each condensing point is set as relative depth information from the reference surface when the surface of the sample A having a predetermined depth is used as the reference surface. ing. The reference plane is preferably set to the focal plane of the objective lens 13 when the objective lens 13 is fixed at a predetermined position with respect to the specimen A, but is not limited thereto.
Moreover, the condensing point in the sample A may be set at an arbitrary position or may be set at a position that responds to light stimulation. When it is desired to set the focal point to a position that responds to light stimulation, a monitor (not shown) is a two-dimensional image or a three-dimensional image acquired by the microscope device 3 so that the observer can easily designate the focal point. Is preferably displayed.

The wavefront calculation unit 24, for each set condensing point, the position information set from the input unit 23, the entire characteristic data of the objective lens 13 stored in the storage unit 22, and the refractive index of the sample A Is used to calculate the wavefront corresponding to each condensing point.
Specifically, a point light source is assumed at a position specified by position information set with respect to the condensing point, and the refractive index of the sample A and all of the objective lens 13 from the point light source to the entrance pupil position of the objective lens 13 are assumed. The wavefront is calculated by performing backward ray tracing of the laser beam using the system characteristic data. For the condensing points arranged on the focal plane which is the reference plane, the wavefront at the entrance pupil position is a plane wave in which the angle with respect to the optical axis of the objective lens 13 is varied according to the position of the condensing point in the reference plane. is there.
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 that simultaneously condenses laser light at a plurality of positions with different depths in the specimen A using the hologram image projector 1 according to the present embodiment configured as described above. explain.
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. A three-dimensional fluorescence image can be acquired by acquiring a plurality of slice images while relatively moving the objective lens 13 and the specimen A in the optical axis direction.

  The observer designates the position of the condensing point where the laser beam is to be condensed in the three-dimensional fluorescent image displayed on a monitor (not shown). For example, in the case where the specimen A is a nerve cell and the behavior is observed by applying a stimulus by laser light, the focal points to which the stimulus is desired are distributed three-dimensionally. In this case, the observer sets the three-dimensional position information of the condensing points by designating all the condensing points.

By fixing the objective lens 13 to the specimen A, the focal plane of the objective lens 13 is fixed to the specimen A. Of the position information of the condensing point, for the depth information, a relative distance in the depth direction from the reference surface is set when the surface of the sample A having a predetermined depth is used as the reference surface. This reference plane is set to the focal plane of the objective lens 13, for example.
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 three-dimensional fluorescence image.

  Next, the wavefront calculation unit 24 performs reverse ray tracing using the position information set by the input unit 23, the entire characteristic data of the objective lens 13 stored in the storage unit 22, and the refractive index of the sample A. 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). Then, 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.
A sample of such a three-dimensional hologram image that is simultaneously focused on a plurality of focusing points arranged at different positions in the depth direction when the laser beam is focused by the objective lens 13. A can be projected into A.

  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, the laser beam is simultaneously condensed on a plurality of target portions arranged at different positions in the depth direction of the specimen A. Can do. 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 a hologram image projection method according to the second embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, the same reference numerals are given to portions having the same configurations as those of the hologram image projection apparatus and the hologram image projection method according to the first embodiment described above, and description thereof is omitted.

  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 forward ray is traced from each condensing point to a predetermined reference plane to obtain a spot image, and a wavefront is generated by converting the spot image.

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 has a storage unit 22 that stores the characteristic data of the entire system of the objective lens 13 and the refractive index of the sample A, and an input unit that sets three-dimensional position information of the laser beam condensing point in the sample A ( (Position information setting unit) 23, the position information of each condensing point set by the input unit 23, the entire characteristic data of the objective lens 13 stored in the storage unit 22, and the refractive index of the sample A, A virtual image generation unit 27 that generates a virtual image obtained by projecting each condensing point on a predetermined reference plane, and a wavefront calculation unit 28 that calculates a wavefront at the entrance pupil position of the objective lens 13 by Fourier transforming the generated virtual image. And a phase pattern setting unit 26 that sets a phase pattern to be applied to the wavefront modulation element 12 based on the wavefront calculated by the wavefront calculation unit 28.
Here, the wavefront calculation unit 28 uses the characteristic data of the entire system of the objective lens 13 stored in the storage unit 22 and the refractive index of the sample A to Fourier transform the virtual image generated by the virtual image generation unit 27. is doing.

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

First, as shown in FIG. 4, planar position information within the reference plane of each condensing point is set in the input unit 23 (step S <b> 11).
Next, the virtual image generation unit 27 is arranged at different positions in the depth direction with respect to the reference plane based on the planar position information in the reference plane of each condensing point set by the input unit 23. A first virtual image is generated when it is assumed that all the condensing points are condensed on the reference plane to form spots (step S12). The first virtual image is generated by designating each condensing point using only one slice image acquired on an arbitrary reference plane. This reference plane is preferably set to the focal plane of the objective lens 13, but is not limited thereto. Thus, in the generation of the first virtual image, since the actual depth information of the condensing point is not used, the first virtual image does not reflect an actual spot image, It is a spot image when it is assumed that all the condensing points are on the reference plane.

Next, depth information from the reference plane at each condensing point is input from the input unit 23 (step S13).
Then, in the virtual image generation unit 27, the reference plane is corrected by adding the depth information set by the input unit 23 to the spot corresponding to the condensing point condensed at a position different from the reference plane. A second virtual image is generated (step S14). Thereafter, the generated second virtual image is Fourier-transformed to calculate a wavefront at the entrance pupil position of the objective lens 13 that can project a spot in the second virtual image (step S15). Then, the calculated 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 wavefront at the entrance pupil position of the objective lens 13 or a phase-wrapped pattern. It is set (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).

  As shown in FIG. 5, the second virtual image is generated by assuming a point light source at a condensing point P that does not exist on the reference plane Q, from the point light source to the reference plane Q in the order (a). The wavefront constituting the new spots R1 and R2 on the reference plane Q is calculated by ray tracing or (b) reverse ray tracing. In the obtained wavefront, as shown in FIG. 6, the depth information included in the wavefront is inverted between (a) spot R1 in the case of forward ray tracing and (b) spot R2 in the case of reverse ray tracing. Yes. The spot R existing on the reference plane Q is not corrected.

As another method for generating the second virtual image, first, as shown in FIG. 7A, the objective lens 13 is fixed to the specimen A, and laser light is collected on the reference plane Q. Fluorescent image is acquired by light. Next, as shown in FIG. 7B, the objective lens 13 is moved by Δd in the optical axis direction. Thereby, since the condensing point P shift | deviates from the reference plane Q, the light quantity of a fluorescence image changes. Fluorescence in this state, as shown in FIG. 7 (c), when continuously changing the fourth term coefficient Z ₄ of the Zernike polynomials is a vector information representing the aberration in the focal plane Q of the objective lens 13 observing the light intensity of the image, determining the value of the coefficient Z _4, such as the same amount and the first amount of light is obtained. Then, the variation of the coefficient Z ₄ in this case as a [Delta] Z, calculates a proportional coefficient K = ΔZ / Δd. Thus, the proportional coefficient K calculated, based on the depth information from the reference plane Q to the converging point P, Zernike coefficient Z ₄ and the incident pupil position of the objective lens 13 (black triangles in FIG. 6 The wavefront W at the position indicated by

A cylindrical function w (r, θ) that gives a wavefront shape 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 of the objective lens 13 when normalized by the radius from the pupil center, and θ is on the surface of the wavefront modulation element 12 or the entrance pupil surface of the objective lens 13. The angle with respect to a predetermined reference line in the polar coordinates above is shown. Zn is a Zernike coefficient.

The wavefront W at the entrance pupil position of the objective lens 13 uses the fourth term that is the defocus term of the Zernike polynomial.
W = Z ₄ × (2ρ ² −1)
It can be expressed as. In addition, Zernike coefficient _{Z 4} are,
Z ₄ = K × d
It can be expressed as.
Here, d is depth information set in the input unit 23 from the reference plane Q to the condensing point P.

  After the wavefront W is obtained in this way, the wavefront at the reference plane Q when the laser light of this wavefront is incident on the entrance pupil position of the objective lens 13 is obtained, and the corresponding spot in the first virtual image is obtained Correct by replacing with wavefront. By repeating the same operation for all the condensing points P that are displaced in the depth direction with respect to the reference plane Q, a second virtual image is generated.

  In the present embodiment, the second virtual image corrected based on the depth information is generated after generating the first virtual image when it is assumed that all the condensing points are condensed on the reference plane. Instead of generating a virtual image, instead of this, a virtual image in which each condensing point is projected on a predetermined reference plane may be generated once based on the three-dimensional position information of the condensing point. . In this case, in the input unit 23, three-dimensional position information of each condensing point is set. Further, the virtual image generation unit 27 determines each condensing point based on the three-dimensional position information, the entire characteristic data of the objective lens 13 stored in the storage unit 22, and the refractive index of the sample A. A virtual image projected on the reference plane is generated.

  Further, in the first and second embodiments, the segment type MEMS mirror that changes the surface shape is exemplified as the wavefront modulation element 12, but instead of this, any other wavefront modulation element 12, For example, a liquid crystal element, a deformable 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.

A Sample Δd Predetermined distance P Condensing point Q Reference plane R, R1, R2 Spot ΔZ Vector information variation S1, S11 Step for setting position information S2 Step for calculating wavefront at the entrance pupil position of laser light S3 Compound wavefront Steps for calculation S4, S16 Step for setting phase pattern S5 Step for condensing laser light on the specimen S12 Step for generating first virtual image S14 Step for generating second virtual image 1,100 Hologram image projector 12 Wavefront modulation element 13 Objective lens 23 Input section (position information setting section)
24 Wavefront Calculation Unit 25 Wavefront Synthesis Unit 26 Phase Pattern Setting Unit

Claims (9)

A position setting step for setting three-dimensional position information in the sample of a plurality of condensing points on which the laser light is to be condensed via the objective lens;
Using the position information, the refractive index of the specimen, and the characteristic data of the entire system of the objective lens of the set focal point, from the focused point assumed at the set position, the A wavefront calculating step of performing reverse 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 combining step of calculating a combined wavefront by combining a 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 A hologram image projection method including:   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 three-dimensional position information in the sample of a plurality of condensing points on which the laser light is to be condensed via the objective lens;
A virtual image generating step for generating a virtual image obtained by projecting each focused point on a predetermined reference plane in the sample, using the position information of each set focused point in the sample;
A wavefront calculating step of calculating a wavefront of the laser light at the entrance pupil position of the objective lens by Fourier transforming the generated virtual image using the refractive index of the sample and the characteristic data of the entire system of the objective lens When,
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 A hologram image projection method including: The position setting step includes a planar position setting step for setting planar position information in the sample of the plurality of focusing points, and a depth information setting for setting depth information for each focusing point with respect to the reference plane. Including steps,
The virtual image generation step uses the planar position information of each condensing point in the sample to condense each condensing point on a predetermined reference surface in the sample to form a plurality of spots. A first virtual image generating step for generating a first virtual image when it is assumed to be, and a corresponding spot in the first virtual image based on the set depth information of each condensing point A hologram image projection method according to claim 4, further comprising: a second virtual image generation step of correcting the error and generating a second virtual image.   The second virtual image generating step replaces the spot in the first virtual image with the spot of the laser beam obtained by ray tracing the laser beam condensed at each condensing point to the reference plane. The hologram image projecting method according to claim 5, wherein the corresponding spot in the first virtual image is corrected.   In the second virtual image generating step, the objective lens is focused on the reference plane in a state where the objective lens is arranged so that the focal position of the objective lens is shifted from the reference plane by a predetermined distance in the optical axis direction. Dividing the vector information change amount when the vector information representing the aberration in the focal plane is divided by the predetermined distance to calculate a proportional coefficient, and multiplying the proportional coefficient and the depth information for each spot And correcting the spot using the vector information obtained as described above. 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 specimen;
A position information setting unit for setting three-dimensional position information in a sample of a plurality of condensing points on which the laser light is to be condensed via the objective lens;
Each position assumed in the set position using the position information in the sample of each condensing point set by the position information setting unit, the refractive index of the sample, and the characteristic data of the entire system of the objective lens A wavefront calculation unit that performs reverse ray tracing from the condensing point to the entrance pupil position of the objective lens, and calculates a wavefront at the entrance pupil position of the laser light from each condensing point;
A wavefront synthesizing unit that calculates a synthesized wavefront by synthesizing a plurality of wavefronts calculated corresponding to each of the condensing points by the wavefront calculating unit;
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. 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 three-dimensional position information in a sample of a plurality of condensing points on which the laser light is to be condensed via the objective lens;
Virtual image generation for generating a virtual image in which each condensing point is projected on a predetermined reference plane in the sample using the position information in the sample of each condensing point set by the position information setting unit And
The wavefront generated at the entrance pupil position of the objective lens of the objective lens by Fourier transforming the virtual image generated by the virtual image generator using the refractive index of the specimen and the characteristic data of the entire objective lens system A wavefront calculating unit for calculating
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 wavefront.

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