JP6770616B2 - Image acquisition device, image acquisition method, and spatial optical modulation unit - Google Patents

Description

The present invention relates to an image acquisition device, an image acquisition method, and a spatial light modulation unit that acquire an image by capturing the detection light emitted from a sample when irradiated with excitation light.

As such an image acquisition device, there is a light sheet microscope that irradiates a sample with a sheet-shaped excitation light and images the detection light emitted from the sample by the irradiation of the excitation light (see, for example, Non-Patent Document 1 below). .. In the light sheet microscope described in Non-Patent Document 1, a Bessel beam is generated by a spatial light modulator (SLM), and the focused point of the generated Bessel beam is scanned along the optical axis. , Creates a pseudo sheet-like excitation light.

FlorianO. Fahrbach and Alexander Rohrbach, “A line Scanned light-sheetmicroscope withphase shaped self-reconstructing beams”, November2010 / Vol.18, No.23 / OPTICS EXPRESS pp.24229-24244

In the light sheet microscope described in Non-Patent Document 1, a pseudo sheet-like excitation light is generated by scanning the focusing point of the Bessel beam along the optical axis. Optical elements and the like are required. Therefore, the device configuration may be complicated.

Therefore, an object of the present invention is to provide an image acquisition device, an image acquisition method, and a spatial light modulation unit capable of generating sheet-shaped excitation light with a simple configuration.

The image acquisition device according to one aspect of the present invention has a light source that outputs excitation light including a wavelength that excites a sample, and a plurality of pixels arranged in two dimensions, and determines the phase of the excitation light output from the light source. A spatial light modulator that modulates each of a plurality of pixels, a first objective lens that irradiates the sample with excitation light modulated by the spatial light modulator, and a sample that accompanies irradiation of excitation light from the first objective lens. A second objective lens that guides the detection light emitted from the second objective lens, an optical detector that captures the detection light guided by the second objective lens, and a phase value corresponding to each of a plurality of pixels are distributed in two dimensions. A control unit that controls the amount of phase modulation for each of a plurality of pixels according to the phase pattern to be performed is provided, the phase pattern is a phase pattern generated based on a predetermined basic phase pattern, and the basic phase pattern is a predetermined direction. A first region in which the phase value continuously increases along the predetermined direction and a second region facing the first region in a predetermined direction and in which the phase value continuously decreases along the predetermined direction. Have.

In this image acquisition device, a basic phase having a first region in which the phase value continuously increases along a predetermined direction and a second region in which the phase value continuously decreases along a predetermined direction. The phase pattern is calculated based on the pattern. By modulating the excitation light with the spatial light modulator according to this phase pattern, it is possible to irradiate the sample with the excitation light in the form of a sheet from the first objective lens. Therefore, it is not necessary to scan the focusing point of the Bessel beam in order to generate the sheet-shaped excitation light as in the prior art, and the optical element or the like for the scanning can be omitted. Therefore, according to this image acquisition device, it is possible to generate sheet-shaped excitation light with a simple configuration.

In the image acquisition device according to one aspect of the present invention, in the first region, the phase value increases linearly along a predetermined direction, and in the second region, the phase value linearly increases along a predetermined direction. May decrease. According to this, since the basic phase pattern is simplified, it is possible to generate sheet-shaped excitation light with a simpler configuration without using a complicated optical element or the like.

In the image acquisition device according to one aspect of the present invention, the basic phase pattern may be axisymmetric with respect to a straight line passing through the center in a predetermined direction and orthogonal to the predetermined direction. According to this, the sheet-shaped excitation light can be generated on the optical axis of the first objective lens. Therefore, the optical axis adjustment of the first objective lens and the second objective lens becomes easy.

In the image acquisition apparatus according to one aspect of the present invention, the basic phase pattern may be non-axisymmetric with respect to a straight line passing through the center in a predetermined direction and orthogonal to the predetermined direction. According to this, the sheet-shaped excitation light can be generated at a position different from the optical axis of the first objective lens.

In the image acquisition apparatus according to one aspect of the present invention, the first region and the second region may be adjacent to each other and the phase values may be continuous at the boundary thereof. According to this, since the basic phase pattern is simplified, it is possible to generate sheet-shaped excitation light with a simpler configuration without using a complicated optical element or the like.

In the image acquisition apparatus according to one aspect of the present invention, the phase pattern may be a phase pattern in which a diffraction grating-like diffraction grating pattern and a basic phase pattern are superimposed. According to this, the phase of the excitation light can be made into a diffraction grating shape without providing a diffraction grating. Therefore, a sheet-shaped excitation light can be generated with a simpler configuration.

In the image acquisition device according to one aspect of the present invention, the phase pattern may be a phase pattern in which a lens-shaped lens pattern and a basic phase pattern are superimposed. According to this, the phase of the excitation light can be made into a lens shape without providing a lens element. Therefore, a sheet-shaped excitation light can be generated with a simpler configuration.

The image acquisition device according to one aspect of the present invention may further include an optical scanning unit that scans the excitation light with respect to the sample.

In the image acquisition device according to one aspect of the present invention, the photodetector may be a two-dimensional image sensor having a plurality of pixel sequences and capable of rolling readout. According to this, the S / N can be improved as compared with the case of using a two-dimensional image sensor capable of global readout.

The image acquisition method according to one aspect of the present invention is a first method in which the phase of the excitation light including the wavelength for exciting the sample is modulated for each plurality of pixels by a spatial light modulator having a plurality of pixels arranged in two dimensions. Steps, the second step of irradiating the sample with the excitation light modulated by the spatial light modulator, and the detection light emitted from the sample due to the irradiation of the excitation light are guided, and the guided detection light is used. Including the third step of imaging, in the first step, each of a plurality of pixels is generated based on a predetermined basic phase pattern, and the phase values corresponding to each of the plurality of pixels are distributed in two dimensions according to the phase pattern. The phase modulation amount of the above is controlled, and the basic phase pattern faces the first region in which the phase value continuously increases along the predetermined direction and the first region in the predetermined direction, and follows the predetermined direction. It has a second region in which the phase value continuously decreases.

In this image acquisition method, a basic phase having a first region in which the phase value continuously increases along a predetermined direction and a second region in which the phase value continuously decreases along a predetermined direction. The phase pattern is calculated based on the pattern. By modulating the excitation light with a spatial light modulator according to this phase pattern, it is possible to irradiate the sample with sheet-shaped excitation light. Therefore, it is not necessary to scan the focusing point of the Bessel beam in order to generate the sheet-shaped excitation light as in the prior art, and the optical element or the like for the scanning can be omitted. Therefore, according to this image acquisition method, it is possible to generate sheet-shaped excitation light with a simple configuration.

The spatial light modulation unit according to one aspect of the present invention is a spatial light modulation unit used in a light sheet microscope, has a plurality of pixels arranged in two dimensions, and has a plurality of pixels in the phase of input light. A spatial optical modulator that modulates each unit and outputs the modulated light, and a control unit that controls the amount of phase modulation for each of a plurality of pixels according to a phase pattern in which the phase values corresponding to each of the plurality of pixels are distributed in two dimensions. The phase pattern is a phase pattern generated based on a predetermined basic phase pattern, and the basic phase pattern includes a first region in which the phase value continuously increases along a predetermined direction and a predetermined region. It has a second region which faces the first region in the direction of the above and whose phase value continuously decreases along a predetermined direction.

In this spatial light modulation unit, a basic region having a first region in which the phase value continuously increases along a predetermined direction and a second region in which the phase value continuously decreases along a predetermined direction. The phase pattern is calculated based on the phase pattern. By modulating the excitation light with a spatial light modulator according to this phase pattern, it is possible to irradiate the sample with the excitation light in the form of a sheet. Therefore, it is not necessary to scan the focusing point of the Bessel beam in order to generate the sheet-shaped excitation light as in the prior art, and the optical element or the like for the scanning can be omitted. Therefore, according to this spatial light modulation unit, it is possible to generate sheet-shaped excitation light with a simple configuration.

According to the present invention, a sheet-shaped excitation light can be generated with a simple configuration.

It is a block diagram which shows the structure of the light sheet microscope which is one Embodiment of the image acquisition apparatus of this invention. (A) is a diagram showing a light receiving surface of the photodetector of FIG. 1, and (b) is a diagram showing rolling readout in the photodetector. It is a figure which shows the basic phase pattern used in the light sheet microscope of FIG. It is a conceptual diagram explaining how the sheet-shaped excitation light is generated. It is a figure which shows the 1st modification of the basic phase pattern. It is a conceptual diagram explaining how the sheet-shaped excitation light is generated by using the basic phase pattern of FIG. It is a figure which shows the 2nd to 4th modification of the basic phase pattern. It is a figure explaining how the diffraction grating pattern is superposed on the basic phase pattern. It is a figure explaining how the lens pattern is superposed on the basic phase pattern. It is a block diagram which shows the structure of the light sheet microscope of the 5th modification. It is a conceptual diagram explaining how the sheet-shaped excitation light is generated in the light sheet microscope of FIG.

Hereinafter, embodiments relating to the image acquisition device and the image acquisition method of the present invention will be described in detail with reference to the drawings. In the following description, the same reference numerals will be used for the same or equivalent elements, and duplicate description will be omitted.

The light sheet microscope (image acquisition device) 1 shown in FIG. 1 irradiates the sample S with the sheet-shaped excitation light L1 and images the detection light L2 emitted from the sample S with the irradiation of the excitation light L1. , A device for acquiring an image of sample S. In the light sheet microscope 1, the focused position of the excitation light L1 is scanned with respect to the sample S along the direction orthogonal to the optical axis of the excitation light L1, and an image of the sample S is acquired at each focused position. In the light sheet microscope 1, since the region where the excitation light L1 is irradiated to the sample S is narrow, deterioration of the sample S such as photofading or phototoxicity can be suppressed, and image acquisition can be speeded up. ..

The sample S to be observed is, for example, a sample of a cell, a living body, or the like containing a fluorescent substance such as a fluorescent dye or a fluorescent gene. The sample S emits detection light L2 such as fluorescence when it is irradiated with light in a predetermined wavelength range. The sample S is housed in, for example, a holder having at least transparency to the excitation light L1 and the detection light L2. This holder is held, for example, on a stage.

As shown in FIG. 1, the light sheet microscope 1 includes a light source 11, a collimator lens 12, an optical scanning unit 13, an SLM 14, a first optical system 15, a first objective lens 16, and a second objective lens 16. The objective lens 17, the filter 18, the second optical system 19, the light detector 20, and the control unit 21 are provided.

The light source 11 outputs the excitation light L1 including the wavelength that excites the sample S. The light source 11 emits, for example, coherent light or incoherent light. Examples of the coherent light source include a laser light source such as a laser diode (LD). Examples of the incoherent light source include a light emitting diode (LED), a superluminescent diode (SLD), and a lamp-based light source. As the laser light source, a light source that oscillates a continuous wave is preferable, and a light source that oscillates a pulsed light such as an ultrashort pulsed light may be used. As the light source that oscillates the pulsed light, a unit that combines a light source that outputs the pulsed light and an optical shutter or an AOM (Acousto-Optic Modulator) for pulse modulation may be used. The light source 11 may be configured to output excitation light L1 including a plurality of wavelength regions. In this case, a part of the wavelength of the excitation light L1 may be selectively transmitted by an optical filter such as an acoustic-optical variable filter (Acousto-Optic Tunable Filter).

The collimator lens 12 parallelizes the excitation light L1 output from the light source 11 and outputs the parallelized excitation light L1. The optical scanning unit 13 is an optical scanner that scans the excitation light L1 with respect to the sample S by changing the traveling direction of the excitation light L1 output from the collimator lens 12. As a result, the irradiation position of the excitation light L1 irradiated to the sample S via the first optical system 15 and the first objective lens 16 is the optical axis of the first objective lens 16 (the optical axis of the excitation light L1). It is scanned on the surface of sample S along a direction orthogonal to. The optical scanning unit 13 is, for example, a galvano mirror, a resonant scanner, a polygon mirror, a MEMS (Micro Electro Mechanical System) mirror, or an acoustic optical element such as an AOM or AOD (Acousto-Optic Deflector).

The SLM 14 is a phase modulation type spatial light modulator having a plurality of pixels arranged in two dimensions and modulating the phase of the excitation light L1 output from the light source 11 for each of the plurality of pixels. The SLM 14 modulates the excitation light L1 incident from the optical scanning unit 13 and outputs the modulated excitation light L1 toward the first optical system 15 (first step). The SLM 14 is configured, for example, as a transmissive type or a reflective type. In FIG. 1, a transmissive SLM 14 is shown. The SLM14 is, for example, a refractive index changing material type SLM (for example, LCOS (Liquid Crystal on Silicon) type SLM, LCD (LiquidCrystalDisplay)), a variable mirror type SLM (for example, a Segment Mirror type SLM, a Continuous Deformable Mirror type SLM), or An electric address type liquid crystal element or an SLM using an optical address type liquid crystal element. The SLM 14 is electrically connected to the controller 23 of the control unit 21 and constitutes a spatial optical modulation unit. The drive of the SLM 14 is controlled by the controller 23. Details of the control of the SLM 14 by the control unit 21 will be described later.

The first optical system 15 optically couples the SLM 14 and the first objective lens 16 so that the excitation light L1 output from the SLM 14 is guided to the first objective lens 16. The first optical system 15 here has a lens 15a that focuses the excitation light L1 from the SLM 14 with the pupil of the first objective lens 16 and constitutes a bilateral telecentric optical system.

The first objective lens 16 is an objective lens for illumination, and irradiates the sample S with the excitation light L1 modulated by the SLM 14 (second step). The first objective lens 16 is movable along its optical axis by a driving element such as a piezo actuator or a stepping motor. As a result, the focusing position of the excitation light L1 can be adjusted.

The second objective lens 17 is an objective lens for detection, and guides the detection light L2 emitted from the sample S to the photodetector 20 side in association with the irradiation of the excitation light L1 from the first objective lens 16. .. In this example, the second objective lens 17 is arranged so that its optical axis (optical axis of the detection light L2) and the optical axis of the first objective lens 16 intersect (cross). The second objective lens 17 can be moved along its optical axis by a driving element such as a piezo actuator or a stepping motor. As a result, the focal position of the second objective lens 17 can be adjusted.

The filter 18 is an optical filter for separating the excitation light L1 and the detection light L2 from the light guided by the second objective lens 17 and outputting the extracted detection light L2 to the photodetector 20 side. .. The filter 18 is arranged on the optical path between the second objective lens 17 and the photodetector 20. The second optical system 19 optically connects the second objective lens 17 and the photodetector 20 so that the detection light L2 output from the second objective lens 17 is guided to the photodetector 20. It is combined. The second optical system 19 has a lens 19a that forms an image of the detection light L2 from the second objective lens 17 on the light receiving surface 20a (FIG. 2) of the photodetector 20.

The photodetector 20 captures the detection light L2 imaged on the light receiving surface 20a by being guided by the second objective lens 17 (third step). The photodetector 20 is a two-dimensional image sensor that has a plurality of pixel sequences and is capable of rolling readout for each of the plurality of pixel sequences. Examples of such a photodetector 20 include a CMOS image sensor and the like. As shown in FIG. 2A, a plurality of pixel sequences R in which a plurality of pixels are arranged in a direction perpendicular to the reading direction are arranged on the light receiving surface 20a of the photodetector 20 in a plurality of reading directions. ..

In the photodetector 20, as shown in FIG. 2B, exposure and readout are controlled for each pixel string R by inputting a reset signal and a readout start signal based on the drive cycle of the drive clock. .. In the rolling read, the read start signal is sequentially input for each pixel string R with a predetermined time difference. Therefore, unlike the global read-out in which all the pixel trains are read out at the same time, the read-out for each pixel row R is sequentially performed with a predetermined time difference.

The control unit 21 is composed of a computer 22 including a processor, a memory, and the like, and a controller 23 including the processor, the memory, and the like. The computer 22 is, for example, a personal computer or a smart device, and the processor controls the operations of the optical scanning unit 13, the first objective lens 16, the second objective lens 17, the photodetector 20, the controller 23, and the like. , Perform various controls. For example, the computer 22 controls to synchronize the scanning of the excitation light L1 by the optical scanning unit 13 with the imaging of the detection light L2 by the photodetector 20. Specifically, the detection light L2 detected by the photodetector 20 also moves according to the scanning of the excitation light L1 by the optical scanning unit 13. Therefore, the computer 22 controls the photodetector 20 or the optical scanning unit 13 so that the signal is read by rolling reading in accordance with the movement of the detection light L2 in the photodetector 20.

The controller 23 is electrically connected to the computer 22 and controls the phase modulation amount for each of a plurality of pixels in the SLM 14 according to the two-dimensional phase pattern P as shown in FIG. The phase pattern P is a pattern of phase values relating to positions on a two-dimensional plane, and each position in the phase pattern P corresponds to a plurality of pixels of the SLM 14. The phase value of the phase pattern P is defined between 0 and 2π radians. In FIG. 3, the phase value in each part of the phase pattern P is represented by the color density. The upper limit of the phase value of the phase pattern P may be larger than 2π radians.

The controller 23 controls the phase modulation amount of each pixel of the SLM 14 according to the phase value of the position corresponding to the pixel in the phase pattern P. Specifically, for example, in the controller 23, a D / A conversion that converts the phase value of the phase pattern P input as digital data such as DVI (Digital Video Interface) into a drive voltage value applied to each pixel. A unit (digital / analog converter) is provided. When the phase pattern P is input from the computer 22 to the controller 23, the controller 23 converts the phase value of the phase pattern P into a drive voltage value by the D / A conversion unit, and inputs the drive voltage value to the SLM 14. The SLM 14 applies a voltage to each pixel according to the input drive voltage value. For example, the SLM 14 may have a D / A conversion unit, and the controller 23 may input digital data corresponding to the phase pattern P into the SLM 14. In this case, the D / A conversion unit of the SLM 14 converts the phase value of the phase pattern P into a drive voltage value. Further, the SLM 14 may control the voltage value applied to each pixel based on the digital signal output from the controller 23 without performing the D / A conversion.

The phase pattern P is calculated by the computer 22 of the control unit 21 based on a predetermined basic phase pattern 31. The basic phase pattern 31 is stored in advance in the memory of the computer 22, for example. By modulating the excitation light L1 with the SLM 14 according to the phase pattern P calculated based on the basic phase pattern 31, it is possible to irradiate the sheet-shaped excitation light L1 from the first objective lens 16. As will be described later, the phase pattern P may be calculated by further superimposing another pattern on the basic phase pattern 31, but the case where the basic phase pattern 31 is used as it is as the phase pattern P will be described below. To do.

As shown in FIG. 3, the basic phase pattern 31 is set within a rectangular range. The basic phase pattern 31 faces a rectangular first region 32 whose phase value continuously increases along a predetermined direction D1 and a first region 32 in the direction D1 and is continuous along the direction D1. It has a rectangular second region 33 whose phase value decreases. That is, in the first region 32 and the second region 33, the directions in which the phase values increase or decrease are opposite to each other. In addition, "the phase value continuously increases" in a certain region means that the phase value is continuous without interruption over the entire region. Further, the case where the phase value is 0 radians and the case where the phase value is 2π radians mean the same state, and even if the phase value changes across between 0 radians and 2π radians, the phase value remains. It is continuous.

In the first region 32, the phase value increases linearly along the direction D1. In the second region 33, the phase value decreases linearly along the direction D1. In both the first region 32 and the second region 33, the phase value changes by 2π radians. That is, the absolute value of the slope of the phase value (rate of increase) in the first region 32 and the absolute value of the slope of the phase value (rate of decrease) in the second region 33 are equal. In both the first region 32 and the second region 33, the phase value is constant along the direction D2 orthogonal to the direction D1. The first region 32 and the second region 33 are adjacent to each other, and the phase values are continuous at the boundary thereof. In this example, the phase value at the boundary is 0 radians. The basic phase pattern 31 is axisymmetric with respect to a straight line (center line) C that passes through the center in the direction D1 and is orthogonal to the direction D1. In this example, the boundary between the first region 32 and the second region 33 is located on the center line C.

FIG. 4 is a conceptual diagram illustrating how the sheet-shaped excitation light L1 is generated by modulating the excitation light L1 with the SLM 14 according to the basic phase pattern 31. FIG. 4A is a diagram showing an optical path of the excitation light L1 when viewed from the direction d2 corresponding to the direction D2, and FIG. 4B is a diagram showing the excitation light when viewed from the direction d1 corresponding to the direction D1. It is a figure which shows the optical path of light L1. In FIG. 4A, as an example of the optical path of the excitation light L1 incident on the first region 32, the three excitation lights A1, B1, and C1 are arranged in ascending order from the optical axis X of the first objective lens 16. It is shown. Further, as an example of the optical path of the excitation light L1 incident on the second region 33, three excitation lights A2, B2, and C2 are shown in ascending order of distance from the optical axis X.

As shown in FIG. 4A, the excitation lights A1 and A2 are imaged on the optical axis X with their phases delayed by a predetermined amount at SLM14. In the excitation lights B1 and B2, the amount of phase delay in the SLM 14 is larger than that in the excitation lights A1 and A2. Therefore, the excitation lights B1 and B2 are imaged on the optical axis X at a position farther from the first objective lens 16 than the excitation lights A1 and A2. In the excitation lights C1 and C2, the phase delay amount in the SLM 14 is smaller than that in the excitation lights A1 and A2, and the phase is almost unchanged in the SLM 14. Therefore, the excitation lights C1 and C2 are imaged on the optical axis X at a position closer to the first objective lens 16 than the excitation lights A1 and A2.

As shown in FIG. 4B, the phase of the excitation light L1 does not change with the SLM 14 when viewed from the direction d1. From the above, the sheet-shaped excitation light L1 is generated at the predetermined imaging position Y1. In this example, the sheet-shaped excitation light L1 is generated on the optical axis X so that its width direction is along the direction d2.

As described above, in the light sheet microscope 1, the first region 32 in which the phase value continuously increases along the direction D1 and the second region 33 in which the phase value continuously decreases along the direction D1. The phase pattern P is calculated based on the basic phase pattern 31 having the above. By modulating the excitation light with the SLM 14 according to this phase pattern P, it is possible to irradiate the sample S with the sheet-shaped excitation light L1 from the first objective lens 16. Therefore, it is not necessary to scan the focusing point of the Bessel beam in order to generate the sheet-shaped excitation light L1 as in the prior art, and the optical element or the like for the scanning can be omitted. Therefore, according to this light sheet microscope 1, a sheet-shaped excitation light L1 can be generated with a simple configuration. Further, unlike the conventional technique, it is not necessary to scan the focusing point of the Bessel beam in order to generate the sheet-shaped excitation light L1, so that the control can be facilitated and the time required for image acquisition can be shortened. be able to.

In the light sheet microscope 1, in the first region 32, the phase value increases linearly along the direction D1, and in the second region 33, the phase value decreases linearly along the direction D1. .. As a result, the basic phase pattern 31 is simplified, so that the sheet-shaped excitation light L1 can be generated with a simpler configuration without using a complicated optical element or the like. That is, when the phase value does not increase linearly along the direction D1 in at least one of the first region 32 and the second region 33, the first optical system for generating the sheet-shaped excitation light L1. The configuration of the optical system such as the 15 or the first objective lens 16 may be complicated. On the other hand, in the light sheet microscope 1, since the phase value increases linearly along the direction D1 in both the first region 32 and the second region 33, the sheet-shaped excitation light L1 is generated. The configuration of the optical system for this purpose can be simplified.

In the light sheet microscope 1, the basic phase pattern 31 is axisymmetric with respect to the straight line C. As a result, the sheet-shaped excitation light L1 can be generated on the optical axis X of the first objective lens 16. Therefore, the optical axis adjustment of the first objective lens 16 and the second objective lens 17 becomes easy.

In the light sheet microscope 1, the first region 32 and the second region 33 are adjacent to each other, and the phase values are continuous at the boundary thereof. As a result, the basic phase pattern 31 is simplified, so that the sheet-shaped excitation light L1 can be generated with a simpler configuration without using a complicated optical element or the like.

Since the light sheet microscope 1 includes an optical scanning unit 13 that scans the excitation light L1 with respect to the sample S, the irradiation position of the excitation light L1 emitted from the first objective lens 16 is scanned with respect to the sample S. can do.

In the light sheet microscope 1, the photodetector 20 is a two-dimensional image sensor having a plurality of pixel rows R and capable of rolling readout. As a result, the S / N can be improved as compared with the case of using a two-dimensional image sensor capable of global readout.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited to the above embodiment. For example, the basic phase pattern 31A of the first modification shown in FIG. 5 may be used. In the second region 33A of the basic phase pattern 31A, the phase value is linearly reduced by 4π radians along the direction D1. That is, the absolute value of the slope of the phase value in the first region 32 and the absolute value of the slope of the second region 33A are different. The basic phase pattern 31A is non-axisymmetric with respect to the center line C. In this example as well, the boundary between the first region 32 and the second region 33A is located on the center line C.

Even when such a basic phase pattern 31A is used, as shown in FIG. 6, the excitation light L1 is modulated by the SLM 14 according to the basic phase pattern 31A, so that the sheet-shaped excitation light L1 is generated. In this case, as shown in FIG. 6A, in the excitation light A2 incident on the second region 33A, the phase delay amount in the SLM 14 is larger than the excitation light A1 incident on the first region 32. .. Similarly, in the excitation light B2, the phase delay amount in the SLM 14 is larger than that in the excitation light B1, and in the excitation light C2, the phase delay amount in the SLM 14 is larger than that in the excitation light C1. As a result, the sheet-shaped excitation light L1 is generated at the imaging position Y2 farther from the first objective lens 16 than the imaging position Y1 in the case of the above embodiment. The sheet-shaped excitation light L1 is generated at a position different from that on the optical axis X.

As described above, even in the first modification, it is possible to irradiate the sample S with the sheet-shaped excitation light L1 from the first objective lens 16 as in the case of the above embodiment, which is a simple configuration. Can generate a sheet-shaped excitation light L1. Further, in the first modification, since the basic phase pattern 31 is non-axisymmetric with respect to the straight line C, the sheet-shaped excitation light L1 is generated at a position different from that on the optical axis X of the first objective lens 16. can do.

Further, the basic phase pattern 31B of the second modification, the basic phase pattern 31C of the third modification, or the basic phase pattern 31D of the fourth modification shown in FIG. 7 may be used. In the first region 33B of the basic phase pattern 31B, the phase value increases linearly along the direction D1 by 4π radians. That is, the absolute value of the slope of the phase value in the first region 32B and the absolute value of the slope of the phase value in the second region 33A are equal.

In the basic phase pattern 31C, the positional relationship between the first region 32 and the second region 33 in the direction D1 is opposite to that in the above embodiment. That is, in the above-mentioned basic phase pattern 31, in the first region 32, the phase value increases as the distance from the boundary (center line C) increases, and in the second region 33, the distance from the boundary decreases. Whereas the phase value decreased, in the basic phase pattern 31C, in the first region 32, the phase value increased as the distance from the boundary decreased, and in the second region 33, the distance from the boundary increased. The larger the value, the smaller the phase value.

In the basic phase pattern 31D, the positional relationship between the first region 32D and the second region 33D in the direction D1 is opposite to that in the case of the above embodiment, as in the third modification. Further, in the basic phase pattern 31D, in the first region 32D, the phase value increases linearly by 4π radians along the direction D1, and in the second region 33D, the phase value linearly increases along the direction D1. It is reduced by 4π radians. The basic phase patterns 31B to 31D are line symmetric with respect to the center line C. Even in the basic phase patterns 31B to 31D, the boundary between the first region 32 and the second region 33A is located on the center line C.

Even when these basic phase patterns 31B to 31D are used, it is possible to irradiate the sample S with the sheet-shaped excitation light L1 from the first objective lens 16 as in the case of the above embodiment. A sheet-shaped excitation light L1 can be generated with a simple configuration.

Further, as shown in FIG. 8, the phase pattern P may be calculated by superimposing the diffraction grating pattern 41 on the basic phase pattern 31. The diffraction grating pattern 41 has a diffraction grating shape in the direction D2. Further, as shown in FIG. 9, the phase pattern P may be calculated by superimposing a lens-shaped lens pattern 42 such as a Fresnel lens on the basic phase pattern 31. The phase pattern P may be calculated by superimposing the diffraction grating pattern 41 or the lens pattern 42 on the basic phase patterns 31A to 31C.

Even in these cases, it is possible to irradiate the sample S with the sheet-shaped excitation light L1 from the first objective lens 16 as in the case of the above embodiment, and the sheet-shaped excitation light L1 can be irradiated with a simple configuration. Can be generated. Further, the phase of the excitation light can be made into a diffraction grating or a lens without providing a diffraction grating or a lens element. Therefore, the sheet-shaped excitation light L1 can be generated with a simpler configuration.

Further, it may be configured like the light sheet microscope 1D of the fifth modification shown in FIG. The light sheet microscope 1D is different from the light sheet microscope 1 of the above embodiment in that it includes a first optical system 15D constituting a telescope optical system. The first optical system 15D has two lenses 15Da and 15Db that focus the excitation light L1 from the SLM 14 with the pupil of the first objective lens 16, and constitutes a telescope optical system. Examples of such a telescope optical system include a 4f optical system, a Kepler type optical system, a Galileo type optical system, and the like.

Even with such a light sheet microscope 1D, as shown in FIG. 11, the excitation light L1 is modulated by the SLM 14 according to the basic phase pattern 31, so that the sheet-shaped excitation light L1 is generated. As described above, even in the fifth modification, it is possible to irradiate the sample S with the sheet-shaped excitation light L1 from the first objective lens 16 as in the case of the above embodiment, which is a simple configuration. Can generate a sheet-shaped excitation light L1.

Further, in the basic phase pattern 31 of the above embodiment, a third region in which the phase value is constant in the direction D1 may be further provided. Such a third region is provided, for example, between the first region 32 and the second region 33. In this case, the first region 32 and the second region 33 are not adjacent to each other.

Further, in the above embodiment, the photodetector 20 may be a globally readable area image sensor. The first optical system 15 may be omitted, and the excitation light L1 output from the SLM 14 may be directly input to the first objective lens 16. The optical scanning unit 13 may be arranged so as to receive the excitation light L1 output from the SLM 14. The optical axis of the first objective lens 16 and the optical axis of the second objective lens 17 may not be orthogonal to each other, or may not intersect with each other.

Further, the control unit 21 may control the wavelength, the thickness, the condensing position, the shape, and the like of the sheet-shaped excitation light L1 to be generated by appropriately changing the phase pattern P. The control unit 21 may calculate the phase pattern P by superimposing the pattern for aberration correction on the basic phase pattern 31. Further, for example, an optical element having a slit for cutting the 0th-order light or the higher-order light of the excitation light L1 may be provided between the lens 15a and the first objective lens 16. As a result, unnecessary light (light that can become noise) can be blocked.

Further, the control unit 21 may set the number of pixel rows N to be simultaneously exposed by the photodetector 20 capable of rolling and reading out according to the phase pattern P. At this time, the control unit 21 sets the reading period T2 for rolling reading based on the set number of pixel rows N and the exposure period T1 for each pixel row. In this case, the exposure period T1 of each pixel string is set by the user or the like. Further, the control unit 21 may set the exposure period T1 of each pixel sequence based on the set number of pixel sequences N and the reading period T2 of the rolling readout. In this case, the read period T2 for rolling read is set by the user or the like. In either case, the control unit 21 may use the optical scanning unit 13 or the optical scanning unit 13 or the control unit 21 so that the scanning of the excitation light L1 by the optical scanning unit 13 and the signal readout of each pixel sequence R by the photodetector 20 capable of rolling readout are synchronized. Controls the photodetector 20.

1 ... Light sheet microscope (image acquisition device), 11 ... Light source, 13 ... Optical scanning unit, 14 ... Spatial light modulator, 16 ... First objective lens, 17 ... Second objective lens, 20 ... Photodetector, 21 ... Control unit, 22 ... Computer, 23 ... Controller, 31 ... Basic phase pattern, 32 ... First region, 33 ... Second region, 41 ... Diffraction grating pattern, 42 ... Lens pattern, C ... Straight line (center line) ), D1 ... predetermined direction, L1 ... excitation light, L2 ... detection light, P ... phase pattern, R ... pixel series, S ... sample.

Claims (10)

A light source that outputs excitation light containing a wavelength that excites the sample,
A spatial light modulator having a plurality of pixels arranged in two dimensions and modulating the phase of the excitation light output from the light source for each of the plurality of pixels.
A first objective lens that irradiates the sample with the excitation light modulated by the spatial light modulator, and
A second objective lens that guides the detection light emitted from the sample in association with the irradiation of the excitation light from the first objective lens.
A photodetector that captures the detection light guided by the second objective lens, and
A control unit that controls the phase modulation amount for each of the plurality of pixels according to a phase pattern in which the phase values corresponding to the plurality of pixels are distributed in two dimensions is provided.
The phase pattern is a phase pattern generated based on a predetermined basic phase pattern.
The basic phase pattern includes a first region in which the phase value increases continuously and linearly along a predetermined direction and the phase value is constant in a direction orthogonal to the predetermined direction, and the predetermined region. The phase value faces the first region in the direction of the above, and the phase value decreases continuously and linearly along the predetermined direction, and the phase value is constant in the direction orthogonal to the predetermined direction . An image acquisition device having two regions. The basic phase pattern, the predetermined has about the line that passes through the center and perpendicular to the predetermined direction line symmetry in the direction, the image acquisition apparatus according to claim 1. The basic phase pattern, the predetermined in the non-axisymmetrical with respect to a straight line and perpendicular to the predetermined direction passes through the center in the direction, the image acquisition apparatus according to claim 1. The image acquisition device according to claim 1 or 2 , wherein the first region and the second region are adjacent to each other and the phase values are continuous at the boundary thereof. The image acquisition device according to any one of claims 1 to 3 , wherein the phase pattern is a phase pattern in which a diffraction grating-like diffraction grating pattern and the basic phase pattern are superimposed. The image acquisition device according to any one of claims 1 to 4 , wherein the phase pattern is a phase pattern in which a lens-shaped lens pattern and the basic phase pattern are superimposed. The image acquisition device according to any one of claims 1 to 5 , further comprising an optical scanning unit that scans the excitation light with respect to the sample. The image acquisition device according to any one of claims 1 to 6 , wherein the photodetector is a two-dimensional image pickup element having a plurality of pixel sequences and capable of rolling readout. The first step of modulating the phase of the excitation light including the wavelength for exciting the sample for each of the plurality of pixels by a spatial light modulator having a plurality of pixels arranged in two dimensions.
A second step of irradiating the sample with the excitation light modulated by the spatial light modulator,
A third step of guiding the detection light emitted from the sample with the irradiation of the excitation light and imaging the guided detection light is included.
In the first step, the phase modulation amount for each of the plurality of pixels is controlled according to the phase pattern generated based on a predetermined basic phase pattern and the phase values corresponding to the plurality of pixels are distributed in two dimensions.
The basic phase pattern includes a first region in which the phase value increases continuously and linearly along a predetermined direction and the phase value is constant in a direction orthogonal to the predetermined direction, and the predetermined region. The phase value faces the first region in the direction of the above, and the phase value decreases continuously and linearly along the predetermined direction, and the phase value is constant in the direction orthogonal to the predetermined direction . An image acquisition method having two regions. A spatial light modulation unit used in light sheet microscopes.
A spatial light modulator that has a plurality of pixels arranged in two dimensions, modulates the phase of the input light for each of the plurality of pixels, and outputs the modulated light.
A control unit that controls the phase modulation amount for each of the plurality of pixels according to a phase pattern in which the phase values corresponding to the plurality of pixels are distributed in two dimensions is provided.
The phase pattern is a phase pattern generated based on a predetermined basic phase pattern.
The basic phase pattern includes a first region in which the phase value increases continuously and linearly along a predetermined direction and the phase value is constant in a direction orthogonal to the predetermined direction, and the predetermined region. The phase value faces the first region in the direction of the above, and the phase value decreases continuously and linearly along the predetermined direction, and the phase value is constant in the direction orthogonal to the predetermined direction . A spatial light modulation unit having two regions.