CN210142077U

CN210142077U - Novel selective planar illumination microscope with tiled polished section

Info

Publication number: CN210142077U
Application number: CN201920282160.8U
Authority: CN
Inventors: 殷明
Original assignee: Shanghai Biological Science Instrument (shanghai) Co Ltd
Current assignee: Fuhai bioscience instrument (Shanghai) Co.,Ltd.
Priority date: 2019-03-06
Filing date: 2019-03-06
Publication date: 2020-03-13
Anticipated expiration: 2029-03-06

Abstract

The utility model provides a novel selective planar illumination microscope of tiling slide, including laser light source system, expand beam collimation system, spatial light modulation system, first 4f system, the scanning mirror that shakes, the speculum group, left 4f system, right 4f system, left side arouses objective, right side arouses objective, detect objective, the cover glass, the detector, 3D translation platform and the control unit of arranging in the sample room in, the control unit respectively with laser light source system, spatial light modulation system, the scanning mirror that shakes, detector and 3D translation platform control connection, the control unit transmits different phase maps to spatial light modulation system, change excitation beam focal line size and position, in detector visual field interior tiling slide; change scanning galvanometer angular position, to the sample left and right sides 3D image acquisition respectively, 3D formation of image is accomplished to the concatenation method, the utility model overcomes contradiction between spatial resolution, optical chromatography ability and the visual field, improved spatial resolution and optical chromatography ability, optimize real-time imaging performance.

Description

Novel selective planar illumination microscope with tiled polished section

Technical Field

The utility model relates to an optical microscopy technical field especially relates to a novel tiling slide selectivity plane illumination microscope.

Background

Selective Planar Illumination Microscopy (SPIM) is a 3D imaging performed by confining an illuminating planar light sheet to the vicinity of the detection focal plane. SPIM microscopes require a thin, uniform thickness, and large planar size light sheet to maximize the 3D imaging capabilities of the SPIM microscope. SPIM microscopes use different types of planar illumination sheets to achieve different 3D spatial resolutions, optical chromatograms capabilities, and fields of view. However, diffraction of light does not physically produce an ideal light sheet with a thin thickness, uniformity of thickness, good confinement of excitation light, and large size. As the field of view increases to tens of microns or more, balancing the above characteristics becomes very difficult to meet the imaging needs for multi-cellular samples. Since the thickness of the planar illumination light sheet increases with the size of the planar illumination light sheet, and the confinement capability of the illumination light decreases, the spatial resolution and the optical tomography capability decrease with the increase of the field of view, and therefore, the trade-off among the spatial resolution, the optical tomography capability and the field of view size becomes an essential problem limiting the three-dimensional imaging capability of the conventional SPIM microscope. In addition, recalibration and optimization of the SPIM microscope for different biological samples often requires the use of different planar illumination sheets, which is not only inconvenient, but also prevents the SPIM microscope from being optimized for the imaging subject in real time for optimal imaging performance.

SUMMERY OF THE UTILITY MODEL

The utility model aims at providing a novel tiling slide selectivity plane illumination microscope, through the different phase diagram of spatial light modulator loading to incidenting on it excitation beam modulate and reflection output in the detector field of view interior tiling slide, the mode of tiling the slide has overcome the contradiction between SPIM microscope's spatial resolution, optical tomography ability and field of view size, can optimize real-time imaging performance simultaneously; the left side and the right side of the sample are excited and irradiated respectively by changing the angle position of the scanning galvanometer, two groups of 3D images are acquired layer by layer, the left group and the right group of 3D images are spliced into a complete 3D image, the sample light path is shortened by the left group and the right group of 3D images, the spatial resolution is improved by the left group and the right group of 3D images, and meanwhile, the microscope has the function of automatically calibrating the position of a light sheet and further improves the imaging performance.

In order to achieve the above purpose, the utility model adopts the following technical scheme:

a novel flat light sheet selective plane illumination microscope,

the device comprises an excitation light source system, a beam expanding collimation system, a spatial light modulation system, a first 4f system, a scanning galvanometer, a reflector group, a left 4f system, a left exciting objective, a right 4f system, a right exciting objective, a detection objective, a barrel mirror, a detector, a 3D translation table arranged in a sample chamber and a control unit, wherein the excitation light source system generates exciting light beams which are incident into the beam expanding collimation system, are subjected to phase modulation after being expanded by the beam expanding collimation system and then are incident onto the scanning galvanometer through the first 4f system to generate an exciting light sheet, a transmission path of the exciting light sheet is divided into two paths by the scanning galvanometer, one path of the transmitting light sheet sequentially passes through the left reflector, the left 4f system and the left exciting objective of the reflector group to be irradiated onto the left side of a sample on the 3D translation table to generate fluorescence, the other path of the fluorescence is irradiated to the right side of the sample on the 3D translation stage through a right reflector, a right 4f system and a right excitation objective lens of the reflector group in sequence to generate fluorescence, and the fluorescence generated by the sample on the 3D translation stage is collected by the detection objective lens and then focused and imaged to the detector through the cylindrical lens; wherein, left side 4f system and right side 4f system are based on the axis symmetry of speculum group, the optical axis that arouses objective and right side arouses objective is on a straight line on a left side, the optical axis perpendicular to that detects objective arouses the optical axis that objective and right side arouse objective, the control unit respectively with excitation light source system, spatial light modulation system, scanning galvanometer, detector and 3D translation platform control connection.

The width of an excitation light beam emitted by the spatial light modulation system determines the thickness and the length of an excitation light sheet in a detector view field, the control unit transmits different phase diagram information to the spatial light modulation system to perform corresponding phase modulation on the excitation light beam, the geometric size and the position of a focal line of the excitation light beam are changed, a flat polished section in the detector view field is realized, and the mode of the flat polished section overcomes the contradiction among the spatial resolution, the optical tomography capability and the view field size of the SPIM microscope and optimizes the real-time imaging performance. After the excitation light beam passes through the scanning galvanometer, a transmission light path of the excitation light beam is divided into a left path and a right path, wherein one path is that the excitation light beam passes through the left reflecting mirror, the left 4f system, the left excitation objective lens, the detection objective lens, the tube lens and the detector of the reflecting mirror group in sequence after passing through the scanning galvanometer; the other path is that the excitation light beam sequentially passes through a right reflector, a right 4f system, a right excitation objective lens, a detection objective lens, a barrel lens and a detector of the reflector group, the light transmission path realizes 3D image acquisition on the left side and the right side of the sample respectively, and 3D imaging of the sample is completed by splicing the left group of 3D images and the right group of 3D images.

Further, the laser light source system includes a plurality of lasers and dichroic beam combining filters to which laser beams generated by the plurality of lasers are focused to combine to form a single collinear excitation beam.

The excitation light beam is expanded by the beam expanding and collimating system and then enters the first half-wave plate, passes through the rotation angle of the polarization direction of the first half-wave plate and then enters the spatial light modulation system. The first half-wave plate is used for ensuring the polarization of an exciting beam line entering the spatial light modulation system.

Further, the spatial light modulation system comprises a spatial light modulator, a second half-wave plate and a polarization beam splitter, the excitation beam sequentially passes through the polarization beam splitter and the second half-wave plate to irradiate the spatial light modulator, and the excitation beam diffracted by the spatial light modulator sequentially passes through the second half-wave plate and the polarization beam splitter to be transmitted to the first 4f system. The spatial light modulation system only adopts one spatial light modulator, the light path structure is simplified, and the influence of light-induced noise is reduced.

Further, the first 4f system comprises a first lens, a second lens and an optical slit, wherein the back focal point of the first lens is coincident with the front focal point of the second lens, and the optical slit is positioned at the coincident focal point of the first lens and the second lens. The optical slit is used for blocking the unnecessary diffraction orders in the laser beams after phase modulation, and the automatic correction of optical errors is realized.

Further, the left 4f system comprises a first left lens, a first left reflector, a second left lens and a second left reflector, the reflector group is located on the front focal plane of the first left lens, the first left reflector is located at the coincidence point of the rear focal plane of the first left lens and the front focal plane of the second left lens, and the second left reflector is located at the coincidence point of the rear focal plane of the second left lens and the entrance pupil of the left excitation objective lens; the right side 4f system includes first right lens, first right speculum, second right lens and the right speculum of second, the speculum group is located the focal plane before the first right lens, first right speculum is located the back focal plane of first right lens with the coincidence point of the focal plane before the second right lens, the second right speculum be located the second right lens back focal plane with the coincidence point of right side excitation objective entrance pupil. The left and right 4f systems expand the excitation beam into parallel light, i.e., change the excitation beam path, so that the excitation beam is irradiated to the left excitation objective lens through the left 4f system, or the excitation beam is irradiated to the right excitation objective lens through the right 4f system.

Further, the modulation plane of the spatial light modulator is located at the front focal point of the first lens, the scanning galvanometer is located at the back focal point of the second lens, the spatial light modulator is conjugated with the scanning galvanometer through a first 4f system, the scanning galvanometer is conjugated to the entrance pupil of the left excitation objective lens through the left 4f system, and the scanning galvanometer is conjugated to the entrance pupil of the right excitation objective lens through the right 4f system. The conjugate optical system ensures the phase modulation accuracy of the spatial light modulator, thereby accurately controlling the position of the tiled light sheet and further ensuring the realization of higher axial resolution and better optical chromatography capability.

Further, the scanning galvanometer deflects around the central axis of the reflector group, and the deflection angle of the scanning galvanometer is theta. The incident direction of the excitation light beam is changed by changing the deflection angle of the central axis of the scanning galvanometer and the reflector group, and when the scanning galvanometer deflects an angle theta leftwards around the central axis of the reflector group, the excitation light beam scanned by the scanning galvanometer sequentially passes through a left reflector, a left 4f system, a left excitation objective, a detection objective, a barrel lens and a detector of the reflector group, so that the left 3D image acquisition of the sample is realized; when the scanning galvanometer deflects an angle theta rightwards around the central axis of the reflector group, the excitation light beam sequentially passes through a right reflector, a right 4f system, a right excitation objective, a detection objective, a barrel lens and a detector of the reflector group, and the right 3D image acquisition of the sample is realized.

Further, the 3D translation stage drives the sample along the X, Y or Z axis, enabling 3D imaging. The 3D translation stage moves in the X or Y direction so that the samples enter the detector field of view in the desired order, and the 3D translation stage moves in the Z direction so that the samples within the field of view undergo 3D image acquisition.

Further, the control unit controls the spatial light modulator to automatically calibrate the position of an excitation light sheet generated after the excitation light beam passes through the scanning galvanometer. The center of the diffraction pattern of the spatial light modulator is ensured to be positioned on the optical axis, and +1 and-1 order diffraction spots of the excitation light beam are ensured to pass through the optical slit, so that the accuracy of the optical sheet tiling position is ensured.

The utility model provides a novel SPIM microscope of tiling slide, its beneficial effect as follows:

1. the control unit transmits different phase diagrams to the spatial light modulation system to modulate the excitation light beam, changes the geometric dimension and the position of a focal line forming the excitation light beam, further realizes the tiled light sheet in a detector field of view, and simultaneously, the tiled light sheet can be adjusted in real time, so the mode of tiling the light sheet overcomes the contradiction among the spatial resolution, the optical tomography capability and the field of view size of the SPIM microscope, improves the axial resolution and the optical tomography capability, and simultaneously optimizes the real-time imaging performance.

2. The transmission path of the excitation light beam is divided into two paths by the scanning galvanometer, and when the scanning galvanometer deflects an angle theta leftwards around the central axis of the reflector group, the excitation light beam passes through the scanning galvanometer and then sequentially passes through a left reflector, a left 4f system, a left excitation objective, a detection objective, a cylindrical lens and a detector of the reflector group, so that 3D image acquisition is carried out on the left side of the sample; when the scanning galvanometer deflects an angle theta rightwards around the central axis of the reflector group, the excitation light beam is scanned by the scanning galvanometer and then sequentially passes through a right reflector, a right 4f system, a right excitation objective lens, a detection objective lens, a barrel lens and a detector of the reflector group, 3D image acquisition is carried out on the right side of the sample, the incidence direction of the excitation light beam is determined by the offset angle of the scanning galvanometer and the central axis of the reflector group, namely the transmission path of the excitation light beam is determined, two groups of left and right 3D images of the sample are obtained through the transmission path, and 3D imaging of the complete sample is realized through a splicing mode.

3. The spatial light modulator, the first 4f system, the scanning galvanometer, the left 4f system and the left excitation objective form a conjugate optical system; the spatial light modulator, the first 4f system, the scanning galvanometer, the right 4f system and the right excitation objective form a conjugate optical system, and the conjugate optical system ensures the phase modulation accuracy of the spatial light modulator, so that the position of a tiled light sheet is accurately controlled, and higher axial resolution and optical chromatography performance are further ensured.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the technical solutions in the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

Fig. 1 is a schematic structural view of a novel selective planar illumination microscope with tiled polished sections according to an embodiment of the present invention;

fig. 2 is a comparison diagram of a tiled polished section and a conventional polished section provided in an embodiment of the present invention;

fig. 3 is a schematic view of a deflection angle of a scanning galvanometer around a central axis of a mirror group according to an embodiment of the present invention;

fig. 4 is a schematic diagram of imaging of a sample rat brain in an embodiment of the present invention;

reference numerals: 1-excitation light source system; 101-a first laser; 102-a second laser; 103-dichroic beam combining filter; 104-a first mirror; 2-a beam expanding collimation system; 201-a third lens; 202-a fourth lens; 3-a first half wave plate; 4-a spatial light modulator system; 401-a spatial light modulator; 402-a second half-wave plate; 403-a polarizing beam splitter; 5-first 4f system; 501-a first lens; 502-a second lens; 503-optical slit; 6-scanning galvanometer; 7-a reflector group; 701-a left reflector; 702-a right mirror; 8-left 4f system; 801-first left lens; 802-a first left mirror; 803-second left lens; 804 — a second left mirror; 9-left excitation objective lens; 10-right 4f system; 1001-first right lens; 1002-a first right mirror; 1003-second right lens; 1004 — a second right mirror; 11-right excitation objective lens; 12-a detection objective; 13-a cylindrical mirror; 14-a detector; 15-3D translation stage; 16-a control unit; 17-a second mirror; a-tiling a polished section; b-conventional light sheet.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the present invention belongs.

In the description of the present application, it is to be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience of description and simplicity of description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting.

Furthermore, the terms "first", "second", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. In the description of the present invention, "a plurality" means two or more unless specifically limited otherwise.

In this application, unless expressly stated or limited otherwise, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can include, for example, fixed connections, removable connections, or integral parts; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meaning of the above terms in the present invention can be understood according to specific situations by those skilled in the art.

The utility model provides a tiling slide selectivity plane illumination microscope, realized in the detector focal plane along slide length direction fast tiling small and thin laser piece in order to enlarge the field of view and do not influence spatial resolution and optical chromatography ability, the tiling slide method can be adjusted in real time simultaneously, makes SPIM microscope in the utility model realize the space resolution who flexibly adjusts the formation of image; the angle position of the scanning galvanometer determines the incident direction of the excitation light beam, namely determines the transmission path of the excitation light beam, so that the left side and the right side of the sample are respectively subjected to 3D image acquisition, a left group of 3D images and a right group of 3D images are obtained, the left group of 3D images and the right group of 3D images are finally spliced, the 3D imaging of the whole sample is completed, the light path of the sample is shortened, the infection of the sample on the imaging quality is reduced, and the spatial resolution is. Compare with traditional SPIM microscope, the utility model discloses a SPIM microscope has higher spatial resolution, and better optical chromatography ability or both compromise, have improved the 3D imaging ability of SPIM microscope in the aspect of solving the complex structure to realize automatic calibration, optimized real-time imaging performance, more be applicable to the not unidimensional multicellular sample of formation of image.

As shown in fig. 1, a novel tiled polished section selective planar illumination microscope provided by the embodiment of the present invention includes an excitation light source system 1, a beam expanding and collimating system 2, a spatial light modulation system 4, a first 4f system 5, a scanning galvanometer 6, a mirror group 7, a left 4f system 8, a left excitation objective 9, a left excitation objective, a left polarization beam splitter, a first polarization beam splitter, a second polarization,

The system comprises a right 4f system 10, a right excitation objective lens 11, a detection objective lens 12, a cylindrical lens 13, a detector 14, a 3D translation table 15 and a control unit 16, wherein the excitation light source system 1 generates excitation light beams which are incident into the beam expanding and collimating system 2, are expanded by the beam expanding and collimating system 2 and then are incident into the spatial light modulation system 4 for phase modulation, and then are incident onto the scanning galvanometer 6 through the first 4f system 5 to generate an excitation light sheet; the transmission path of the excitation light sheet is divided into two paths by the scanning galvanometer 6, wherein one path of the transmission path sequentially passes through a left reflector 701 of a reflector group 7, a left 4f system 8 and a left excitation objective lens 9 to irradiate the left side of the sample on the 3D translation stage 15 to generate fluorescence; the other path of the fluorescence is irradiated to the right side of the sample on the 3D translation stage 15 through a right reflector 702, a right 4f system 10 and a right excitation objective lens 11 of the reflector group 7 in sequence to generate fluorescence, and the fluorescence generated by the sample on the 3D translation stage 15 is collected by the detection objective lens 12 and then focused and imaged to the detector 14 through the tube lens 13; the left 4f system 8 and the right 4f system 10 are symmetrical based on an axial line of the reflecting mirror group 7, optical axes of the left excitation objective lens 9 and the right excitation objective lens 11 are on the same straight line, an optical axis of the detection objective lens 11 is perpendicular to the optical axes of the left excitation objective lens 9 and the right excitation objective lens 11, and the control unit 16 is respectively in control connection with the excitation light source system 1, the spatial light modulation system 4, the scanning galvanometer 6, the detector 14 and the 3D translation stage 15.

Further, the laser light source system 1 includes a plurality of lasers and a dichroic beam combining filter 103, and laser beams generated by the plurality of lasers are converged to the dichroic beam combining filter 103 and combined to form a single collinear excitation beam.

Further, the laser device further comprises a first half-wave plate 3, the first half-wave plate 3 is located between the beam expanding and collimating system 2 and the spatial light modulation system 4, the excitation light beam is expanded by the beam expanding and collimating system 2 and then enters the first half-wave plate 3, passes through a polarization direction rotation angle of the first half-wave plate 3, and then enters the spatial light modulation system 4.

Further, the spatial light modulation system 4 includes a spatial light modulator 401, a second half-wave plate 402, and a polarization beam splitter 403, the excitation beam sequentially passes through the polarization beam splitter 403 and the second half-wave plate 402 and is irradiated to the spatial light modulator 401, and the excitation beam diffracted by the spatial light modulator 401 sequentially passes through the second half-wave plate 402 and the polarization beam splitter 403 and is transmitted to the first 4f system 5.

Further, the first 4f system 5 includes a first lens 501, a second lens 502 and an optical slit 503, wherein a back focal point of the first lens 501 is coincident with a front focal point of the second lens 502, and the optical slit 503 is located at a focal point where the first lens 501 and the second lens 502 are coincident.

Further, the left 4f system 8 includes a first left lens 801, a first left mirror 802, a second left lens 803, and a second left mirror 804, the mirror group 7 is located at the front focal plane of the first left lens 801, the first left mirror 802 is located at the coincidence point of the rear focal plane of the first left lens 801 and the front focal plane of the second left lens 803, and the second left mirror 804 is located at the coincidence point of the rear focal plane of the second left lens 803 and the entrance pupil of the left excitation objective lens 9; the right 4f system 10 includes a first right lens 1001, a first right reflector 1002, a second right lens 1003 and a second right reflector 1004, the reflector group 7 is located on a front focal plane of the first right lens 1001, the first right reflector 1002 is located at a coincidence point of a rear focal plane of the first right lens 1001 and a front focal plane of the second right lens 1003, and the second right reflector 1004 is located at a coincidence point of a rear focal plane of the second right lens 1003 and an entrance pupil of the right excitation objective lens 11.

Further, the modulation plane of the spatial light modulator 401 is located at the front focal point of the first lens 501, the scanning galvanometer 6 is located at the rear focal point of the second lens 502, the spatial light modulator 401 is conjugated with the scanning galvanometer 6 through the first 4f system 5, the scanning galvanometer 6 is conjugated to the entrance pupil of the left excitation objective lens 9 through the left 4f system 8, and the scanning galvanometer 7 is conjugated to the entrance pupil of the right excitation objective lens 11 through the right 4f system 10.

Further, the scanning galvanometer 6 deflects around a central axis of the mirror group 7, and a deflection angle of the scanning galvanometer 6 is θ.

Further, the 3D translation stage 15 drives the sample along X, Y or the Z axis, enabling 3D imaging.

Further, the control unit 16 controls the spatial light modulator 401 to automatically calibrate the position of the excitation light sheet generated after the excitation light beam passes through the scanning galvanometer.

The following will be based on foretell the utility model discloses selective planar illumination microscope's of tiling slide introduction, transmission path and the imaging process of light path, as follows:

the control unit 16 may be a computer or a control module, and the control technique of the control unit 16 is realized by conventional means and available to those skilled in the art. Referring to fig. 1, the control unit 16 is a computer that controls the operations of the laser light source system 1, the spatial light modulation system 4, the scanning galvanometer 6, the detector 14, and the 3D translation stage 15.

In an embodiment, the excitation light source system 1 includes a first laser 101, a second laser 102, and a dichroic beam combining filter 103, and a first reflector 104 is used to reflect laser light generated by the first laser 101 to a dichroic beam combining filter 103 and a laser beam generated by the second laser to combine into a single collinear excitation beam, and the excitation beam is reflected to the beam expanding and collimating system 2 by a second reflector 17, where the beam expanding and collimating system 2 includes two lenses, which are a third lens 201 and a fourth lens 202 respectively, the excitation beam sequentially passes through the third lens 201 and the fourth lens 202 to expand to a preset size, and the expanded excitation beam passes through a rotation angle of a polarization direction of the first half-wave plate 3 and then enters the spatial light modulation system 4.

The spatial light modulation system comprises a spatial light modulator 401, a second half-wave plate 402 and a polarization beam splitter 403, an excitation beam polarized by the first half-wave plate sequentially passes through the polarization beam splitter 403 and the second half-wave plate 402 to irradiate the spatial light modulator 401, a computer transmits different phase diagram information to the spatial light modulator to perform phase modulation on the incident excitation beam, the excitation beam after phase modulation sequentially passes through the second half-wave plate 402 and the polarization beam splitter 403 to emit out of the spatial light modulation system 4 to emit into a first 4f system 5, the excitation beam passing through the first 4f system 5 emits into a scanning galvanometer 6, and the scanning galvanometer 6 scans the excitation beam to generate the excitation beam. Phase map information is loaded through the spatial light modulator 401, and the geometric size and position of the excitation beam are changed, so that the light sheet is tiled in the field of view of the detector 14 along the length direction of the light sheet, and fig. 2 is a comparison diagram of the tiled light sheet and a traditional light sheet.

The first 4f system 5 includes a first lens 501, a second lens 502 and an optical slit 503, the phase-modulated excitation beam sequentially passes through the first lens 501, the optical slit 503 and the second lens 502, and the optical slit 503 is used to block the unwanted diffraction orders of the excitation beam modulated by the spatial light modulator 403 and can automatically correct the optical errors.

The spatial light modulator 401, the second half-wave plate 402, the polarization beam splitter 403, the first lens 501, the optical slit 503, the second lens 502, and the scanning galvanometer 6 are on one optical axis.

As shown in fig. 1 and fig. 3, the computer further controls the deflection angle between the scanning galvanometer 6 and the central axis of the mirror group 7, changes the incident direction of the excitation light beam, when the scanning galvanometer 6 deflects to the right by θ around the central axis of the mirror group 7, the excitation light beam after being correspondingly phase-modulated by the spatial light modulator 401 is scanned by the scanning galvanometer 6 and reflected to the right mirror 702 of the mirror group 7, and is irradiated to a certain area on the right side of the sample on the 3D translation stage 15 through the right 4f system 10 and the right excitation objective lens 11 in sequence, the generated fluorescence is collected by the detection objective lens 12, is focused and imaged to the detector 14 through the barrel lens 13, and simultaneously controls the 3D translation stage 15 to move along the Z direction through the control unit 16, so as to realize the 3D image collection of a certain area on the right side of the sample, after the image collection of the area is completed, the control unit 16 controls the 3D translation stage 15, enabling the right side of the sample to enter the field of view of the detector 14 in sequence according to the expected sample moving sequence, and repeating the 3D image acquisition step to enable the sample in each field of view to realize 3D image acquisition;

after the right side of the sample finishes 3D image acquisition, the control unit 16 controls the scanning galvanometer 6 to deflect an angle θ leftwards around the central axis of the reflector group 7, a corresponding phase diagram is loaded through the spatial light modulator 401 to modulate an excitation light beam, the modulated excitation light beam is scanned by the scanning galvanometer 6 and reflected to the left reflector 701 of the reflector group 7, and then is irradiated to a certain expected area on the left side of the sample on the 3D translation stage 15 through the left 4f system 8 and the left excitation objective lens 9 in sequence, the generated fluorescence is collected by the detection objective lens 12 and focused and imaged to the detector 14 through the barrel lens 13, and similarly, the above steps are repeated, the control unit 16 controls the 3D translation stage 15 to move along the X, Y or Z direction to finish the left side 3D image acquisition of the sample, so as to obtain two groups of left and right 3D images of the sample, and finally, the 3D imaging of the whole sample is.

A schematic representation of the imaging of a murine brain sample as described in figure 4, with a field of view of about 1 x 1mm, 3D images of the entire sample were obtained using a stacked array of 3D images adjacent to each other. When the scanning galvanometer 6 deflects by an angle theta to the right around the central axis of the reflector group 7, the control unit 16 transmits a corresponding phase diagram to the spatial light modulator 401, the excitation light beam after phase modulation is scanned by the scanning galvanometer 6 and then passes through the right reflector 702, the right 4f system 10 and the right excitation objective lens 11 of the reflector group 7 in sequence to be excited and irradiated to the ROI1 area of the sample, a light sheet is spread in the field of view of the detector 14, the fluorescence generated in the ROI1 area of the sample is collected by the detection objective lens 12 and then focused and imaged to the detector 14 through the tube lens 13, and meanwhile, the control unit 16 controls the 3D translation platform 15 to move along the Z direction, so that the 3D image collection of the ROI1 area is completed; then the control unit controls the 3D translation stage 15 to move along the X or Y direction, so that the ROI2 region enters the field of view of the detector, the 3D image acquisition step of the ROI1 region is repeated, the 3D image acquisition of the ROI2 region is further completed, then the 3D translation stage is controlled to move along the X or Y direction, so that the samples enter the field of view of the detector 14 in sequence according to the expected sequence, namely the ROI1-ROI8 sequence, and the steps are repeated, so that the 3D image acquisition is carried out on the expected region of the samples in each field of view; after the 3D image acquisition of the right side of the sample rat brain is completed, the control unit 16 controls the scanning galvanometer 6 to deflect the angle theta leftwards around the central axis of the reflector group 7, the step of 3D image acquisition of the right side of the sample rat brain is repeated, the left side of the sample rat brain sequentially enters the fields of the detector 14 according to the expected sequence, namely the sequence from ROI9 to ROI16, the 3D image acquisition in each field is completed until the 3D image acquisition of the left side of the sample rat brain is completed, and finally, the left group of 3D images and the right group of 3D images are adjacently spliced to complete the 3D imaging of the whole rat brain. During imaging of the sample, the control unit 16 delivers the selected phase map to the spatial light modulation system 4 also loaded in the desired sequence and synchronized with the detector 14.

The computer controls the excitation light source system 1, the spatial light modulation system 4, the scanning galvanometer 6, the detector 12 and the 3D translation stage 15 to work cooperatively, and in the sample imaging process, the excitation scanning process, the sample exposure area and the image of the sample on the focal plane of the detection objective lens 12 are kept synchronous with the detector 14, so that the detection objective lens 12 can clearly limit the area. In this embodiment, an included angle between the scanning galvanometer 6 and a central axis of the mirror group 7 is θ, which may be 3 °, and the detector 14 may employ an SCMOS detection camera.

The control unit further controls the spatial light modulator 401 to achieve automatic calibration of the position of the light-emitting sheet, so that the center of a diffraction pattern of the spatial light modulator 401 is located on an optical axis, and +1 and-1 order diffraction spots of the excitation light beam can pass through the optical slit, and the accuracy of the tiling position of the light sheet is further guaranteed.

The width of the excitation beam diffracted by the spatial light modulator 403 determines the thickness and the length of the excitation sheet generated after scanning by the scanning galvanometer 6, and the spatial light modulator 403 loads different phase diagram information to modulate the excitation beam, changes the geometric size and the position of the focal line of the excitation beam, realizes the light sheet tiling in the length direction (y direction) of the excitation sheet, and controls the position of the light sheet tiling. The mode of tiling the light sheets overcomes the contradiction among the spatial resolution, the optical tomography capability and the field size of the SPIM microscope, and simultaneously improves the axial resolution and the optical tomography capability.

After the excitation light beam passes through the scanning galvanometer 6, the transmission path of the excitation light beam is divided into a left path and a right path, wherein one path is that the excitation light beam passes through the scanning galvanometer 6 and then sequentially passes through a left reflector 701, a left 4f system 8, a left excitation objective 9, a detection objective 12, a cylindrical lens 13 and a detector 14 of a reflector group 7; the other path is that the excitation light beam passes through a right reflector 702, a right 4f system 10, a right excitation objective lens 11, a detection objective lens 12, a tube lens 13 and a detector 14 of the reflector group 7 in sequence. The angle position of the scanning galvanometer 6 determines the incident direction of the excitation light beam, namely determines the transmission path of the excitation light beam, the two transmission paths can obtain two groups of left and right 3D images of the sample, and finally the 3D imaging of the whole sample is completed by a splicing method. The different angles of the scanning galvanometer also determine the focal line array of the sample in the horizontal direction, namely the width of the excited light sheet is controlled.

The spatial light modulator 401, the first 4f system 5, the scanning galvanometer 6, the left 4f system 8 and the left excitation objective 9 form a conjugate optical system; the spatial light modulator 401, the first 4f system 5, the scanning galvanometer 6, the right 4f system 10 and the right excitation objective lens 11 form a conjugate optical system, and the conjugate optical system ensures the phase modulation accuracy of the spatial light modulator 401, so that the position of a tiled light sheet is accurately controlled, and higher axial resolution and optical chromatography performance are further ensured.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention.

Claims

1. A novel tiled light sheet selective planar illumination microscope is characterized by comprising an excitation light source system, a beam expanding collimation system, a spatial light modulation system, a first 4f system, a scanning vibrating mirror, a reflector group, a left 4f system, a left exciting objective lens, a right 4f system, a right exciting objective lens, a detection objective lens, a cylindrical lens, a detector, a 3D translation table arranged in a sample chamber and a control unit, wherein the excitation light source system generates exciting light beams which are incident into the beam expanding collimation system, the exciting light beams are subjected to phase modulation after being expanded by the beam expanding collimation system and then incident onto the scanning vibrating mirror through the first 4f system to generate an exciting light sheet, a transmission path of the exciting light sheet is divided into two paths by the scanning vibrating mirror, one path of the two paths is irradiated onto the left side of a sample on the 3D translation table through the left reflector, the left 4f system and the left exciting objective lens of the reflector group in sequence to generate fluorescence, the other path of the fluorescence is irradiated to the right side of the sample on the 3D translation stage through a right reflector, a right 4f system and a right excitation objective lens of the reflector group in sequence to generate fluorescence, and the fluorescence generated by the sample on the 3D translation stage is collected by the detection objective lens and then focused and imaged to the detector through the cylindrical lens; wherein, left side 4f system and right side 4f system are based on the axis symmetry of speculum group, the optical axis that arouses objective and right side arouses objective is on a straight line on a left side, the optical axis perpendicular to that detects objective arouses the optical axis that objective and right side arouse objective, the control unit respectively with excitation light source system, spatial light modulation system, scanning galvanometer, detector and 3D translation platform control connection.

2. The novel tiled light slice selective flat illumination microscope according to claim 1, wherein the excitation light source system comprises a plurality of lasers and dichroic beam combining filters, wherein the laser beams generated by the plurality of lasers converge to the dichroic beam combining filters and combine to form a single collinear excitation beam.

3. The novel tiled light sheet selective planar illumination microscope of claim 1, further comprising a first half-wave plate, wherein the first half-wave plate is located between the beam expanding and collimating system and the spatial light modulation system, and the excitation light beam is expanded by the beam expanding and collimating system, then enters the first half-wave plate, passes through a polarization direction rotation angle of the first half-wave plate, and then enters the spatial light modulation system.

4. The novel tiled light sheet selective planar illumination microscope according to claim 1, wherein the spatial light modulation system comprises a spatial light modulator, a second half-wave plate and a polarization beam splitter, the excitation beam sequentially passes through the polarization beam splitter and the second half-wave plate to illuminate the spatial light modulator, and the excitation beam diffracted by the spatial light modulator sequentially passes through the second half-wave plate and the polarization beam splitter to be transmitted into the first 4f system.

5. The novel tiled light sheet selective flat illumination microscope of claim 4, wherein the first 4f system comprises a first lens, a second lens, and an optical slit, wherein the first lens back focal point and the second lens front focal point coincide, and wherein the optical slit is located at the focal point where the first lens and the second lens coincide.

6. The novel tiled light sheet selective planar illumination microscope of claim 1, wherein the left 4f system comprises a first left lens, a first left mirror, a second left lens and a second left mirror, the mirror group is located at the front focal plane of the first left lens, the first left mirror is located at the point where the rear focal plane of the first left lens coincides with the front focal plane of the second left lens, and the second left mirror is located at the point where the rear focal plane of the second left lens coincides with the entrance pupil of the left excitation objective lens; the right side 4f system includes first right lens, first right speculum, second right lens and the right speculum of second, the speculum group is located the focal plane before the first right lens, first right speculum is located the back focal plane of first right lens with the coincidence point of the focal plane before the second right lens, the second right speculum be located the second right lens back focal plane with the coincidence point of right side excitation objective entrance pupil.

7. The novel tiled light sheet selective flat illumination microscope of claim 5, wherein the modulation plane of the spatial light modulator is located at the front focal point of the first lens, the scanning galvanometer is located at the back focal point of the second lens, the spatial light modulator is conjugated with the scanning galvanometer through a first 4f system, the scanning galvanometer is conjugated to the entrance pupil of the left excitation objective through the left 4f system, and the scanning galvanometer is conjugated to the entrance pupil of the right excitation objective through the right 4f system.

8. The novel tiled light sheet selective planar illumination microscope of claim 1, wherein the scanning galvanometer is deflected about a central axis of the mirror group, and the deflection angle of the scanning galvanometer is θ.

9. The novel tiled light sheet selective planar illumination microscope of claim 1, wherein the 3D translation stage drives the sample in X, Y or Z-axis direction for 3D imaging.

10. The novel tiled light sheet selective planar illumination microscope of claim 4, wherein the control unit further controls the spatial light modulator to automatically calibrate the position of the excitation light sheet generated by the excitation light beam after passing through the scanning galvanometer.