CN113552709A - Microprism-based reflective axial light sheet fluorescence microscopic imaging device and method - Google Patents
Microprism-based reflective axial light sheet fluorescence microscopic imaging device and method Download PDFInfo
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a microprism-based reflective axial light sheet fluorescence microscopic imaging device and method. The invention utilizes the 45-degree microprism to simplify a light sheet system with a complex inclined objective lens into a transmission type microscopic imaging system, has simple light path adjustment and can be combined with a plurality of imaging modes; the sample is only needed to be injected into the microtube, or the biological samples such as cells and the like can grow in the microtube, and the sample does not need to be subjected to complex treatment; the complete axial plane can be excited once, the observation of an axial special distribution sample can be quickly realized, and the device can realize the use of an objective lens with a large numerical aperture, so that the limitation that the resolution cannot be further improved in the traditional light sheet fluorescence microscopic imaging is improved; and the light path design is simple and easy, and the subsequent industrialized development is convenient to realize.
Description
Technical Field
The invention relates to a light sheet fluorescence microscopic imaging technology, in particular to a reflective axial light sheet fluorescence microscopic imaging device based on a microprism and an imaging method thereof.
Background
The optical microscopic imaging technology has important application in living biological research by virtue of the characteristics of non-contact and low damage. The fluorescent dye or genetic engineering can be used for specifically marking various molecules and organelles in the cell, the fluorescent microscopic imaging technology can reduce the fine structure of the fluorescent mark in the cell, and the single molecule or the organelle is tracked and detected, thereby realizing the exploration and the regulation of the life essence. However, the images taken by the conventional fluorescence microscope cannot shield the fluorescence signals emitted by the afocal fluorophores, so that very high background noise exists. If the whole sample is imaged in three dimensions, the fluorophores have to be excited multiple times, resulting in severe photobleaching and phototoxicity. The fluorescence microscopic imaging technology of selectively exciting one plane of light sheet at a time can well solve the problem.
Compared with the traditional fluorescence microscope, the light sheet fluorescence microscopy adopts a selective sheet excitation mode to carry out one-time plane excitation on the sample, and the rest areas outside the plane are not excited, so that ineffective exposure is obviously reduced, and photobleaching and photodamage are effectively reduced. Meanwhile, the excitation plane is simultaneously positioned on the focal plane of the detection objective lens, and the detection objective lens and the excitation objective lens are orthogonally arranged, so that compared with the point scanning imaging mode of the traditional fluorescence microscope, the wide-field imaging mode of the light sheet fluorescence microscopy greatly improves the imaging speed of the system.
The light sheet fluorescence microscopy technology is rapidly developed since being proposed, however, due to the characteristic that the excitation light sheet is located in the focal plane of the detection objective lens, two inclined objective lenses are mostly adopted to be orthogonally arranged to respectively carry out excitation of a sample and collection of fluorescence information. The objective lens is usually a high-numerical-aperture objective lens, and the working distance is limited, so that the optical path adjustment is very complicated. And the complexity of the optical path can reduce the applicability of simultaneous imaging of the optical path and other modes, and further development of the light sheet microscopic imaging technology is limited. The single objective lens imaging light sheet imaging technology is proposed, but the technology has low light utilization rate and complicated light path adjustment.
Disclosure of Invention
In order to solve the limitation problem of the optical sheet fluorescence microscopic imaging, the invention provides a reflective axial optical sheet fluorescence microscopic imaging device based on a microprism and an imaging method thereof.
The invention aims to provide a reflective axial light sheet fluorescence microscopic imaging device based on a microprism.
The invention relates to a microprism-based reflective axial light sheet fluorescence microscopic imaging device, which comprises: the device comprises a laser light source, a Gaussian light beam expanding and collimating device, a spatial light field regulating and controlling device, a focusing lens, a digital scanning device, a 4f relay system, an excitation objective lens, a receiving objective lens, a translation table, a first tube lens, a first camera, a sample frame, a piezoelectric scanning system, a white light illuminating system and a control device; wherein, the sample rack comprises a flat plate, a light through hole, a microprism fixing device, a micropipe and a 45-degree microprism, the middle of the flat plate is provided with the light through hole penetrating through the upper surface and the lower surface of the flat plate, the upper surface of the flat plate and the upper part of the light through hole are provided with the microprism fixing device, two sides of the microprism fixing device are respectively provided with the micropipe fixing device, the bottom of the light through hole is an excitation objective lens which is vertically arranged, the micropipe is horizontally arranged on the upper surface of the flat plate through the micropipe fixing device and is positioned at the top of the light through hole, the 45-degree microprism is arranged on the upper surface of the flat plate through the microprism fixing device, the bevel edge surface of the 45-degree microprism is a reflecting surface, the first right-angle edge surface of the 45-degree microprism is positioned on an x-y plane and is parallel to the upper surface of the flat plate and is parallel to the lower surface of the micropipe, the second right-angle surface of the flat plate, the cross section of the microtube is rectangular or circular, the section of the micro-tube is rectangular, the bottom edge of the first right-angle side surface of the 45-degree micro-prism is attached to the bottom edge of one side wall of the micro-tube and is tightly close to the micro-tube, or the section of the micro-tube is circular, and the bottom edge of the first right-angle side surface of the 45-degree micro-prism and the outermost tangent plane of the side wall of the micro-tube are on the same plane; a vertically arranged receiving objective lens is arranged above the reflecting surface of the 45-degree micro prism, and a sample is positioned in the micro tube; the sample is positioned at the front focal plane of the exciting objective lens, and the sample frame is arranged on the piezoelectric scanning system; the receiving objective lens is placed on the translation table, and the piezoelectric scanning system, the digital scanning device and the first camera are connected to the control device; the laser light source emits a linear polarization Gaussian beam; after passing through the Gaussian beam expanding and collimating device, the linearly polarized Gaussian beam enters the spatial light field regulating and controlling device; the spatial light field regulating and controlling device generates a non-diffraction light beam, and performs phase difference correction on the generated non-diffraction light beam and changes the spatial distribution characteristic of the non-diffraction light beam; the diffraction-free light beams are focused to the digital scanning device through the focusing lens, the spectrum distribution of the diffraction-free light beams is generated at the digital scanning device due to the Fourier transform effect of the focusing lens, uniform light sheets are generated by scanning the diffraction-free light beams, then the spectrum distribution on the digital scanning device is relayed to a back focal plane of the vertically-arranged excitation objective lens by a 4f relay system to be used as excitation light, the excitation light enters an entrance pupil of the excitation objective lens, and the optical axis is along the z direction; the focusing lens, the 4f relay system and the exciting objective lens meet the confocal condition to form a confocal system, after the diffraction-free light beam is transmitted in a confocal way through the focusing lens and the 4f relay system, the focusing light spot at the position of the focusing plane of the exciting objective lens is axial line light, the axial line light is changed into axial plane light by a light beam scanning system to form a digital exciting light sheet, the digital exciting light sheet is focused by the vertically-arranged exciting objective lens, the square distribution of the back focal plane and the front focal plane of the exciting objective lens is in a Fourier transform conjugate relation, the frequency spectrum distribution at the back focal plane of the exciting objective lens is under the Fourier transform action of the exciting objective lens, generating a y-z plane light sheet without diffraction beams on a front focal plane of the excitation objective lens to form a virtual digital excitation light sheet, wherein the digital excitation light sheet is vertical to the surface of the sample along the axial direction and performs sheet excitation on the sample positioned in the microtube to generate fluorescence; the fluorescence is incident to the reflecting surface of the 45-degree microprism and then reflected into an x-y plane, and is directly collected by a vertically placed receiving objective lens; the image is converged and imaged onto a first camera through a first tube lens; a first camera directly receives a two-dimensional image of a sample; meanwhile, the white light illumination system emits white light to irradiate the surface of the sample, the information at the sample is observed, and the sample in the microtube is determined to be positioned on the front focal plane of the excitation objective lens; the control device controls the digital scanning device and the first camera to be synchronous; the piezoelectric scanning system performs one-dimensional moving scanning on the sample along the direction vertical to the axial direction and the direction of the digital excitation light sheet to obtain the whole three-dimensional fluorescence distribution, so that the three-dimensional distribution of the sample is obtained.
The Gaussian beam expanding and collimating device comprises two lenses with different focal lengths, wherein the two lenses with different focal lengths modulate the collimating state and the spot diameter of a linearly polarized Gaussian beam emitted by the laser, so that collimated light is emitted, and the diameter of the spot is matched with the liquid crystal panel of the spatial light modulator.
The spatial light field regulation and control device comprises a half wave plate, a 96-degree prism and a spatial light modulator; the half wave plate modulates the polarization state of the linear polarization Gaussian beam after beam expansion and collimation to enable the linear polarization Gaussian beam to be matched with the polarization direction of a silicon-based liquid crystal of the spatial light modulator; the 96-degree prism folds a light path, linearly polarized light with a horizontal polarization state is reflected by the 96-degree reflecting prism and then is projected onto a silicon-based liquid crystal panel of the spatial light modulator, different phase holograms are loaded on the spatial light modulator to generate a non-diffraction light beam, the non-diffraction light beam is a Bessel (Bessel) light beam or an Airy (Airy) light beam, a modulated light field is reflected into the light path by the 96-degree reflecting prism again, and an included angle between an incident angle and a reflecting angle is 6 degrees, so that the highest modulation efficiency of the spatial light modulator is achieved. The spatial light modulator is a pure phase type silicon-based liquid crystal spatial light modulator, and liquid crystal molecules are nematic liquid crystal molecules which are arranged in parallel, so that the whole spatial light modulator has uniaxial crystal properties. The liquid crystal spatial light modulator realizes a pixelized uniform-thickness liquid crystal panel with refractive index variation by modulating the main axis direction of liquid crystal molecules in each pixel. Therefore, the spatial light modulator has a high requirement for the polarization state of incident light. Before loading the hologram, the relationship between the voltage duty cycle and the phase modulation value, i.e. gamma correction, needs to be determined. And loading a proper phase hologram to the incident light field to further obtain expected emergent light field distribution. The calculation of the phase hologram of the light field to be generated is completed through a Gerchberg-Saxton (GS) algorithm, and the phase hologram is loaded on the spatial light modulator to generate the expected light field distribution.
The digital scanning device is a one-dimensional scanning galvanometer, and the control device controls the scanning range, the stepping distance and the number of sampling points of the one-dimensional scanning galvanometer to realize the rapid scanning of light beams so that diffraction-free light beams focused on a sample form light sheets.
The 4f relay system comprises two lenses with the same focal length, and the light field distribution of the plane where the digital scanning device is located and the back focal plane of the excitation objective lens is completely consistent.
The white light illumination system comprises an LED white light source, a second tube lens and a second camera, wherein the LED white light source irradiates a sample, the sample is collected by an excitation objective lens and imaged on the second camera through the second tube lens, x-y plane information of the sample is observed, and the sample in the microtube is determined to be in a front focal plane of the excitation objective lens, namely in a Bessel light beam area.
The control device is a digital acquisition system DAQ and controls the scanning range and the sampling frequency of the digital scanning device by outputting an analog signal; triggering and controlling line-by-line exposure, interval between every two exposed lines and triggering delay of the first camera by outputting a digital signal; analog signals and digital signals are synchronously output, and each scanning position of the digital scanning device is ensured to be matched with an exposure line of the first camera by setting the exposure time, the stepping distance and the scanning range of each plane, so that high-resolution imaging is ensured.
The invention also aims to provide a reflective axial light sheet fluorescence microscopic imaging method based on the microprism.
The invention relates to a microprism-based reflective axial light sheet fluorescence microscopic imaging method, which comprises the following steps:
1) device installation:
a) arranging a light through hole penetrating through the upper surface and the lower surface of a flat plate in the middle of the flat plate, arranging a micro-prism fixing device on the upper surface of the flat plate and above the light through hole, respectively arranging micro-tube fixing devices on two sides of the micro-prism fixing device, arranging a vertically-arranged excitation objective lens at the bottom of the light through hole, horizontally arranging a micro-tube on the upper surface of the flat plate through the micro-tube fixing device and at the top of the light through hole, arranging a 45-degree micro-prism on the upper surface of the flat plate through the micro-prism fixing device, wherein the oblique angle side surface of the 45-degree micro-prism is a reflecting surface, the first right angle side surface of the 45-degree micro-prism is positioned on an x-y plane and parallel to the upper surface of the flat plate and parallel to the lower surface of the micro-tube, the second right angle side surface is vertical to the upper surface of the flat plate, the cross section of the micro-tube is rectangular or circular, the cross section of the micro-tube is rectangular, and the bottom edge of the first right angle side surface of the 45-prism is attached to and tightly attached to the micro-tube, or the cross section of the microtube is circular, the bottom edge of the first right-angle side surface of the 45-degree microprism and the outermost side tangent plane of the side wall of the microtube are in the same plane at 45 degrees
A vertically arranged receiving objective lens is arranged above the reflecting surface of the microprism and is arranged on the translation platform to complete the arrangement of the sample rack;
b) a sample is positioned in the microtube, the sample is positioned at a front focal plane of the exciting objective lens, and a sample frame is placed on the piezoelectric scanning system;
c) the receiving objective lens is placed on the translation table, and the piezoelectric scanning system, the digital scanning device and the first camera are connected to the control device;
2) the laser light source emits a linear polarization Gaussian beam;
3) after passing through the Gaussian beam expanding and collimating device, the linearly polarized Gaussian beam enters the spatial light field regulating and controlling device; the spatial light field regulating and controlling device generates a non-diffraction light beam, and performs phase difference correction on the generated non-diffraction light beam and changes the spatial distribution characteristic of the non-diffraction light beam;
4) the diffraction-free light beams are focused to the digital scanning device through the focusing lens, the spectrum distribution of the diffraction-free light beams is generated at the digital scanning device due to the Fourier transform effect of the focusing lens, uniform light sheets are generated by scanning the diffraction-free light beams, then the spectrum distribution on the digital scanning device is relayed to a back focal plane of the vertically-arranged excitation objective lens by a 4f relay system to be used as excitation light, the excitation light enters an entrance pupil of the excitation objective lens, and the optical axis is along the z direction;
5) the focusing lens, the 4f relay system and the exciting objective lens meet the confocal condition to form a confocal system, after the diffraction-free light beam is transmitted in a confocal way through the focusing lens and the 4f relay system, the focusing light spot at the position of the focusing plane of the exciting objective lens is axial line light, the axial line light is changed into axial plane light by a light beam scanning system to form a digital exciting light sheet, the digital exciting light sheet is focused by the vertically-arranged exciting objective lens, the square distribution of the back focal plane and the front focal plane of the exciting objective lens is in a Fourier transform conjugate relation, the frequency spectrum distribution at the back focal plane of the exciting objective lens is under the Fourier transform action of the exciting objective lens, generating a y-z plane light sheet without diffraction beams on a front focal plane of the excitation objective lens to form a virtual digital excitation light sheet, wherein the digital excitation light sheet is vertical to the surface of the sample along the axial direction and performs sheet excitation on the sample positioned in the microtube to generate fluorescence;
6) the fluorescence is incident to the reflecting surface of the 45-degree microprism and then reflected into an x-y plane, and is directly collected by a vertically placed receiving objective lens;
7) the image is converged and imaged onto a first camera through a first tube lens;
8) a first camera directly receives a two-dimensional image of a sample;
9) meanwhile, the white light illumination system emits white light to irradiate the surface of the sample, the information at the sample is observed, and the sample in the microtube is determined to be positioned on the front focal plane of the excitation objective lens;
10) the control device controls the digital scanning device and the first camera to be synchronous;
11) the piezoelectric scanning system performs one-dimensional moving scanning on the sample along the direction vertical to the axial direction and the digital excitation light sheet,
obtaining the whole three-dimensional fluorescence distribution, thereby obtaining the three-dimensional distribution of the sample.
In step 6), adjusting the position of the receiving objective lens to make the center of the receiving objective lens correspond to the reflection surface of the 45 ° micro prism, arranging the sample holder on a piezoelectric translation stage of the piezoelectric scanning system, where the plane of the piezoelectric translation stage is an x-y plane, the direction perpendicular to the piezoelectric translation stage is a z direction, the long side direction of the micro tube is a y direction, and the short side direction is an x direction, and the specific adjustment method is as follows:
i. the sample holder is positioned on a piezoelectric translation table of a piezoelectric scanning system, the position of the micro-tube is observed, the position of the micro-tube is moved through the piezoelectric translation table, the micro-tube is approximately positioned at the center of an exciting objective lens, fluorescent microspheres are placed in the micro-tube, a white light illuminating system is turned on, the z direction of the sample holder is adjusted, the sample holder is observed in a camera, the x direction position of the sample holder is adjusted until a sample in the micro-tube is observed to be in a focus state in a second camera, the x direction position of the sample holder is continuously adjusted, a 45-degree micro prism, the sample and the tube wall of the micro-tube can be observed, and the sample holder is moved to the position of the tube wall of the micro-tube on the premise that the y direction is kept motionless
The sample is located at the position of the front focal plane;
and ii.the distance between the 45-degree micro prism and the tube wall of the micro tube is 0.1-0.5 mm, when the detection objective lens is adjusted, the distance in the x direction of the receiving objective lens is finely adjusted in a small range, the optical path of light reflected by the 45-degree micro prism is larger than that of light directly transmitted by the micro tube, when the detection objective lens is actually adjusted, the detection objective lens is transversely moved by determining the reflecting surface of the 45-degree micro prism as a focal plane, no focal image exists when the axial position of the detection objective lens is not adjusted, and the information of the tube wall of the micro tube can be observed in a visual field to judge whether the detection plane of the first camera is the axial plane information of the sample reflected by the 45-degree micro prism.
In step 7), the receiving plane of the first camera is adjusted to be perpendicular to the light incidence direction, and meanwhile, the light incidence point is ensured to be located at the right middle position of the receiving plane.
In step 9), the control device is a digital acquisition system DAQ, and controls the scanning range and the sampling frequency of the digital scanning device by outputting an analog signal; triggering and controlling line-by-line exposure, interval between every two exposed lines and triggering delay of the first camera by outputting a digital signal; analog signals and digital signals are synchronously output, and each scanning position of the digital scanning device is ensured to be matched with an exposure line of the first camera by setting the exposure time, the stepping distance and the scanning range of each plane, so that high-resolution imaging is ensured.
The invention has the advantages that:
the invention utilizes the 45-degree microprism to simplify a light sheet system with a complex inclined objective lens into a transmission type microscopic imaging system, has simple light path adjustment and can be combined with a plurality of imaging modes; the sample is only needed to be injected into the microtube, or the biological samples such as cells and the like can grow in the microtube, and the sample does not need to be subjected to complex treatment; the complete axial plane can be excited once, the observation of an axial special distribution sample can be quickly realized, and the device can realize the use of an objective lens with a large numerical aperture, so that the limitation that the resolution cannot be further improved in the traditional light sheet fluorescence microscopic imaging is improved; and the light path design is simple and easy, and the subsequent industrialized development is convenient to realize.
Drawings
FIG. 1 is a light path diagram of a first embodiment of a microprism-based reflective axial-ray sheet fluorescence microscopy imaging apparatus of the present invention;
FIG. 2 is a top view of a sample holder of a first embodiment of the microprism based reflective axial light sheet fluorescence microscopy imaging apparatus of the present invention;
FIG. 3 is a schematic view of the light direction of an excitation objective, a micro tube, a 45-degree micro prism and a detection objective according to a first embodiment of the present invention;
FIG. 4 is a graph of experimental results of a first embodiment of the microprism-based reflective axial-ray sheet fluorescence microscopy imaging apparatus of the present invention;
fig. 5 is a schematic diagram of a position relationship between a microtube and a 45 ° microprism in a second embodiment of the microprism-based reflective axial light sheet fluorescence microscopic imaging apparatus of the present invention.
Detailed Description
The invention will be further elucidated by means of specific embodiments in the following with reference to the drawing.
Example one
As shown in fig. 1, the microprism-based reflective axial light sheet fluorescence microscopic imaging apparatus of the present embodiment includes: the system comprises a laser light source 1, a Gaussian beam expanding and collimating device 2, a spatial light field regulating and controlling device 3, a focusing lens 4, a first reflector 5, a digital scanning device 6, a 4f relay system, a first beam splitter 9, a first tube lens 19, a filter 20, a first camera 21, a second reflector 12, a second beam splitter 13, an LED white light source 14, an excitation objective lens 15, a piezoelectric scanning system 16, a receiving objective lens 17, a reflector 18, a second tube lens 10, a second camera 11 and a control device; wherein, as shown in fig. 2, the sample holder comprises a flat plate 22, a light through hole, a microprism fixing device 23, a micropipe fixing device 24, a 45-degree microprism 25 and a micropipe 26, the middle of the flat plate is provided with the light through hole penetrating through the upper surface and the lower surface of the flat plate, the upper surface of the flat plate and above the light through hole are provided with the microprism fixing device, two sides of the microprism fixing device are respectively provided with the micropipe fixing device, the bottom of the light through hole is an excitation objective lens vertically (along the z axis), the cross section of the micropipe is square, the micropipe is horizontally arranged on the upper surface of the flat plate and is positioned on the top of the light through hole through the micropipe fixing device, the 45-degree microprism is arranged on the upper surface of the flat plate through the microprism fixing device, the oblique angle side surface of the 45-degree microprism is a reflecting surface, the first right angle side surface of the 45-degree microprism is positioned on the x-y plane and is parallel to the upper surface of the flat plate and is flush with the lower surface of the micropipe, the second right-angle side surface is vertical to the upper surface of the flat plate, the cross section of the micro-tube is rectangular, the bottom edge of the first right-angle side surface of the 45-degree micro prism is attached to the bottom edge of one side wall of the micro-tube and is tightly close to the micro-tube, a receiving objective lens vertically arranged (along the z axis) is arranged above the reflecting surface of the 45-degree micro prism, and the sample is positioned in the micro-tube; the sample is located at the front focal plane of the excitation objective 15, and the sample holder is placed on the piezoelectric scanning system 16; the receiving objective 17 is placed on a translation stage, the piezoelectric scanning system 16, the digital scanning device 6 and the first camera 21 being connected to the control device; the laser light source 1 emits a polarized Gaussian beam, and the optical axis of emergent light is along the x direction; after passing through the Gaussian beam expanding and collimating device 2, the linearly polarized Gaussian beam enters the spatial light field regulating and controlling device 3; the spatial light field regulating device 3 generates a non-diffraction light beam, and performs phase difference correction on the generated non-diffraction light beam and changes the spatial distribution characteristic of the non-diffraction light beam; after being focused by the focusing lens 4, the diffraction-free light beam is reflected by the first reflector 5 to enter the digital scanning device 6, a uniform light sheet is generated by scanning the diffraction-free light beam, then the light field distribution on the digital scanning device 6 is relayed to the focal plane behind the vertically placed excitation objective lens 15 by the 4f relay system to be used as excitation light, the excitation light enters the entrance pupil of the excitation objective lens 15, in order to enable the light beam of the optical axis along the x direction to enter the vertically placed objective lens, the light beam is reflected by the second reflector 12, and the reflected optical axis is along the z direction; the focusing lens 4, the 4f relay system and the exciting objective lens 15 meet the confocal condition to form a confocal system, the 4f relay system comprises a first lens 7 and a second lens 8 with the same focal length, after the non-diffracted light beam is transmitted by the confocal of the focusing lens 4 and the 4f relay system, the focusing light spot at the focusing plane of the exciting objective lens 15 is axial line light, the axial line light is changed into axial plane light by a light beam scanning system to form a digital exciting light sheet, the digital exciting light sheet is focused by the vertically arranged exciting objective lens 15, the wide field distribution of the rear focal plane and the front focal plane of the exciting objective lens 15 is in a Fourier transform conjugate relation, the frequency spectrum distribution at the rear focal plane of the exciting objective lens 15 is subjected to the Fourier transform action of the exciting objective lens 15, the y-z plane light sheet without the diffracted light beam is generated at the front focal plane of the exciting objective lens 15, a virtual digital exciting light sheet is formed, the digital exciting light sheet is vertical to the surface of the sample along the axial direction, carrying out sheet excitation on a sample positioned in the microtube to generate fluorescence; the fluorescence is incident to the reflecting surface of the 45-degree micro prism and then reflected into an x-y plane, and is directly collected by a vertically placed receiving objective lens 17; after being reflected by the third reflector 18, the light is converged by the first tube mirror 19 and filtered by the filter 20 to be imaged on the first camera 21; the first camera 21 directly receives a two-dimensional image of the sample; meanwhile, the LED white light source 14 irradiates a sample after being reflected by the second beam splitter 13, is collected by the exciting objective lens 15, is reflected by the second reflector 12 and the first beam splitter 9, is imaged on the second camera 11 by the second tube lens 10, observes x-y plane information of the sample, and determines that the sample in the microtube is positioned on a front focal plane of the exciting objective lens 15, namely in a Bessel light beam area; determining the focal plane of the excitation objective 15 in which the sample is located in the microtube; the control device controls the digital scanning device 6 and the first camera 21 to be synchronous; the piezoelectric scanning system 16 performs one-dimensional moving scanning on the sample along the direction perpendicular to the axial direction and the direction of the digital excitation light sheet to obtain the whole three-dimensional fluorescence distribution, so as to obtain the three-dimensional distribution of the sample.
The Gaussian beam expanding and collimating device 2 comprises two lenses with different focal lengths, wherein the two lenses with different focal lengths modulate the collimation state and the spot diameter of the linearly polarized Gaussian beam emitted by the laser device, so that collimated light is emitted, and the diameter of the spot is matched with the liquid crystal panel of the spatial light modulator.
The spatial light field regulation and control device 3 comprises a half wave plate 3-1, a 96-degree prism 3-2 and a spatial light modulator 3-3; the half wave plate modulates the polarization state of the linear polarization Gaussian beam after beam expansion and collimation to enable the linear polarization Gaussian beam to be matched with the polarization direction of a silicon-based liquid crystal of the spatial light modulator; the 96-degree prism folds a light path, linearly polarized light with a horizontal polarization state is reflected by the 96-degree reflection prism and then is projected to a silicon-based liquid crystal panel of the spatial light modulator, different phase holograms are loaded on the spatial light modulator to generate Bessel (Bessel) light beams, the modulated light field is reflected to the light path by the 96-degree reflection prism again, and an included angle between an incident angle and a reflection angle is 6 degrees, so that the highest modulation efficiency of the spatial light modulator is achieved.
In this embodiment, the laser light source 1 is a 561 nm continuous light semiconductor laser, and linearly polarized gaussian light passes through the beam expanding and collimating system 2 and then is expanded to a diameter of about 12 mm; the spatial light modulator 3-4 adopts a pure phase type silicon-based liquid crystal spatial light modulator; the distance between the spatial light modulator and the focusing lens 4 is 100 mm; in the light beam scanning system, the scanning amplitude of a galvanometer 5 is 1V when the voltage is increased or reduced, and the maximum scanning angle is increased or reduced by 1 degree; in accordance with the confocal relationship of the 4f relay system 6 to the excitation objective 158, to obtain a sheet width of 100 microns, the voltage applied to the galvanometer was chosen to be in the range-4.5V to 4.5V, with a scanning galvanometer response frequency of 10000 Hz.
As shown in fig. 2, the microprism fixing device is used for placing and fixing a 45 ° microprism, and is abutted against the microtube, wherein a microprism opening for fixing the 45 ° microprism is arranged in the middle of the microprism fixing device, the microprism opening is square in shape, the size is 1mm × 1mm, and one surface of the 45 ° microprism is abutted against the back surface of the square hole; the model of the screw holes at the left end and the right end of the microprism fixing device is M1, and the microprism fixing device is tightly installed with the sample rack main body; a micro-tube opening for fixing the micro-tube is formed in the middle of the micro-tube fixing device, the micro-tube opening is square, the size of the micro-tube opening is 0.85mm multiplied by 0.85mm, and the section of the micro-mirror is 0.8mm multiplied by 0.8mm and penetrates through the micro-tube opening; the model of the screw holes at the left end and the right end of the microtube fixing device is M1 and is used for being tightly installed with the sample rack main body. The model of the screw hole above the microtube fixing device is M2, and the screw hole is used for fixing the microtube so that the microtube cannot move.
The reflection type axial light sheet fluorescence microscopic imaging method based on the 45-degree micro prism comprises the following steps:
1) device installation:
a) a light through hole penetrating through the upper surface and the lower surface of a flat plate is formed in the middle of the flat plate, a microprism fixing device is arranged on the upper surface of the flat plate and above the light through hole, a micropipe fixing device is respectively arranged on two sides of the microprism fixing device, an excitation objective lens 15 which is vertically arranged is arranged at the bottom of the light through hole, the micropipe is horizontally arranged on the upper surface of the flat plate through the micropipe fixing device and is positioned at the top of the light through hole, a 45-degree microprism is arranged on the upper surface of the flat plate through the microprism fixing device, an oblique angle side surface of the 45-degree microprism is a reflecting surface, a first right angle side surface of the 45-degree microprism is positioned on an x-y plane and is parallel to the upper surface of the flat plate and is flush with the lower surface of the micropipe, a second right angle side surface is vertical to the upper surface of the flat plate, the cross section of the micropipe is rectangular, the bottom edge of the first right angle side surface of the 45-degree microprism is attached to one side wall of the micropipe and is tightly close to the micropipe, a receiving objective lens 17 which is vertically (along the z axis) arranged is arranged above the reflecting surface of the 45-degree micro prism, and the receiving objective lens 17 is arranged on a translation table to complete the arrangement of the sample rack;
b) the sample is positioned in the microtube, the sample is positioned at the front focal plane of the exciting objective 15, and the sample holder is placed on the piezoelectric scanning system 16;
c) the receiving objective 17 is placed on a translation stage, the piezoelectric scanning system 16, the translation stage, the digital scanning device 6 and the first camera 21 being connected to the control device;
2) the laser light source 1 emits a linear polarization Gaussian beam, and the optical axis of the linear polarization Gaussian beam is along the z direction;
3) after passing through the Gaussian beam expanding and collimating device 2, the linearly polarized Gaussian beam enters the spatial light field regulating and controlling device 3; the spatial light field regulating device 3 generates a non-diffraction light beam, and performs phase difference correction on the generated non-diffraction light beam and changes the spatial distribution characteristic of the non-diffraction light beam;
4) the diffraction-free light beams are focused to the digital scanning device 6 through the focusing lens 4, the spectrum distribution of the diffraction-free light beams is generated at the digital scanning device 6 due to the Fourier transform effect of the focusing lens 4, uniform light sheets are generated by scanning the diffraction-free light beams, then the spectrum distribution on the digital scanning device 6 is relayed to a focal plane behind the vertically-arranged exciting objective lens 15 through a 4f relay system to be used as exciting light, the exciting light enters an entrance pupil of the exciting objective lens 15, and the optical axis is along the z direction;
5) the focusing lens 4, the 4f relay system and the exciting objective lens 15 meet the confocal condition to form a confocal system, after the non-diffracted light beam is transmitted in a confocal way through the focusing lens 4 and the 4f relay system, a focusing light spot at the focusing plane of the exciting objective lens 15 is axial line light, the axial line light is changed into axial plane light by the light beam scanning system to form a digital exciting light sheet, the digital exciting light sheet is focused by the vertically arranged exciting objective lens 15, the wide field distribution of the back focal plane and the front focal plane of the exciting objective lens 15 is in a Fourier transform conjugate relation, the frequency spectrum distribution at the back focal plane of the exciting objective lens 15 is subjected to the Fourier transform action of the exciting objective lens 15 to generate a y-z plane light sheet of the non-diffracted light beam at the front focal plane of the exciting objective lens 15 to form a virtual digital exciting light sheet, the digital exciting light sheet is perpendicular to the surface of the sample along the axial direction and the surface of the sample to perform sheet excitation on the sample positioned in a micro tube, generating fluorescence;
6) the fluorescence is incident to the reflecting surface of the 45-degree microprism and then reflected into an x-y plane, and is directly received by a vertically arranged receiving objective lens 17
Collecting;
7) focused and imaged on a first camera 21 through a first tube lens 19;
8) the first camera 21 directly receives a two-dimensional image of the sample;
9) meanwhile, the LED white light source 14 irradiates a sample, the sample is collected by the exciting objective lens 15 and imaged on the second camera 11 through the second tube lens 10, the x-y plane information of the sample is observed, and the sample in the microtube is determined to be in the front focus of the exciting objective lens 15
Planar, i.e., in the bessel beam region;
10) the control device controls the digital scanning device 6 and the first camera 21 to be synchronous;
11) the piezoelectric scanning system 16 performs one-dimensional moving scanning on the sample along the direction perpendicular to the axial direction and the direction of the digital excitation light sheet to obtain the whole three-dimensional fluorescence distribution, so as to obtain the three-dimensional distribution of the sample.
Fig. 3 is a schematic diagram of the light direction of the sample holder accessory of this embodiment, and the scanning light sheet generated by the excitation objective 15 excites the sample in the microtube 26, and the excitation plane is reflected by the 45 ° micro prism 25 and then received by the receiving objective 17.
Fig. 4 is an experimental imaging result of the microprism-based reflective axial light sheet fluorescence micro-imaging device of the present embodiment on a fluorescent bead sample with a diameter of 10 micrometers, wherein the length of the ruler is 10 micrometers.
Example two
In this embodiment, the cross-section of the microtube is circular, and the bottom edge of the first right-angle side surface of the 45 ° microprism is in the same plane as the outermost tangent plane of the side wall of the microtube, as shown in fig. 5.
Finally, it is noted that the disclosed embodiments are intended to aid in further understanding of the invention, but those skilled in the art will appreciate that: various substitutions and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the invention should not be limited to the embodiments disclosed, but the scope of the invention is defined by the appended claims.
Claims (10)
1. A microprism-based reflective axial light sheet fluorescence microscopic imaging device is characterized by comprising: the device comprises a laser light source, a Gaussian light beam expanding and collimating device, a spatial light field regulating and controlling device, a focusing lens, a digital scanning device, a 4f relay system, an excitation objective lens, a receiving objective lens, a translation table, a first tube lens, a first camera, a sample frame, a piezoelectric scanning system, a white light illuminating system and a control device; wherein, the sample rack comprises a flat plate, a light through hole, a microprism fixing device, a micropipe and a 45-degree microprism, the middle of the flat plate is provided with the light through hole penetrating through the upper surface and the lower surface of the flat plate, the upper surface of the flat plate and the upper part of the light through hole are provided with the microprism fixing device, two sides of the microprism fixing device are respectively provided with the micropipe fixing device, the bottom of the light through hole is an excitation objective lens which is vertically arranged, the micropipe is horizontally arranged on the upper surface of the flat plate through the micropipe fixing device and is positioned at the top of the light through hole, the 45-degree microprism is arranged on the upper surface of the flat plate through the microprism fixing device, the bevel edge surface of the 45-degree microprism is a reflecting surface, the first right-angle edge surface of the 45-degree microprism is positioned on an x-y plane and is parallel to the upper surface of the flat plate and is parallel to the lower surface of the micropipe, the second right-angle surface of the flat plate, the cross section of the microtube is rectangular or circular, the section of the micro-tube is rectangular, the bottom edge of the first right-angle side surface of the 45-degree micro-prism is attached to the bottom edge of one side wall of the micro-tube and is tightly close to the micro-tube, or the section of the micro-tube is circular, and the bottom edge of the first right-angle side surface of the 45-degree micro-prism and the outermost tangent plane of the side wall of the micro-tube are on the same plane; a vertically arranged receiving objective lens is arranged above the reflecting surface of the 45-degree micro prism, and a sample is positioned in the micro tube; the sample is positioned at the front focal plane of the exciting objective lens, and the sample frame is arranged on the piezoelectric scanning system; the receiving objective lens is placed on the translation table, and the piezoelectric scanning system, the digital scanning device and the first camera are connected to the control device; the laser light source emits a linear polarization Gaussian beam; after passing through the Gaussian beam expanding and collimating device, the linearly polarized Gaussian beam enters the spatial light field regulating and controlling device; the spatial light field regulating and controlling device generates a non-diffraction light beam, and performs phase difference correction on the generated non-diffraction light beam and changes the spatial distribution characteristic of the non-diffraction light beam; the diffraction-free light beams are focused to the digital scanning device through the focusing lens, the spectrum distribution of the diffraction-free light beams is generated at the digital scanning device due to the Fourier transform effect of the focusing lens, uniform light sheets are generated by scanning the diffraction-free light beams, then the spectrum distribution on the digital scanning device is relayed to a back focal plane of the vertically-arranged excitation objective lens by a 4f relay system to be used as excitation light, the excitation light enters an entrance pupil of the excitation objective lens, and the optical axis is along the z direction; the focusing lens, the 4f relay system and the exciting objective lens meet the confocal condition to form a confocal system, after the diffraction-free light beam is transmitted in a confocal way through the focusing lens and the 4f relay system, the focusing light spot at the position of the focusing plane of the exciting objective lens is axial line light, the axial line light is changed into axial plane light by a light beam scanning system to form a digital exciting light sheet, the digital exciting light sheet is focused by the vertically-arranged exciting objective lens, the square distribution of the back focal plane and the front focal plane of the exciting objective lens is in a Fourier transform conjugate relation, the frequency spectrum distribution at the back focal plane of the exciting objective lens is under the Fourier transform action of the exciting objective lens, generating a y-z plane light sheet without diffraction beams on a front focal plane of the excitation objective lens to form a virtual digital excitation light sheet, wherein the digital excitation light sheet is vertical to the surface of the sample along the axial direction and performs sheet excitation on the sample positioned in the microtube to generate fluorescence; the fluorescence is incident to the reflecting surface of the 45-degree microprism and then reflected into an x-y plane, and is directly collected by a vertically placed receiving objective lens; the image is converged and imaged onto a first camera through a first tube lens; a first camera directly receives a two-dimensional image of a sample; meanwhile, the white light illumination system emits white light to irradiate the surface of the sample, the information at the sample is observed, and the sample in the microtube is determined to be positioned on the front focal plane of the excitation objective lens; the control device controls the digital scanning device and the first camera to be synchronous; the piezoelectric scanning system performs one-dimensional moving scanning on the sample along the direction vertical to the axial direction and the direction of the digital excitation light sheet to obtain the whole three-dimensional fluorescence distribution, so that the three-dimensional distribution of the sample is obtained.
2. The microprism-based reflective axial light sheet fluorescence microscopy imaging apparatus of claim 1, wherein the gaussian beam expanding and collimating device comprises two lenses with different focal lengths, wherein the two lenses with different focal lengths modulate the collimation state and the spot diameter of the linearly polarized gaussian beam emitted from the laser to emit collimated light, and the spot diameter is adapted to the liquid crystal panel of the spatial light modulator.
3. The microprism-based reflective axial light sheet fluorescence microscopy imaging device of claim 1, wherein the spatial light field modulation device comprises a half wave plate, a 96 ° prism, and a spatial light modulator; the half wave plate modulates the polarization state of the linear polarization Gaussian beam after beam expansion and collimation to enable the linear polarization Gaussian beam to be matched with the polarization direction of a silicon-based liquid crystal of the spatial light modulator; the 96-degree prism folds a light path, linearly polarized light with a horizontal polarization state is reflected by the 96-degree reflection prism and then is projected onto a silicon-based liquid crystal panel of the spatial light modulator, different phase holograms are loaded on the spatial light modulator to generate a non-diffraction light beam, the non-diffraction light beam is a Bessel light beam or an Airy light beam, a modulated light field is reflected into the light path by the 96-degree reflection prism again, and an included angle between an incident angle and a reflection angle is 6 degrees, so that the highest modulation efficiency of the spatial light modulator is achieved.
4. The microprism-based reflective axial light sheet fluorescence microscopy imaging device of claim 1, wherein the digital scanning device is a one-dimensional scanning galvanometer, and the control device controls the scanning range, the step distance and the number of sampling points of the one-dimensional scanning galvanometer to realize rapid scanning of the light beam, so that the diffraction-free light beam focused on the sample forms the light sheet.
5. The microprism-based reflective axial light sheet fluorescence microscopy imaging device of claim 1, wherein the 4f relay system comprises two lenses with the same focal length, and the digital scanning device is positioned on a plane completely consistent with the optical field distribution of the excitation objective lens back focal plane.
6. The microprism-based reflective axial light sheet fluorescence microscopy imaging apparatus of claim 1, wherein the white light illumination system comprises an LED white light source, a second tube lens and a second camera, wherein the LED white light source illuminates the sample, is collected by the excitation objective lens, is imaged onto the second camera through the second tube lens, observes x-y plane information of the sample, and determines that the sample in the microtube is in a front focal plane of the excitation objective lens, i.e., in the bessel beam region.
7. The microprism-based reflective axial light sheet fluorescence microscopy imaging device of claim 1, wherein the control device is a digital acquisition system (DAQ) that controls the scanning range and sampling frequency of the digital scanning device by outputting an analog signal; triggering and controlling line-by-line exposure, interval between every two exposed lines and triggering delay of the first camera by outputting a digital signal; analog signals and digital signals are synchronously output, and each scanning position of the digital scanning device is ensured to be matched with an exposure line of the first camera by setting the exposure time, the stepping distance and the scanning range of each plane, so that high-resolution imaging is ensured.
8. A microprism-based reflective axial light sheet fluorescence microscopic imaging method is characterized by comprising the following steps:
1) device installation:
a) arranging a light through hole penetrating through the upper surface and the lower surface of a flat plate in the middle of the flat plate, arranging a micro-prism fixing device on the upper surface of the flat plate and above the light through hole, respectively arranging micro-tube fixing devices on two sides of the micro-prism fixing device, arranging a vertically-arranged excitation objective lens at the bottom of the light through hole, horizontally arranging a micro-tube on the upper surface of the flat plate through the micro-tube fixing device and at the top of the light through hole, arranging a 45-degree micro-prism on the upper surface of the flat plate through the micro-prism fixing device, wherein the oblique angle side surface of the 45-degree micro-prism is a reflecting surface, the first right angle side surface of the 45-degree micro-prism is positioned on an x-y plane and parallel to the upper surface of the flat plate and parallel to the lower surface of the micro-tube, the second right angle side surface is vertical to the upper surface of the flat plate, the cross section of the micro-tube is rectangular or circular, the cross section of the micro-tube is rectangular, and the bottom edge of the first right angle side surface of the 45-prism is attached to and tightly attached to the micro-tube, or the section of the micro-tube is circular, the bottom edge of the first right-angle side surface of the 45-degree micro-prism and the outermost tangent plane of the side wall of the micro-tube are on the same plane, a vertically-placed receiving objective lens is arranged above the reflecting surface of the 45-degree micro-prism, and the receiving objective lens is placed on the translation table to complete the setting of the sample holder;
b) a sample is positioned in the microtube, the sample is positioned at a front focal plane of the exciting objective lens, and a sample frame is placed on the piezoelectric scanning system;
c) the receiving objective lens is placed on the translation table, and the piezoelectric scanning system, the digital scanning device and the first camera are connected to the control device;
2) the laser light source emits a linear polarization Gaussian beam;
3) after passing through the Gaussian beam expanding and collimating device, the linearly polarized Gaussian beam enters the spatial light field regulating and controlling device; the spatial light field regulating and controlling device generates a non-diffraction light beam, and performs phase difference correction on the generated non-diffraction light beam and changes the spatial distribution characteristic of the non-diffraction light beam;
4) the diffraction-free light beams are focused to the digital scanning device through the focusing lens, the spectrum distribution of the diffraction-free light beams is generated at the digital scanning device due to the Fourier transform effect of the focusing lens, uniform light sheets are generated by scanning the diffraction-free light beams, then the spectrum distribution on the digital scanning device is relayed to a back focal plane of the vertically-arranged excitation objective lens by a 4f relay system to be used as excitation light, the excitation light enters an entrance pupil of the excitation objective lens, and the optical axis is along the z direction;
5) the focusing lens, the 4f relay system and the exciting objective lens meet the confocal condition to form a confocal system, after the diffraction-free light beam is transmitted in a confocal way through the focusing lens and the 4f relay system, the focusing light spot at the position of the focusing plane of the exciting objective lens is axial line light, the axial line light is changed into axial plane light by a light beam scanning system to form a digital exciting light sheet, the digital exciting light sheet is focused by the vertically-arranged exciting objective lens, the square distribution of the back focal plane and the front focal plane of the exciting objective lens is in a Fourier transform conjugate relation, the frequency spectrum distribution at the back focal plane of the exciting objective lens is under the Fourier transform action of the exciting objective lens, generating a y-z plane light sheet without diffraction beams on a front focal plane of the excitation objective lens to form a virtual digital excitation light sheet, wherein the digital excitation light sheet is vertical to the surface of the sample along the axial direction and performs sheet excitation on the sample positioned in the microtube to generate fluorescence;
6) the fluorescence is incident to the reflecting surface of the 45-degree microprism and then reflected into an x-y plane, and is directly collected by a vertically placed receiving objective lens;
7) the image is converged and imaged onto a first camera through a first tube lens;
8) a first camera directly receives a two-dimensional image of a sample;
9) meanwhile, the white light illumination system emits white light to irradiate the surface of the sample, the information at the sample is observed, and the sample in the microtube is determined to be positioned on the front focal plane of the excitation objective lens;
10) the control device controls the digital scanning device and the first camera to be synchronous;
11) the piezoelectric scanning system performs one-dimensional moving scanning on the sample along the direction vertical to the axial direction and the direction of the digital excitation light sheet to obtain the whole three-dimensional fluorescence distribution, so that the three-dimensional distribution of the sample is obtained.
9. The imaging method as claimed in claim 8, wherein in step 6), the position of the receiving objective lens is adjusted so that the center of the receiving objective lens corresponds to the reflection surface of the 45 ° micro prism, the sample holder is disposed on a piezoelectric translation stage of the piezoelectric scanning system, the plane of the piezoelectric translation stage is an x-y plane, the direction perpendicular to the piezoelectric translation stage is a z direction, the long side direction of the micro prism is a y direction, and the short side direction is an x direction, and the specific adjustment method is as follows:
i. the sample holder is positioned on a piezoelectric translation table of a piezoelectric scanning system, the position of the micro-tube is observed, the position of the micro-tube is moved through the piezoelectric translation table, the micro-tube is approximately positioned at the center of an excitation objective lens, fluorescent microspheres are placed in the micro-tube, a white light illumination system is turned on, the z direction of the sample holder is adjusted, the sample holder is observed in a camera, the x direction position of the sample holder is adjusted until a sample in the micro-tube is observed to be in a focus state in a second camera, the x direction position of the sample holder is continuously adjusted, a 45-degree micro prism, the sample and the tube wall of the micro-tube can be observed, and the sample holder is moved to the position of the sample positioned at a focal plane on the premise that the y direction is kept motionless;
and ii.the distance between the 45-degree micro prism and the tube wall of the micro tube is 0.1-0.5 mm, when the detection objective lens is adjusted, the distance in the x direction of the receiving objective lens is finely adjusted in a small range, the optical path of light reflected by the 45-degree micro prism is larger than that of light directly transmitted by the micro tube, when the detection objective lens is actually adjusted, the detection objective lens is transversely moved by determining the reflecting surface of the 45-degree micro prism as a focal plane, no focal image exists when the axial position of the detection objective lens is not adjusted, and the information of the tube wall of the micro tube can be observed in a visual field to judge whether the detection plane of the first camera is the axial plane information of the sample reflected by the 45-degree micro prism.
10. The imaging method as claimed in claim 8, wherein in step 9), the control means is a digital acquisition system DAQ which controls a scanning range and a sampling frequency of the digital scanning means by outputting an analog signal; triggering and controlling line-by-line exposure, interval between every two exposed lines and triggering delay of the first camera by outputting a digital signal; analog signals and digital signals are synchronously output, and each scanning position of the digital scanning device is ensured to be matched with an exposure line of the first camera by setting the exposure time, the stepping distance and the scanning range of each plane, so that high-resolution imaging is ensured.
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