CN117647879A - Single-objective optical sheet fluorescent scanning imaging system - Google Patents

Abstract

The invention discloses a single-objective light sheet fluorescent scanning imaging system, which comprises a dichroic mirror, a main objective, a beam shaping module, an exposure camera and an axial scanning module, wherein the dichroic mirror is arranged on the main objective; the dichroic mirror and the main object mirror are simultaneously positioned on an excitation light path and a detection light path: the illumination light sheet generated by the beam shaping module is projected on the edge of the rear pupil of the main objective lens through a dichroic mirror, so as to generate the illumination light sheet; fluorescent light emitted by the sample excitation plane is collected by the main object lens and then projected on the roller shutter exposure camera to form images by the dichroic mirror; the axial scanning module based on optical path adjustment is arranged between the dichroic mirror and the main object mirror on the optical path; and the axial scanning module adjusts the optical path of the excitation optical path and the optical path of the detection optical path by the same period and the same displacement, so that the sample excitation plane is exposed on the exposure camera line by line along the axial direction of the light sheet for imaging. The invention improves the axial resolution and solves the problem of extra synchronization of the excitation light and the detection optical axial scanning.

Description

Single-objective optical sheet fluorescent scanning imaging system

Technical Field

The invention belongs to the field of light sheet fluorescence imaging, and particularly relates to a single-objective light sheet fluorescence scanning imaging system.

Background

A light sheet fluorescence microscope is a completely new fluorescence microscope that irradiates a sample from the side of the sample by generating a sheet of light beam by a pattern of illumination beams through a cylindrical mirror or gaussian beam. The sheet-shaped side illumination mode has the advantages that the excitation area is large, the imaging speed is high, meanwhile, the illumination is limited to the focusing plane of the detector as much as possible by the light sheet, only fluorescent substances near the focal plane can be excited, interference of non-focal plane fluorescent signals is reduced, the imaging slicing capacity and the longitudinal resolution are improved, the phototoxicity and the photobleaching performance of a sample are reduced, and the sheet-shaped side illumination mode has important significance for life science research.

Under a light sheet microscope in a conventional mode, two objective lenses are generally required to be vertically arranged, one for exciting an illumination sample and one for receiving detection fluorescence, and the arrangement limits the types of samples that can be positioned and observed, and in order to prevent collision of the two objective lenses, a low-power objective lens with a longer working distance is required, so that the light sheet microscope is not suitable for a high-power objective lens with a high NA value. An innovative device design is therefore proposed, i.e. simultaneous illumination and detection with one objective, i.e. a single objective light sheet microscope.

As shown in fig. 4, a conventional single-objective optical path diagram in the prior art is shown, and a single-objective optical sheet uses the same objective to form an optical sheet and detect fluorescence. The incident light is incident from the edge of the rear pupil of the objective lens (O1), obliquely illuminates the sample at an angle theta, and collects fluorescent signals with the same objective lens. Since the excitation optical axis and the detection optical axis are not orthogonal to each other, the plane on which information on the inclined plane excited by the optical sheet is imaged by the objective lens is also the inclined plane. In order to obtain an aberration-free image with high resolution, correction of the oblique image is required. Another objective lens (O2) is selected, O1, O2 and a lens in the middle are coaxially arranged, an inclined light sheet illumination plane is perfectly conjugated to the focal plane of the second objective lens, a third objective lens (O3) is selected, and a certain angle (theta) is formed between the third objective lens and the optical axis of the second objective lens (O2), so that the focal plane of the O3 coincides with the excitation plane of the light sheet, and the inclined image is corrected, so that object plane information is collected and imaged on the camera plane by the third objective lens.

However, the objective lens O3 of the conventional single objective lens system is inclined at a certain angle to realize vertical detection, so that a part of the light receiving angle is lost, the lateral resolution and the axial resolution cannot be considered, and the design of the inclined objective lens increases the complexity of the whole system construction.

Disclosure of Invention

Aiming at the defects or improvement demands of the prior art, the invention provides a single-objective optical sheet fluorescent scanning imaging system, which adjusts the optical path length of an excitation optical path and the optical path length of a detection optical path in the same period and the same displacement, and is matched with roller shutter exposure imaging, thereby breaking through the imaging limit of the single-objective optical sheet, improving the axial resolution and realizing microscopic imaging which does not need oblique correction and has equivalent transverse axial resolution.

According to one aspect of the present invention, there is provided a single objective light sheet fluorescence scanning imaging system comprising a dichroic mirror and a main objective, a beam shaping module, an exposure camera, and an axial scanning module;

the dichroic mirror and the main object mirror are simultaneously positioned on an excitation light path and a detection light path:

the illumination light sheet generated by the beam shaping module is projected on the edge of the rear pupil of the main objective lens through a dichroic mirror, so as to generate the illumination light sheet;

fluorescent light emitted by the sample excitation plane is collected by the main object lens and then projected on the roller shutter exposure camera to form images by the dichroic mirror;

the axial scanning module based on optical path adjustment is arranged between the dichroic mirror and the main object mirror;

and the axial scanning module adjusts the optical path of the excitation optical path and the optical path of the detection optical path by the same period and the same displacement, so that the sample excitation plane is exposed and imaged on the exposure camera along the axial direction of the light sheet.

Preferably, the single objective lens fluorescent scanning imaging system, when imaging, makes the exposure camera complete one frame exposure in one scanning stroke of the axial scanning module;

when the axial scanning module has symmetrical positive and negative strokes, the exposure camera completes one-frame exposure in the positive and/or negative strokes; when the axial scanning module has asymmetrical positive and negative strokes, the axial scanning module can be used in a longer scanning stroke, so that the exposure camera can complete one-frame exposure.

The ratio of the reciprocating frequency of the axial scanning module to the imaging frame rate is 1:2 or 1:1.

Preferably, the single objective lens fluorescent scanning imaging system is started to sense light at the same time, and the number of lines of light is smaller than the depth of field range of the main objective lens.

Preferably, in the single objective optical sheet fluorescence scanning imaging system, the axial scanning module is based on a reflection principle, the illumination optical sheet adopts polarized light, and a polarization beam splitter is arranged on a composite optical path of incident light and reflected light of the axial scanning module, and is used for separating the reflected light from the incident light and projecting the reflected light to the illumination objective.

Preferably, the axial scanning module of the single-objective optical sheet fluorescence scanning imaging system comprises a reflecting mirror and a scanning objective lens, wherein the plane of the reflecting mirror is perpendicular to the main optical axis of the scanning objective lens, and the optical path is changed by changing the distance between the reflecting mirror and the scanning objective lens.

Preferably, the axial scanning module of the single-objective light sheet fluorescence scanning imaging system comprises a galvanometer, a scanning objective lens and a reflecting mirror, wherein the reflecting mirror is perpendicular to the incident light, and the rotation angle of the galvanometer is used for scanning the matched objective lens to form an optical path difference.

Preferably, the axial scanning module of the single-objective optical sheet fluorescence scanning imaging system is based on a transmission principle, and the axial scanning module is arranged between the dichroic mirror and a main object lens or assembled on the main object lens; the axial scanning module is a turntable lens, an electrically adjustable lens, an adjustable acoustic gradient lens or an objective lens scanner.

Preferably, the single objective lens fluorescent scanning imaging system, the axial scanning module comprises a scanning lens group and a turntable lens arranged between the scanning lens group; the scanning lens of the scanning lens group is arranged in a confocal mode, and the turntable lens is arranged at a shared focal plane of the scanning lens group.

Preferably, the beam shaping module of the single-objective optical sheet fluorescence scanning imaging system comprises an optical sheet position adjusting assembly, and the position of the optical sheet position adjusting assembly is moved in a horizontal direction in a one-dimensional way so that an illumination optical sheet irradiates at the edge position of the rear pupil of the main objective; the light sheet position adjusting component is a reflecting mirror.

Preferably, in the single objective lens fluorescent scanning imaging system, an inclination angle of the main objective lens emergent lens relative to the optical axis of the main objective lens is equal to a light receiving angle of the main objective lens.

In general, the above technical solutions conceived by the present invention, compared with the prior art, enable the following beneficial effects to be obtained:

the invention provides a single-objective optical sheet fluorescent scanning imaging system, which is characterized in that an axial scanning module is used for adjusting the optical path length of an excitation optical path and the optical path length of a detection optical path in the same period and the same displacement, so that a sample excitation plane is exposed and imaged on an exposure camera along the axial direction of an optical sheet, and on the premise of realizing illumination and imaging synchronization without extra cost, the axial resolution is improved, and the problem of extra synchronization of the excitation light and the detection optical axis scanning is solved.

Meanwhile, the excitation light path and the imaging light path are axially scanned along the illuminating light sheet to be matched with line-by-line imaging, and the inclined image is not required to be corrected by improving the axial resolution imaging of the light sheet, so that a second object lens and a third object lens for correction are not required in the light path; in addition, the roller shutter exposure and the axial sampling are improved, the Rayleigh range display of the static light sheet can be broken through, and a larger field of view is provided.

Drawings

FIG. 1 is a first embodiment of a single objective light sheet fluorescence scanning imaging system provided by the present invention;

FIG. 2 is a schematic diagram of a second embodiment of a single objective light sheet fluorescence scanning imaging system according to the present invention;

FIG. 3 is a third embodiment of a single objective light sheet fluorescence scanning imaging system provided by the present invention;

fig. 4 is a conventional single objective optical path diagram in the prior art.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The invention provides a single-objective light sheet fluorescence scanning imaging system, which comprises a dichroic mirror, a main objective, a beam shaping module and an exposure camera, wherein the dichroic mirror is arranged on the main objective;

the dichroic mirror and the main object mirror are simultaneously positioned on an excitation light path and a detection light path:

the illumination light sheet generated by the beam shaping module is projected on the edge of the rear pupil of the main objective lens through a dichroic mirror, so as to generate the illumination light sheet; the beam shaping module comprises a light sheet position adjusting assembly, a light source module and a light source module, wherein the light sheet position adjusting assembly is used for adjusting the posture of an inclined light sheet, and the position of the light sheet position adjusting assembly is moved in one dimension in the horizontal direction so that an illumination light sheet irradiates at the edge position of the rear pupil of the main object lens; the light sheet position adjusting component is a reflecting mirror.

And the inclination angle of the main objective emergent light sheet relative to the optical axis of the main objective is equal to the light receiving angle of the main objective.

Fluorescent light emitted by the sample excitation plane is collected by the main object lens and then projected on the roller shutter exposure camera to form images by the dichroic mirror;

an axial scanning module based on optical path adjustment between the dichroic mirror and the main objective;

and the axial scanning module adjusts the optical path of the excitation optical path and the optical path of the detection optical path by the same period and the same displacement, so that the sample excitation plane is subjected to roller shutter exposure imaging on the exposure camera along the axial direction of the light sheet. The shutter exposure mode can realize the line-by-line exposure of the camera photosensitive chip, only the exposed lines receive photons and the unexposed lines do not receive signals, and the influence of the signals outside the depth of field on the imaging quality is prevented. The diaphragm type exposure of the progressive exposure is matched with the scanning of the excitation light path and the detection light-emitting path, the axial resolution is improved, the remote focusing system consisting of the second objective lens and the third objective lens is not required to perform oblique correction, and the single objective lens fluorescent imaging can be realized.

During imaging, the exposure camera completes one-frame exposure in one scanning stroke of the axial scanning module. The axial scanning module controls the excitation light path and the detection light path to reciprocate from a starting point (usually a position with the shortest light path or the longest light path) to a final end point (corresponding to the starting point, usually a position with the longest light path or the shortest light path), or returns to the starting point from the final end point to form a scanning stroke. The stroke from the start point to the end point is generally taken as a positive stroke, and the stroke from the end point to the start point is taken as a negative stroke. In one scanning stroke of the axial scanning module, the exposure camera completes one-frame exposure, which can be specifically: thereby, when the axial scanning module is reset to a first position (the position at the beginning of the period, namely the starting point), the row at the topmost layer of the camera, which is matched with the depth of field of the main object lens, starts exposure within the field of view; when the axial scanning module moves to a second position (the position at the end of the period, namely the end point), the line exposure of the bottommost layer of the camera, which is matched with the depth of field of the main object lens, is in the field of view. After one period is finished, the axial scanning module is quickly reset, and the process is repeated by matching with the three-dimensional imaging displacement table to image the next frame, so that three-dimensional imaging is realized.

The ratio of the reciprocating frequency of the axial scanning module to the imaging frame rate is 1:2 or 1:1.

In some embodiments, the positive and negative strokes of the axial scan module are symmetrical, i.e., the instantaneous speeds of the points are the same and opposite when one positive or negative stroke is completed, so that the time for completing one positive or negative stroke is the same, e.g., the optical sheet rayleigh range reciprocates at a constant speed. The ratio of the reciprocating frequency of the axial scanning module to the imaging frame rate can be selected to be 1:2 or 1:1. When the ratio is 1:2, imaging the positive stroke and the negative stroke once respectively, and when the ratio is 1:1; positive-stroke or negative-stroke imaging is typically selected, while the other stroke camera is delayed for waiting.

In some embodiments, the positive and negative strokes of the axial scan module are asymmetric, i.e., the time to complete the positive or negative stroke is different. For example, the roller shutter exposure is carried out in the positive stroke stage, and the negative stroke rapidly returns to the initial position, so that the time for completing the negative stroke is shorter; at the extreme, the time of the negative stroke may be 0 when using a turret lens to construct an axial scan. The ratio of the reciprocating frequency of the axial scanning module to the imaging frame rate is 1:1, and the camera is imaged in a positive stroke and correspondingly delayed in a negative stroke.

And simultaneously, the number of lines for starting the sensitization is smaller than the depth of field range of the main objective lens, and in the adjustable range, the sampling resolution is higher as the number of lines is smaller, and the signal-to-noise ratio of imaging is higher as the number of lines is larger.

The axial scan module may be based on either reflection principles or transmission principles.

When the axial scanning module is based on the reflection principle, the axial scanning module is arranged on the outer sides of the dichroic mirror and the main object lens, in order to realize the arrangement between the dichroic mirror and the main object lens on an optical path, the illumination light sheet adopts polarized light, and a polarization beam splitter is arranged on a composite optical path of incident light and reflected light of the axial scanning module and used for separating the reflected light from the incident light and projecting the reflected light to the illumination objective lens. An axial scan module employing reflection principles may be implemented using an objective lens and mirror assembly (e.g., embodiment 1) or using a galvanometer (e.g., embodiment 2).

The axial scanning module based on the reflecting mirror comprises the reflecting mirror and a scanning objective lens, wherein the plane of the reflecting mirror is perpendicular to the main optical axis of the objective lens, and the optical path is changed by changing the distance between the reflecting mirror and the objective lens. The typical structure is as follows: the axial scanning module sequentially comprises a half wave plate, a polarization beam splitter, a quarter wave plate, a galvanometer, a scanning lens, a tube lens, a scanning objective lens and a first reflecting mirror along a detection light path; the light sheet reflected by the first reflector returns to enter the polarization beam splitter along an original path, and then sequentially passes through the first achromatic double-cemented lens, the second achromatic double-cemented lens and the second reflector to enter the main object lens; the focus of the light focused by the main object lens moves along the propagation direction through the scanning and inverse scanning of the vibrating lens, so that the axial scanning is realized.

The axial scanning module comprises a vibrating mirror, a scanning objective lens and a reflecting mirror, wherein the reflecting mirror is perpendicular to incident light, and the rotating angle scanning of the vibrating mirror is matched with the scanning objective lens to form optical path difference. The typical structure is as follows: the axial scanning module sequentially comprises a half wave plate, a polarization beam splitter, a quarter wave plate, a scanning objective lens and a first reflecting mirror along a detection light path; the light sheet reflected by the first reflecting mirror returns to enter the polarization beam splitter along an original path, and then enters the main object lens through the conjugate correction achromatic double-cemented lens group and the second reflecting mirror in sequence;

the axial scanning module comprises a first reflecting mirror which is loaded on piezoelectric ceramics, and the first reflecting mirror is driven by the piezoelectric ceramics to reciprocate near the focus of the scanning objective lens so as to realize axial scanning of excitation light and detection light; the ratio of focal lengths between the main objective lens and the conjugate correction achromatic double-cemented lens group and the scanning objective lens is equal to the ratio of refractive indexes of media where the main objective lens and the scanning objective lens are located, namely, the perfect imaging law is satisfied.

When the axial scanning module is based on a transmission principle, the light path is matched with the physical position between the dichroic mirror and the main object mirror, and the light path is not required to be turned back, so that the illumination light is widely applicable to the light source, and the polarization light source is not required to be particularly used, and the axial scanning module is arranged between the dichroic mirror and the main object mirror. The axial scanning module adopting the transmission principle can drive the main objective lens to scan by using the motion mechanism, and can also be realized by using the turntable lens (embodiment 3).

And the axial scanning module is based on a movement mechanism, and when the movement mechanism drives the main objective lens, illumination light moves along the optical axis through a focus focused by the main objective lens, so that axial scanning is realized. An axial scanning module based on a turntable lens, wherein the axial scanning module comprises a scanning lens group and turntable lenses arranged between the scanning lens groups; the scanning lens of the scanning lens group is arranged in a confocal mode, and the turntable lens is arranged at a shared focal plane of the scanning lens group. The typical structure is as follows: the axial scanning module sequentially comprises a first reflecting mirror, a first scanning lens, a turntable lens with gradient thickness change, a second scanning lens and a second reflecting mirror along a detection light path; the turntable lens rotates to change the optical path, preferably rotates at a constant speed, so that the position of the focusing focus of the main object lens is changed, and axial scanning of excitation light and detection light is realized.

The following are examples:

embodiment one: mirror axial scanning system

As shown in fig. 1, the laser light source device includes a beam shaping module 1200, an excitation detection sharing module 1100, a detection module 1400, and a stage 1300.

Beam shaping module 1200: the laser 11 emits laser light, and the laser light is collimated by a collimator and then emitted in parallel. The cylindrical mirrors 12, 13 shape the light beam in the height dimension, reflect the light beam by the mirror 14, reach the slit 15, and adjust the thickness of the light sheet by adjusting the size of the slit. The beam is shaped in the thickness dimension by the cylindrical mirrors 16 and 17, and the height of the light sheet is shaped by the cylindrical mirror 18; the mirror 14 is moved in one dimension in the horizontal direction so that the illumination light sheet irradiates the edge position of the rear pupil of the main object lens, and the mirror position where the illumination light sheet irradiates the rear pupil of the main object lens is not limited to being placed in front of the axial scanning module, but can also be placed behind the axial scanning module.

Excitation detection common module 1100: the shaped gaussian light sheet reaches the half wave plate 110 after being reflected by the dichroic mirror 19, the polarization state of the light emitted from the half wave plate 110 is S-linear polarized light, the light is reflected by the polarization beam splitter 111 and then enters the scanning objective lens 113 through the quarter wave plate 112, the light enters the scanning objective lens 113 again after being reflected by the reflecting mirror 114, the polarization state of the light is P-polarized light after passing through the quarter wave plate 112 again, the light passes through the polarization beam splitter 111 and the conjugate correction achromatic double cemented lens groups 115 and 116, and then reaches the main objective lens 118 after being reflected by the reflecting mirror 117, and the light sheet is obliquely irradiated on a sample. Wherein, the reflecting mirror 114 is loaded on the piezoelectric ceramic, and the piezoelectric ceramic drives the reflecting mirror 114 to reciprocate near the focus of the scanning objective 113, so as to realize the axial scanning of the excitation light; the position of the reflecting mirror 14 is moved in one dimension in the horizontal direction, so that the illuminating light sheet irradiates the edge position of the rear pupil of the main objective lens 118, and the final light sheet is formed by the main objective lens 118. The parameters of the optical sheet are determined by the cylindrical lenses 12, 13, 16, 17, 18, the scanning objective lens 113, the achromatic double cemented lens groups 115, 116, the pupil position of the objective lens 118 where the light is incident, and the parameters of the objective lens 118, and the inclination angle of the optical sheet with respect to the optical axis of the objective lens 118 is approximately the acceptance angle of the objective lens 118.

The generated fluorescence passes through the illumination objective 118, is reflected by the reflecting mirror 117, reaches the polarization beam splitter 111 through the conjugate correction lens groups 116 and 115, and the polarization state of the polarized light passing through the polarization beam splitter 111 is P polarized light, then enters the scanning objective 113 through the quarter wave plate 112, enters the scanning objective 113 again after being reflected by the reflecting mirror 114, and enters the detection module through the half wave plate 110 and the dichroic mirror 19 after being reflected by the polarization beam splitter 111 after being polarized light in the polarization state of S polarized light again passing through the quarter wave plate 112. The piezoelectric ceramic drives the reflecting mirror 114 to reciprocate near the focus of the scanning objective lens 113, so as to realize the axial scanning of the probe light. Since the excitation light and the detection light share the mirror 114, synchronization of the scanning of the excitation light and the detection light can be achieved. The ratio of focal lengths between the main objective 118 and the conjugate correction achromatic double cemented lens group and the scanning objective 113 is equal to the ratio of refractive indexes of the medium where the main objective 118 and the scanning objective 113 are located, that is, the perfect imaging law is satisfied.

The axial scanning module is based on a reflection principle, the illumination light sheet adopts polarized light, and a polarization beam splitter is arranged on a composite light path of incident light and reflected light of the axial scanning module and used for separating the reflected light from the incident light and projecting the reflected light to the illumination objective lens. The axial scanning module comprises a reflecting mirror and a scanning objective lens, wherein the plane of the reflecting mirror is perpendicular to the main optical axis of the scanning objective lens, the optical path is changed by changing the distance between the reflecting mirror and the scanning objective lens, and the reflecting mirror is driven by a motion mechanism, such as a displacement table and piezoelectric ceramics. Specifically, the embodiment is as follows: the piezoelectric ceramic drives the reflecting mirror to reciprocate in a specified stroke, and the optical path is changed by changing the divergence and focusing degree of light, so that the axial scanning of the optical path is realized.

Detection module 1400: the axially scanned probe light passes through the tube lens 119 and the filter, and finally is projected on the camera 120. Wherein the tube lens 119 can be replaced according to the required magnification.

Stage 1300: the stage 1300 is composed of a three-axis electric displacement table, and a sample is placed on the stage, and three-dimensional high-speed imaging can be realized by electrically controlling the three-axis displacement table.

Embodiment two: vibrating mirror axial scanning system

As shown in fig. 2, includes a beam shaping module 2200, an excitation detection common module 2100, a detection module 2400, and a stage 2300.

Beam shaping module 2200: the laser 21 emits laser light, and the laser light is collimated by a collimator and then emitted in parallel. The cylindrical mirrors 22, 23 shape the light beam in the height dimension, reflect the light beam by the mirror 24, reach the slit 25, and adjust the thickness of the light sheet by adjusting the size of the slit. The beam is shaped in the thickness dimension by continuing to pass through cylindrical mirrors 26, 27 and the height of the light sheet is shaped by cylindrical mirror 28.

Excitation detection common module 2100: the shaped gaussian light sheet reaches the half wave plate 210 after being reflected by the dichroic mirror 29, the polarization state of the light emitted by the half wave plate 210 is S-linear polarized light, the light passes through the quarter wave plate 212 after being reflected by the polarization beam splitter 211, passes through the scanning lens 214 and the tube lens 215 after being reflected by the scanning galvanometer 213, enters the objective lens 216 in sequence, strikes on an inclined reflecting mirror 217, enters the objective lens 216 again after being reflected by the reflecting mirror 217, passes through the tube lens 215, the scanning lens 214 and the scanning galvanometer 213, passes through the quarter wave plate 212 again, the polarization state of the light is P-polarized light, passes through the polarization beam splitter 211 and the conjugate correction achromatic double-cemented lens groups 218 and 219, and then passes through the reflection of the reflecting mirror 220, and reaches the main objective lens 221, and the light sheet is obliquely irradiated on a sample. The scanning galvanometer 213 is conjugated with the main objective 221 and the rear pupil of the objective lens 216, and when the galvanometer scans the beam, the focal point formed by the objective lens 216 moves laterally. For light that is focused exactly at the mirror 217 surface, it will become parallel light at infinity, eventually focusing at the virtual focal plane of the main object lens 221; when the light is scanned laterally to other positions, it is not focused on the surface of the mirror 217, and it becomes a converging or diverging beam at infinity, and finally the focal point formed on the main mirror 221 is moved axially, thereby realizing axial scanning of the excitation light. The position of the reflecting mirror 24 is moved in one dimension in the horizontal direction so that the illumination light sheet irradiates the edge position of the rear pupil of the objective lens 221, and then the final light sheet is formed by the objective lens 221. The optical sheet parameters are determined by the parameters of the cylindrical lenses 22, 23, 26, 27, 28, the scanning lens 214, the tube lens 215, the objective lens 216, the achromatic double cemented lens groups 218, 219, the pupil position where the light is incident on the objective lens 221, and the inclination angle of the optical sheet with respect to the optical axis of the objective lens 221 is approximately the acceptance angle of the objective lens 221.

The generated fluorescence passes through the illumination objective 221, after being reflected by the reflecting mirror 220, reaches the polarization beam splitter 211 through the conjugate correction achromatic double cemented lens groups 219 and 218, the polarization state of the polarized light passing through the polarization beam splitter 211 is P polarized light, then passes through the quarter wave plate 212, and then sequentially passes through the scanning lens 214 and the tube lens 215 to enter the objective 216 after being reflected by the scanning galvanometer 213, strikes an inclined reflecting mirror 217, then enters the objective 216 again after being reflected by the reflecting mirror 217, passes through the tube lens 215, the scanning lens 214 and the scanning galvanometer 213, and then passes through the quarter wave plate 212 again, and the polarization state of the polarized light is S polarized light, and then passes through the half wave plate 210 and the dichroic mirror 29 after being reflected by the polarization beam splitter 211 to enter the detection module. Wherein the inverse scanning of the probe light Jing Zhen mirror ultimately achieves refocusing. Because the excitation light and the detection light share the scanning galvanometer, the synchronization of the axial scanning of the excitation light and the detection light can be realized.

The axial scanning module is based on a reflection principle, the illumination light sheet adopts polarized light, and a polarization beam splitter is arranged on a composite light path of incident light and reflected light of the axial scanning module and used for separating the reflected light from the incident light and projecting the reflected light to the illumination objective lens. The axial scanning module comprises a scanning galvanometer, a scanning lens, a tube lens, a scanning objective lens and a reflecting mirror, wherein the plane of the reflecting mirror is inclined at a certain angle with the main optical axis of the scanning objective lens, and the optical path is changed through the scanning galvanometer. Specifically, the embodiment is as follows: the vibrating mirror changes the divergence and focusing degree of light exiting the inclined reflecting mirror in the scanning process, and changes the optical path, so that the axial scanning of the optical path is realized.

Detection module 2400: the axially scanned probe light passes through the tube lens 222 and the filter, and finally is projected on the camera 223. Wherein the tube lens 222 may be replaced according to the required magnification.

Stage 2300: the objective table 2300 is composed of a three-axis electric displacement table, and a sample is placed on the objective table, and three-dimensional high-speed imaging can be realized by electrically controlling the three-axis displacement table.

Embodiment III: rotary disk axial scanning system

As shown in fig. 3, the laser light source device includes a beam shaping module 3200, an excitation detection common module 3100, a detection module 3400, and a stage 3300.

The beam shaping module 3200: the laser 31 emits laser light, and the laser light is collimated by a collimator and then emitted in parallel. The cylindrical mirrors 32, 33 shape the light beam in the height dimension, reflect the light beam by the mirror 34, reach the slit 35, and adjust the thickness of the light sheet by adjusting the size of the slit. The beam is shaped in the thickness dimension by continuing through the cylindrical lenses 36, 37 and the height of the light sheet is shaped by the cylindrical lens 38.

Excitation detection sharing module: the shaped gaussian light sheet is reflected by the dichroic mirror 39 and the reflecting mirror 310 to the axial scanning unit, including the achromatic double cemented lens groups 311 and 313 serving as scanning lens groups and the turntable lens 312 with a gradient change in thickness, and then reflected by the reflecting mirror 314 to the main objective lens 315, where the light sheet is obliquely irradiated on the sample. Wherein, the turntable with gradient change of thickness rotates at constant speed to change the optical path, thereby changing the position of the focusing focus of the main objective 315, and realizing the axial scanning of the excitation light; the illumination light sheet irradiates the edge position of the rear pupil of the objective lens 315 by moving the position of the reflecting mirror 34 in one dimension in the horizontal direction, and then the final light sheet is formed by the objective lens 315. The optical sheet parameters are determined by the cylindrical lenses 32, 33, 36, 37, 38, the achromatic doublet lens groups 311, 313, the pupil position of the objective lens 315 where the light is incident, and the parameters of the objective lens 315, and the inclination angle of the optical sheet with respect to the optical axis of the objective lens 315 is approximately the acceptance angle of the objective lens 315.

The generated fluorescence passes through the illumination objective 315, is reflected by the reflecting mirror 314, and then sequentially passes through the conjugate correction achromatic double cemented lens 313, the turntable lens 312 with gradient change in thickness, and the conjugate correction achromatic double cemented lens 311, is reflected by the reflecting mirror 310, and then enters the detection module through the dichroic mirror 39. The axial scanning of the detection light is realized through the turntable lens with the thickness gradient change. Because the excitation light and the detection light share the turntable lens, synchronization of the axial scanning of the excitation light and the detection light can be realized.

The axial scanning module is based on a transmission principle and comprises a turntable lens and an achromatic double-cemented lens group. Specifically, the embodiment is as follows: the turntable is placed at the common focal plane of two lenses of the 4f system, and the optical path is changed by changing the divergence and focusing degree of light, so that the axial scanning of the optical path is realized.

The detection module 3400: the axially scanned probe light passes through tube lens 316 and filters and is ultimately projected onto camera 317. Wherein the tube lens 316 may be replaced according to the required magnification.

Stage 3300: the stage 3300 is composed of a three-axis electric displacement table, and a sample is placed on the stage, and three-dimensional high-speed imaging can be realized by electrically controlling the three-axis displacement table.

The reciprocating frequency of the axial scanning module is consistent with the imaging frame rate during imaging, so as to prevent the sceneThe camera end adopts a shutter exposure mode (the mode can realize the line-by-line exposure of a camera photosensitive chip, only the exposed lines receive photons and the unexposed lines do not receive signals) which is built in the camera, and simultaneously opens the photosensitive line number and the objective lensO1Is matched with the depth of field range of (c). Therefore, when the axial scanning module is reset to the initial position, the row, which is matched with the depth of field of the objective lens O1, of the topmost layer of the camera in the field range is exposed; when the axial scanning module moves to the end position, the bottom layer of the camera in the field range is exposed by the row matched with the depth of field of the objective lens O1. After one period is finished, the piezoelectric ceramics in the axial scanning module are quickly reset, and the process is repeated to image the next frame by matching with the three-dimensional imaging displacement table, so that three-dimensional imaging is realized.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (10)

1. The single-objective light sheet fluorescence scanning imaging system is characterized by comprising a dichroic mirror, a main objective, a beam shaping module, an exposure camera and an axial scanning module;

the dichroic mirror and the main object mirror are simultaneously positioned on an excitation light path and a detection light path:

the illumination light sheet generated by the beam shaping module is projected on the edge of the rear pupil of the main objective lens through a dichroic mirror, so as to generate the illumination light sheet;

fluorescent light emitted by the sample excitation plane is collected by the main object lens and then projected on the roller shutter exposure camera to form images by the dichroic mirror;

the axial scanning module based on optical path adjustment is arranged between the dichroic mirror and the main object mirror on the optical path;

and the axial scanning module adjusts the optical path of the excitation optical path and the optical path of the detection optical path by the same period and the same displacement, so that the sample excitation plane is exposed on the exposure camera line by line along the axial direction of the light sheet for imaging.

2. The single objective light sheet fluorescence scanning imaging system of claim 1, wherein during imaging, the exposure camera completes one frame exposure within one scanning stroke of the axial scanning module;

when the axial scanning module has symmetrical positive and negative strokes, the exposure camera completes one-frame exposure in the positive and/or negative strokes; when the axial scanning module has asymmetrical positive and negative strokes, the axial scanning module can be used in a longer scanning stroke, so that the exposure camera can complete one-frame exposure.

The ratio of the reciprocating frequency of the axial scanning module to the imaging frame rate is 1:2 or 1:1.

3. The single objective light sheet fluorescence scanning imaging system of claim 1, wherein the number of lines simultaneously on-sensitive is less than the depth of field range of the primary objective.

4. The single objective light sheet fluorescence scanning imaging system of claim 1, wherein the axial scanning module is based on reflection principle, the illumination light sheet adopts polarized light, and a polarization beam splitter is arranged on a composite light path of the incident light and the reflected light of the axial scanning module, and is used for separating the reflected light from the incident light and projecting the reflected light to the illumination objective.

5. The single objective light sheet fluorescence scanning imaging system of claim 4, wherein the axial scanning module comprises a mirror and a scanning objective, the plane of the mirror being perpendicular to the primary optical axis of the scanning objective, the optical path being altered by changing the distance between the mirror and the scanning objective.

6. The single objective light sheet fluorescence scanning imaging system of claim 4, wherein the axial scanning module comprises a galvanometer, a scanning objective lens and a reflecting mirror, wherein the reflecting mirror is perpendicular to the incident light, and the rotation angle of the galvanometer is used for scanning the matched objective lens to form an optical path difference.

7. The single objective light sheet fluorescence scanning imaging system of claim 1, wherein the axial scanning module is based on transmission principle, the axial scanning module is disposed between the dichroic mirror and a main objective or mounted on the main objective; the axial scanning module is a turntable lens, an electrically adjustable lens, an adjustable acoustic gradient lens or an objective lens scanner.

8. The single objective light sheet fluorescence scanning imaging system of claim 7, wherein said axial scanning module comprises a scanning lens group and a turret lens disposed between said scanning lens groups; the scanning lens of the scanning lens group is arranged in a confocal mode, and the turntable lens is arranged at a shared focal plane of the scanning lens group.

9. The single objective light sheet fluorescence scanning imaging system of claim 1, wherein the beam shaping module comprises a light sheet position adjustment assembly, the light sheet position adjustment assembly being moved in one dimension in a horizontal direction such that the illuminating light sheet impinges on a back pupil edge position of the main objective; the light sheet position adjusting component is a reflecting mirror.

10. The single objective light sheet fluorescence scanning imaging system of claim 1, wherein the angle of inclination of the primary objective exit light sheet with respect to the primary objective optical axis is equal to the acceptance angle of the primary objective.