CN219810844U - Single-objective light sheet microscopic imaging system - Google Patents
Single-objective light sheet microscopic imaging system Download PDFInfo
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- CN219810844U CN219810844U CN202320842361.5U CN202320842361U CN219810844U CN 219810844 U CN219810844 U CN 219810844U CN 202320842361 U CN202320842361 U CN 202320842361U CN 219810844 U CN219810844 U CN 219810844U
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- 238000003384 imaging method Methods 0.000 title claims abstract description 50
- 238000005286 illumination Methods 0.000 claims abstract description 14
- 238000000386 microscopy Methods 0.000 claims 2
- 230000005284 excitation Effects 0.000 abstract description 15
- 230000003287 optical effect Effects 0.000 abstract description 15
- 239000000523 sample Substances 0.000 description 29
- 238000001514 detection method Methods 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000001917 fluorescence detection Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012788 optical film Substances 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
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- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The utility model discloses a single-objective light sheet microscopic imaging system, which comprises a laser lighting module, a dichroic mirror, an objective, a triaxial moving platform, a tube lens and an imaging module, wherein the laser lighting module comprises a collimating laser and a modulator; the dichroic mirror is arranged on an emergent light path of the laser illumination module; the triaxial moving platform for mounting the sample is arranged on an emergent light path of the objective lens, so that a light sheet irradiates the sample to excite the sample to generate fluorescence; the imaging module comprises an area array detector; the dichroic mirror is arranged between the objective lens and the tube lens, and the tube lens is arranged between the dichroic mirror and the imaging module, so that fluorescence sequentially passes through the objective lens, the dichroic mirror and the tube lens and then reaches the area array detector to be imaged. The utility model can continuously acquire the projection of the sample excitation surface on the plane orthogonal to the main optical axis of the objective lens through the continuous movement of the triaxial moving platform and the imaging module.
Description
Technical Field
The utility model belongs to the technical field of microscopic imaging, and particularly relates to a single-objective optical sheet microscopic imaging system.
Background
The traditional light sheet microscope is provided with an orthogonal light sheet illumination light path and a fluorescence detection light path which are respectively built by a detection objective lens and an illumination objective lens, the whole light path is complex, and the construction difficulty is high. The light sheet microscope is used for coupling the illumination light path and the detection light path into one path, and simultaneously illuminating and detecting a sample by adopting one objective lens, and is called a single objective lens light sheet microscope.
However, the single objective lens is adopted for simultaneous illumination and imaging, and due to oblique intersection of the illumination light path of the light sheet and the fluorescence detection light path, phase difference is caused on one hand, and on the other hand, the single objective lens is limited to be capable of selecting only the objective lens with high numerical aperture. In order to correct the phase difference and realize perfect imaging, three objective lenses are adopted to carry out remote focusing at present, and the system is complex and has low applicability. And because it is necessary to use an objective with a high numerical aperture, which generally corresponds to a higher magnification and smaller field of view, limits the field of view of the microscope.
Disclosure of Invention
In order to meet the above defects or improvement requirements of the prior art, the utility model provides a single-objective light sheet microscopic imaging system, which can continuously acquire the projection of a sample excitation surface on an orthogonal plane of a main optical axis of an objective through continuous movement of a triaxial moving platform and replace the traditional perfect imaging of the sample excitation surface, thereby eliminating two remote imaging objective lenses for correcting phase difference and eliminating the limitation of optical parameters of the objective lenses.
In order to achieve the above object, according to the present utility model, there is provided a single objective optical sheet microscopic imaging system, which is characterized by comprising a laser illumination module, a dichroic mirror, an objective lens, a triaxial moving platform, a tube lens and an imaging module, wherein:
the laser lighting module comprises a collimation laser and a modulator, wherein the modulator comprises a mask plate so as to modulate laser emitted by the collimation laser into a preset light sheet;
the dichroic mirror is arranged on an emergent light path of the laser illumination module so as to reflect the light sheet to the objective lens;
the triaxial moving platform for mounting the sample is arranged on an emergent light path of the objective lens, so that a light sheet irradiates the sample to excite the sample to generate fluorescence;
the imaging module comprises an area array detector;
the dichroic mirror is arranged between the objective lens and the tube lens, and the tube lens is arranged between the dichroic mirror and the imaging module, so that fluorescence sequentially passes through the objective lens, the dichroic mirror and the tube lens and then reaches the area array detector to be imaged.
Preferably, a tube lens assembly for collimating and correcting fluorescent signals collected by the objective lens is arranged between the dichroic mirror and the objective lens.
Preferably, the thickness of the optical sheet is 0.3 um-5 um.
Preferably, the imaging module further comprises a converging lens, and the area array detector is arranged on a focal plane of the converging lens.
Preferably, the lens further comprises a depth-of-field expanding assembly, wherein the depth-of-field expanding assembly is arranged between the dichroic mirror and the tube lens so as to expand the depth of field of the optical sheet along the axial direction of the objective lens.
Preferably, the depth of field expansion component is a phase modulation mask, a spatial light modulator, an axicon or a set of prisms.
Preferably, the three-axis moving platform has an X-axis moving platform, a Y-axis moving platform, and a Z-axis moving platform, and a moving direction of the Z-axis moving platform is parallel to an axial direction of the objective lens, wherein the X-axis, the Y-axis, and the Z-axis constitute a cartesian coordinate system.
In general, the above technical solutions conceived by the present utility model, compared with the prior art, enable the following beneficial effects to be obtained:
1) The utility model mainly uses the movement of the triaxial moving platform to collect projection information, uses the projection of the sample excitation surface on the orthogonal plane of the main optical axis of the objective lens, finally images on the imaging module, can obtain three-dimensional fluorescence data of the sample by three-dimensional reconstruction, can realize the illumination and fluorescence signal collection of the sample by only one objective lens, and can quickly and conveniently obtain the three-dimensional structure information of the biological sample by combining a three-dimensional reconstruction algorithm.
2) The single objective lens is adopted to realize three-dimensional fluorescence imaging of the light sheet, meanwhile, the traditional two remote imaging objective lenses used for correcting phase difference are omitted, the optical path is built without limitation of the magnification of the objective lenses, when the magnification switching is needed to be realized, the objective lens is directly replaced in situ, the in-situ magnification switching is conveniently realized, the imaging speed is not influenced, the presentation speed is equivalent to that of the existing light sheet fluorescence microscope and is far higher than that of a confocal microscope, the application field of the light sheet fluorescence microscope is greatly widened, and the application range of the light sheet fluorescence microscope is widened from scientific research imaging to commercial application of detection and image.
Drawings
Fig. 1 is a schematic view of the optical path of the present utility model.
Detailed Description
The present utility model will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present utility model 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 utility model. In addition, the technical features of the embodiments of the present utility model described below may be combined with each other as long as they do not collide with each other.
Referring to fig. 1, a single objective lens 1 optical film microscopic imaging system comprises a laser illumination module 4, a dichroic mirror 5, an objective lens 1, a triaxial moving platform 7, a tube lens 2 and an imaging module, wherein:
the laser lighting module 4 comprises a collimating laser and a modulator, wherein the modulator comprises a mask plate so as to modulate laser emitted by the collimating laser into a preset light sheet.
The dichroic mirror 5 is disposed on the outgoing light path of the laser lighting module 4 to reflect the light sheet to the objective lens 1, wherein an included angle between the dichroic mirror 5 and the outgoing light path of the laser lighting module 4 is greater than 0 and less than 90 °, i.e. not perpendicular to the outgoing light path, preferably 45 °.
The triaxial moving platform 7 for mounting the sample 8 is arranged on the emergent light path of the objective lens 1, so that a light sheet irradiates the sample 8 to excite the sample 8 to generate fluorescence.
The imaging module comprises an area array detector 3.
The dichroic mirror 5 is disposed between the objective lens 1 and the tube lens 2, and the tube lens 2 is disposed between the dichroic mirror 5 and the imaging module, so that fluorescence passes through the objective lens 1, the dichroic mirror 5, and the tube lens 2 in order to reach the area array detector 3 for imaging. The area array detector 3 obtains the projection of the excitation surface of the sample 8 on the orthogonal plane of the main optical axis of the objective lens 1 according to the time sequence of the control signal. The dichroic mirror reflects the laser with shorter wavelength, transmits the fluorescent light with longer wavelength, and finally the parallel light is converged on the photosensitive surface of the area array detector 3.
After passing through the objective lens 1, illumination laser enters the rear pupil surface of the objective lens 1, a light sheet with an inclination angle theta is formed and projected on the excitation surface of the sample 8, and the sample 8 is excited to generate fluorescence; the excitation surface of the sample 8 is obliquely crossed with the main optical axis of the objective lens 1; fluorescence emitted by the excitation surface of the sample 8 is collected by the objective lens 1, enters the dichroic mirror 5 and the tube lens 2 along the opposite direction of the illumination light path, and finally enters the imaging module for imaging, and the projection of the excitation surface of the sample 8 on the plane orthogonal to the main optical axis of the objective lens 1 is obtained on the imaging module.
The three-axis moving stage 7 has an X-axis moving stage, a Y-axis moving stage, and a Z-axis moving stage, and the moving direction of the Z-axis moving stage is parallel to the axial direction of the objective lens 1, wherein the X-axis, the Y-axis, and the Z-axis constitute a cartesian coordinate system. The Z-axis moving platform of the three-axis moving platform 7 drives the sample 8 to move along the axial direction of the objective lens 1, and the axial position of the sample can be changed stepwise or continuously.
Under the drive of the triaxial moving platform 7, the excitation surface of the sample 8 also moves, fluorescent signals sequentially pass through the objective lens 1, the dichroic mirror 5 and the tube lens 2, the projection of the excitation surface of the sample 8 on a plane orthogonal to the main optical axis of the objective lens 1 is obtained according to time sequence at the same position of the area array detector 3 of the imaging module, the imaging module transmits projection information of the excitation surface of the sample 8 to the microprocessor, and the microprocessor can obtain an actual three-dimensional fluorescent image of the sample 8 through reconstruction stacking. The position of the sample on the three-dimensional moving platform can be calibrated by an encoder, for example, and then the corresponding relationship between the position of the sample and the imaging on the area array detector 3 can be established.
The triaxial moving platform 7 is adjusted to move and match with the imaging speed of the camera, the excitation surface of the sample 8 corresponding to the adjacent projection is controlled, the phenomenon that sampling is missed due to too sparse sampling or fluorescence information crosstalk due to too dense sampling of the camera is avoided, and the imaging quality of the three-dimensional reconstructed image can be improved.
The system scans the excitation surface of the sample 8 through the movement of the triaxial moving platform 7, acquires projection data instead of perfect imaging, and directly forms a three-dimensional image, so that the requirement of perfect imaging on the excitation surface of the sample 8 is avoided, the second objective lens 1 and the third objective lens 1 for correcting phase difference are canceled, the setting restriction on NA value of the objective lens 1 is relieved, and in-situ zoom can be conveniently realized by replacing the objective lens 1. When the magnification is required to be switched, the objective lens 1 is only required to be switched into the objective lens 1 with other magnifications, other parts of the system do not need to be replaced and re-registered, and the light path does not need to be built again.
Further, a tube lens assembly (not shown) for collimating and correcting the fluorescent signal collected by the objective lens 1 is provided between the dichroic mirror 5 and the objective lens 1. The tube lens component and the position of the tube lens 2 are relatively fixed and are used for carrying out collimation correction on fluorescent signals collected by the objective lens 1, so that the objective lens 1 adapting to different multiplying powers is compatible, and the imaging quality of different objective lenses 1 is improved.
Further, the thickness of the light sheet is 0.3 um-5 um. Too thick a light sheet can result in severe projection crosstalk, too thin a light sheet can result in unfavorable three-dimensional imaging stacking, and the thinner the light sheet, the shorter the rayleigh distance, increasing the cost of axial scanning. In design, the relation between the Rayleigh distance of the light sheet and the depth of field of the objective lens 1 is considered.
Further, the imaging module further comprises a converging lens, and the area array detector 3 is arranged on the focal plane of the converging lens.
Further, a depth of field expansion assembly 6 is further included, and the depth of field expansion assembly 6 is arranged between the dichroic mirror 5 and the tube lens 2, so as to expand the depth of field of the optical sheet along the axial direction of the objective lens 1. The depth of field expansion component 6 may be a phase modulation mask, a spatial light modulator, axicon, or a prism group. The depth of field of the light sheet along the axial direction of the objective lens 1 is expanded through the depth of field expansion assembly 6, the field of view range of a single-frame image is expanded, and the camera obtains more depth information through one exposure, so that the displacement step length of the objective lens 1 in the axial direction is increased, and the axial scanning times are reduced. Preferably, the depth of field expansion component 6 is a phase modulation mask, a spatial light modulator, an axicon or a set of prisms.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the utility model and is not intended to limit the utility model, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the utility model are intended to be included within the scope of the utility model.
Claims (7)
1. The single-objective light sheet microscopic imaging system is characterized by comprising a laser illumination module, a dichroic mirror, an objective lens, a triaxial moving platform, a tube lens and an imaging module, wherein:
the laser lighting module comprises a collimation laser and a modulator, wherein the modulator comprises a mask plate so as to modulate laser emitted by the collimation laser into a preset light sheet;
the dichroic mirror is arranged on an emergent light path of the laser illumination module so as to reflect the light sheet to the objective lens;
the triaxial moving platform for mounting the sample is arranged on an emergent light path of the objective lens, so that a light sheet irradiates the sample to excite the sample to generate fluorescence;
the imaging module comprises an area array detector;
the dichroic mirror is arranged between the objective lens and the tube lens, and the tube lens is arranged between the dichroic mirror and the imaging module, so that fluorescence sequentially passes through the objective lens, the dichroic mirror and the tube lens and then reaches the area array detector to be imaged.
2. The single objective light sheet microscopic imaging system according to claim 1, wherein a tube lens assembly for collimating and correcting fluorescent signals collected by the objective is arranged between the dichroic mirror and the objective.
3. The single objective light sheet microimaging system of claim 1, wherein the light sheet has a thickness of 0.3um to 5um.
4. The single objective light sheet microscopy imaging system of claim 1, wherein the imaging module further comprises a converging lens, the area array detector being disposed at a focal plane of the converging lens.
5. The single objective light sheet microscopy imaging system of claim 1, further comprising a depth of field expansion assembly disposed between the dichroic mirror and the tube lens to expand the depth of field of the light sheet along the axial direction of the objective.
6. The single objective light sheet microimaging system of claim 5, wherein the depth expansion assembly is a phase modulation mask, a spatial light modulator, an axicon or a set of prisms.
7. The single objective light sheet microscopic imaging system of claim 1 wherein the three-axis moving stage has an X-axis moving stage, a Y-axis moving stage, and a Z-axis moving stage, and the moving direction of the Z-axis moving stage is parallel to the axial direction of the objective lens, wherein the X-axis, the Y-axis, and the Z-axis form a cartesian coordinate system.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202320842361.5U CN219810844U (en) | 2023-04-17 | 2023-04-17 | Single-objective light sheet microscopic imaging system |
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| CN202320842361.5U CN219810844U (en) | 2023-04-17 | 2023-04-17 | Single-objective light sheet microscopic imaging system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117554340A (en) * | 2023-10-13 | 2024-02-13 | 北京大学 | Laser-induced fluorescence detection system and detection method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117554340A (en) * | 2023-10-13 | 2024-02-13 | 北京大学 | Laser-induced fluorescence detection system and detection method |
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