CN114019667B - Double-arm illumination system, light sheet fluorescence microscope and use method thereof - Google Patents
Double-arm illumination system, light sheet fluorescence microscope and use method thereof Download PDFInfo
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- CN114019667B CN114019667B CN202111270994.5A CN202111270994A CN114019667B CN 114019667 B CN114019667 B CN 114019667B CN 202111270994 A CN202111270994 A CN 202111270994A CN 114019667 B CN114019667 B CN 114019667B
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
- G02B21/025—Objectives with variable magnification
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
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Abstract
The invention is suitable for the technical field of light sheet fluorescence microscopes, and provides a double-arm illumination system, which comprises a light source, a polarization beam splitter, a quarter wave plate and a zooming system capable of focusing along a first direction and a second direction, wherein the light source, the polarization beam splitter and the quarter wave plate are sequentially arranged; the double-arm illumination system further comprises a first illumination system for receiving the first light beam emitted by the spectroscope and for illuminating one side of the sample when the zoom system focuses along the first direction; the double-arm illumination system further comprises a second illumination system which receives the second light beam emitted by the spectroscope when the zoom system focuses along the second direction and is used for illuminating the other side of the sample; the first illumination system and the second illumination system are symmetrically arranged with respect to the beam splitter. The invention also provides a light sheet fluorescence microscope and a using method thereof.
Description
Technical Field
The invention belongs to the technical field of light sheet fluorescence microscopes, and particularly relates to a double-arm illumination system, a light sheet fluorescence microscope and a use method thereof.
Background
The light sheet fluorescence microscope is a novel optical microscopy technology, and the technology has the advantages of smaller photobleaching and phototoxicity of the system, higher axial resolution of imaging of the system, stronger contrast and wide application in the biomedical imaging field due to the unique illumination mode of the unique illumination.
According to the working principle of the light sheet microscope, the axial resolution of the light sheet microscope depends on the thickness of the light sheet, the thickness and focal depth of the light sheet are in a pair of balanced relation, and the NA value of an illumination beam is related, when the illumination system adopts a larger NA value to obtain the light sheet with smaller thickness, the focal depth of the light sheet is also reduced, and the imaging field of view of the system is reduced. In order to have a larger imaging field of view at higher resolutions, one combines zoom technology with a high NA light sheet microscope illumination system so that the system has a larger imaging field of view while having higher resolution.
In order to improve the illumination depth of the system, the light sheet microscope uses two illumination arms to illuminate two sides of the sample, and the imaging view field of the system is improved by improving the illumination depth, but when the illumination arms are introduced into the zoom system, the light path structure of the illumination arms becomes more complex, the zoom systems of the two illumination arms are synchronous with the camera at the same time, the control and operation of the system are also complex, and the light sheet microscope of the two arms has high manufacturing cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a double-arm illumination system, a light sheet fluorescent microscope and a using method thereof, which simplify the light path structure of the system, are simple to operate, improve the zooming speed and the imaging speed and have low manufacturing cost.
The technical scheme of the invention is that the double-arm illuminating system comprises a light source, a polarization beam splitter, a quarter wave plate, a zooming system, a 4f system, a galvanometer and a spectroscope, wherein the light source, the polarization beam splitter, the quarter wave plate and the zooming system are sequentially arranged and used for emitting light beams, the zooming system can focus along a first direction and a second direction, the 4f system is sequentially arranged and used for dividing the light beams reflected by the galvanometer into a first light beam and a second light beam, and the 4f system is arranged on one side of the polarization beam splitter; the double-arm illumination system further comprises a first illumination system for receiving the first light beam emitted by the spectroscope and for illuminating one side of the sample when the zoom system focuses along a first direction; the double-arm illumination system further comprises a second illumination system which receives the second light beam emitted by the spectroscope when the zoom system focuses along a second direction and is used for illuminating the other side of the sample; the first illumination system and the second illumination system are symmetrically disposed with respect to the beam splitter.
As a further improvement of the technical scheme, the 4f system comprises two first lenses and two second lenses which are coaxially arranged; the first lens is disposed coaxially with the polarizing beam splitter.
As a further improvement of the technical scheme, the first illumination system comprises a first illumination light path front lens, a first illumination light path reflecting mirror, a first illumination light path rear lens and a first illumination light path objective lens which are sequentially arranged;
The second illumination system comprises a second illumination light path front lens, a second illumination light path reflecting mirror, a second illumination light path rear lens and a second illumination light path objective lens; the first illumination light path objective lens and the second illumination light path objective lens are coaxially arranged.
As a further improvement of the present technical solution, the zoom system includes a zoom mirror movable in a first direction or a second direction and a zoom objective lens for guiding the light beam transmitted through the quarter wave plate to be reflected by the zoom mirror; the quarter wave plate is arranged on one side of the zoom objective lens, and the zoom reflecting mirror is arranged on the other side of the zoom objective lens.
As a further improvement of the present technical solution, the first lens and the second lens are confocal.
As a further improvement of the technical scheme, the front lens of the first illumination light path and the rear lens of the first illumination light path are confocal; and the second illumination light path front lens and the second illumination light path rear lens are confocal.
As a further improvement of the technical scheme, the polarization beam splitter is a polarization beam cube.
The invention also provides a light sheet fluorescence microscope, which comprises an imaging device, wherein the imaging device is vertically arranged in the sample placement direction; the light sheet fluorescence microscope adopts the double-arm illumination system and is used for illuminating a sample.
The invention also provides a use method of the light sheet fluorescence microscope, which adopts the light sheet fluorescence microscope and comprises the following steps:
the light source emits a light beam; adjusting the position of the zoom mirror;
Wherein adjusting the position of the zoom mirror comprises:
Adjusting the zoom reflector along a first direction, wherein a light beam enters the zoom system through the polarization beam splitter and the quarter wave plate; the light beam received by the zooming system returns to the polarization beam splitter in a primary path and penetrates through the polarization beam splitter to the 4f system; the galvanometer receives the light beam transmitted through the 4f system and then reaches the spectroscope; the first light beam emitted by the spectroscope is captured by the first illumination system, and the first light beam irradiates one side of the sample;
Or adjusting the zoom reflector along a second direction, wherein the light beam enters the zoom system through the polarization beam splitter and the quarter wave plate; the light beam received by the zooming system returns to the polarization beam splitter in a primary path and penetrates through the polarization beam splitter to the 4f system; the galvanometer receives the light beam transmitted through the 4f system and then reaches the spectroscope; the second light beam emitted by the spectroscope is captured by the second illumination system, and the second light beam irradiates the other side of the sample.
Compared with the double-arm illumination system in the prior art, the double-arm illumination system, the light sheet fluorescence microscope and the use method thereof provided by the invention have the advantages that the hardware used by one zoom system is reduced, and the overall cost of the double-arm illumination system is reduced; furthermore, the use of two illumination arms together using one zoom system simplifies the optical path structure, and when the optical path structure is determined, the zoom states of the two illumination systems are naturally synchronized, which makes the control system and the use operation simple. When using dual-arm illumination, the zoom distance of dual-arm illumination is half that of single-arm illumination while ensuring the same zoom distance, so that the dual-arm illumination system can achieve a faster zoom speed, and thus a faster imaging speed.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a light sheet fluorescence microscope provided by an embodiment of the present invention;
Fig. 2 is a diagram showing a relationship between a relative position between a zoom mirror and a zoom objective and a focal position of a light sheet in a dual-arm illumination system according to an embodiment of the present invention.
Reference numerals in the drawings:
1-a light source; a 2-polarizing beam splitter; a 3-quarter wave plate; 4-zooming objective lens; a 5-zoom mirror; a 6-4f system, 61-first lens, 62-second lens;
7-vibrating mirror; 8-spectroscope;
91-first illumination system, 911-first illumination light path front lens, 912-first illumination light path mirror, 913-first illumination light path rear lens, 914-first illumination light path objective;
92-a second illumination system, 921-a second illumination light path front lens, 922-a second illumination light path mirror, 923-a second illumination light path rear lens, 924-a second illumination light path objective;
10-imaging device.
Detailed Description
The present invention 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 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.
It should be noted that the terms "disposed" and "connected" should be construed broadly, and may be, for example, directly disposed or connected, or indirectly disposed or connected through a central element or a central structure.
In addition, in the embodiments of the present invention, terms of directions or positional relationships indicated by "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., are directions or positional relationships based on the directions or positional relationships shown in the drawings or the conventional placement state or use state, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the structures, features, devices or elements to be referred to must have specific directions or positional relationships nor must be constructed and operated in specific directions, and thus should not be construed as limiting the present invention. In the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The various features and embodiments described in the detailed description may be combined in any suitable manner, for example, different embodiments may be formed by different combinations of features/embodiments, where not contradictory, and various possible combinations of features/embodiments in the present invention are not described further in order to avoid unnecessary repetition.
As shown in fig. 1, the dual-arm illumination system provided by the embodiment of the invention comprises a light source 1, a polarization beam splitter 2, a quarter wave plate 3 and a zoom system, wherein the light source 1, the polarization beam splitter 2, the quarter wave plate 3 and the zoom system are sequentially arranged, the zoom system can focus along a first direction and a second direction, the dual-arm illumination system also comprises a 4f system 6, a galvanometer 7 and a beam splitter 8, wherein the beam splitter 8 is used for dividing a light beam reflected by the galvanometer 7 into a first light beam and a second light beam, and the 4f system 6 is arranged on one side of the polarization beam splitter 2; the dual-arm illumination system further comprises a first illumination system 91, wherein when the zoom system focuses along a first direction (direction a in the present embodiment), the first illumination system 91 receives the first light beam emitted by the beam splitter 8 and irradiates one side of the sample; the dual-arm illumination system further comprises a second illumination system 92, wherein the second illumination system 92 irradiates the other side of the sample when the zoom system receives the second light beam emitted from the beam splitter 8 during focusing along the second direction (direction B in the present embodiment); the first illumination system 91 and the second illumination system 92 are symmetrically arranged with respect to the beam splitter 8. Compared with the double-arm illumination system in the prior art, the double-arm illumination system provided by the embodiment of the invention needs two zoom systems, so that the hardware used by one zoom system is reduced, and the overall cost of the double-arm illumination system is reduced; furthermore, the use of two illumination arms together using one zoom system simplifies the optical path structure, and when the optical path structure is determined, the zoom states of the two illumination systems are naturally synchronized, which makes the control system and the use operation simple. When using dual-arm illumination, the zoom distance of dual-arm illumination is half that of single-arm illumination while ensuring the same zoom distance, so that the dual-arm illumination system can achieve a faster zoom speed, and thus a faster imaging speed.
Further, the 4f system 6 comprises two coaxially arranged first and second lenses 61, 62; the first lens 61 is disposed coaxially with the polarizing beam splitter 2. The first lens 61 and the second lens 62 are confocal. In a specific application, the first lens 61, the second lens 62 and the polarizing beam splitter 2 are all coaxially arranged, so that the installation of the whole structure is simpler.
Further, the first illumination system 91 includes a first illumination light path front lens 911, a first illumination light path reflecting mirror 912, a first illumination light path rear lens 913, and a first illumination light path objective lens 914, which are sequentially arranged;
The second illumination system 92 includes a second illumination light path front lens 921, a second illumination light path mirror, a second illumination light path rear lens 923, and a second illumination light path objective 924; the first illumination light path objective 914 and the second illumination light path objective 924 are coaxially disposed. As an alternative embodiment, the included angle between the first light beam and the second light beam split by the beam splitter 8 is 90 degrees, the distance between the first illumination light path front lens 911 and the beam splitter 8, the distance between the second illumination light path front lens 921 and the beam splitter 8 are equal, and the distances between the first illumination light path reflecting mirror 912 and the first illumination light path front lens 911, and the distances between the second illumination light path reflecting mirror and the second illumination light path front lens 921 are equal; the first light beam reflected by the first illumination light path mirror 912 is respectively incident from one side of the sample through the first illumination light path rear lens 913 and the first illumination light path objective 914, the second light beam reflected by the second illumination light path mirror is respectively incident from the other side of the sample through the second illumination light path rear lens 923 and the second illumination light path objective 924, and therefore, the two sides of the sample are respectively incident by the first light beam and the second light beam, the zooming distance is half of that of single-arm illumination, the zooming speed and the imaging speed are doubled, the operation is easy, and the illumination is easy to penetrate the sample.
Further, the zoom system comprises a zoom mirror 5 and a zoom objective 4, the zoom mirror 5 being movable in a first direction or a second direction; the light beam transmitted through the quarter wave plate 3 passes through the zoom objective 4 and then is guided to the zoom reflector 5 for reflection; the quarter wave plate 3 is arranged on one side of the zoom objective 4, and the zoom mirror 5 is arranged on the other side of the zoom objective 4. The polarizing beam splitter 2 is a polarizing beam cube.
In specific application, one side of the polarization beam splitter 2 in the horizontal direction is a light source 1, and the opposite side is provided with the quarter wave plate 3 and the zooming system in sequence; the 4f system 6 is arranged in the longitudinal direction of the polarization beam splitter 2, and the polarization beam splitter is simple in overall structure and convenient to install and maintain. As shown in fig. 2, when the relative position between the zoom mirror 5 and the zoom objective 4 in the dual-arm illumination system is changed, the focal position of the light sheet is also changed. When the zoom mirror 5 moves close to the zoom objective 4 (i.e. the zoom mirror 5 moves along the direction a and is respectively at the position a, the position b and the position c), the focusing area (the area M in fig. 2) of the optical sheet moves away from the zoom objective 4, when the imaging device 10 performs exposure imaging from left to right, the imaging device 10 continuously performs signal acquisition in the focusing area of the optical sheet while ensuring that the exposure speed of the imaging device 10 and the displacement speed of the zoom objective 4 are synchronized, and the system maintains higher resolution; when the zoom mirror 5 moves to the extreme position (position c) close to the zoom objective 4, the zoom mirror 5 moves in the opposite direction and moves away from the zoom objective 4 (i.e., moves in the direction B), and at this time, the focal position of the light sheet (region N in fig. 2) moves correspondingly to the direction close to the zoom objective 4, and the imaging of the left imaging region of the imaging device 10 is completed, the focal region of the light sheet illuminated on the right side just matches the focal region of the light spot on the left side, and at this time, the exposure region of the imaging device 10 is the focal region of the light sheet illuminated on the right side, and the system still maintains good axial resolution. While ensuring high axial resolution of the entire field of view, the zoom distance is only half that of a comparable variable-focus single-arm illumination system, where the zoom distance and zoom speed are a pair of balanced relationships, when the zoom distance is reduced, the zoom system has a faster zoom speed, and typically the imaging speed of the imaging device 10 is related to the zoom speed of the system, thus making the imaging speed of the imaging system faster.
Further, the first illumination light path front lens 911 and the first illumination light path rear lens 913 are confocal; the second illumination light path front lens 921 and the second illumination light path rear lens 923 are confocal.
The invention also provides a light sheet fluorescence microscope, which comprises an imaging device 10, wherein the imaging device 10 is vertically arranged in the sample placement direction; the light sheet fluorescence microscope adopts the double-arm illumination system and is used for illuminating a sample.
The embodiment of the invention also provides a use method of the light sheet fluorescence microscope, which adopts the light sheet fluorescence microscope and comprises the following steps:
the light source 1 emits a light beam; adjusting the position of the zoom mirror 5;
Wherein adjusting the position of the zoom mirror 5 comprises:
Adjusting the zoom reflecting mirror 5 along a first direction, and enabling light beams to enter the zoom system through the polarization beam splitter 2 and the quarter wave plate 3; the light beam received by the zoom system returns to the polarization beam splitter 2 in a primary path and passes through the polarization beam splitter 2 to the 4f system 6; the galvanometer 7 receives the light beam transmitted through the 4f system 6 and reaches the spectroscope 8; the first light beam emitted by the beam splitter 8 is captured by the first illumination system 91, and the first light beam irradiates one side of the sample;
or the zoom reflector 5 is adjusted along the second direction, and the light beam enters the zoom system through the polarization beam splitter 2 and the quarter wave plate 3; the light beam received by the zoom system returns to the polarization beam splitter 2 in a primary path and passes through the polarization beam splitter 2 to the 4f system 6; the galvanometer 7 receives the light beam transmitted through the 4f system 6 and reaches the spectroscope 8; the second light beam emitted from the beam splitter 8 is captured by the second illumination system 92, and the second light beam irradiates the other side of the sample.
The double-arm illumination system, the light sheet fluorescence microscope and the use method thereof provided by the embodiment of the invention not only simplify the light path structure of the double-arm illumination system, reduce the hardware used by one zoom system, and reduce the manufacturing cost of the system; furthermore, the two illumination arms are employed to commonly use one zoom system, and when the optical path structure is determined, the zoom states of the two illumination systems are naturally synchronized, which makes the control system and the use operation simple as well. When using dual-arm illumination, the zoom distance of dual-arm illumination is half that of single-arm illumination while ensuring the same zoom distance, so that the dual-arm illumination system can achieve a faster zoom speed, and thus a faster imaging speed.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (8)
1. The double-arm illumination system comprises a light source, a polarization beam splitter, a quarter wave plate and a zooming system capable of focusing along a first direction and a second direction, which are sequentially arranged, and is characterized by further comprising a 4f system, a galvanometer and a spectroscope, wherein the spectroscope is used for dividing a light beam reflected by the galvanometer into a first light beam and a second light beam, and the 4f system is arranged on one side of the polarization beam splitter; the double-arm illumination system further comprises a first illumination system for receiving the first light beam emitted by the spectroscope and for illuminating one side of the sample when the zoom system focuses along a first direction; the double-arm illumination system further comprises a second illumination system which receives the second light beam emitted by the spectroscope when the zoom system focuses along a second direction and is used for illuminating the other side of the sample; the first illumination system and the second illumination system are symmetrically arranged relative to the spectroscope; the zoom system comprises a zoom reflector capable of moving along a first direction or a second direction and a zoom objective lens for guiding the light beam transmitted through the quarter wave plate to the zoom reflector for reflection; the quarter wave plate is arranged on one side of the zoom objective lens, and the zoom reflecting mirror is arranged on the other side of the zoom objective lens.
2. The dual-arm illumination system of claim 1, wherein the 4f system comprises two coaxially disposed first and second lenses; the first lens is disposed coaxially with the polarizing beam splitter.
3. The dual-arm illumination system of claim 2, wherein the first illumination system comprises a first illumination light path front lens, a first illumination light path mirror, a first illumination light path rear lens, and a first illumination light path objective lens, which are sequentially disposed;
the second illumination system comprises a second illumination light path front lens, a second illumination light path reflecting mirror, a second illumination light path rear lens and a second illumination light path objective lens; the first illumination light path objective lens and the second illumination light path objective lens are coaxially arranged.
4. The dual-arm illumination system of claim 3, wherein said first lens and said second lens are confocal.
5. The dual-arm illumination system of claim 4, wherein said first illumination path front lens and said first illumination path rear lens are confocal; the second illumination light path front lens and the second illumination light path rear lens are confocal.
6. The dual-arm illumination system of any one of claims 1 to 5, wherein the polarizing beam splitter is a polarizing beam cube.
7. The light sheet fluorescence microscope comprises an imaging device, and is characterized in that the imaging device is vertically arranged in the sample placement direction; the light sheet fluorescence microscope employs the dual-arm illumination system as claimed in any one of claims 1 to 6 and is used for illumination of a sample.
8. A method of using the light sheet fluorescence microscope of claim 7, comprising the steps of:
the light source emits a light beam; adjusting the position of the zoom mirror;
Wherein adjusting the position of the zoom mirror comprises:
Adjusting the zoom reflector along a first direction, wherein a light beam enters the zoom system through the polarization beam splitter and the quarter wave plate; the light beam received by the zooming system returns to the polarization beam splitter in a primary path and penetrates through the polarization beam splitter to the 4f system; the galvanometer receives the light beam transmitted through the 4f system and then reaches the spectroscope; the first light beam emitted by the spectroscope is captured by the first illumination system, and the first light beam irradiates one side of the sample;
Or adjusting the zoom reflector along a second direction, wherein the light beam enters the zoom system through the polarization beam splitter and the quarter wave plate; the light beam received by the zooming system returns to the polarization beam splitter in a primary path and penetrates through the polarization beam splitter to the 4f system; the galvanometer receives the light beam transmitted through the 4f system and then reaches the spectroscope; the second light beam emitted by the spectroscope is captured by the second illumination system, and the second light beam irradiates the other side of the sample.
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