CN111879740A - All-optical super-resolution microscopic device based on photon reset technology - Google Patents
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Abstract
The invention provides an all-optical super-resolution microscopic device based on a photon reset technology, which comprises: the device comprises an illumination system, a scanning system and a fluorescence excitation collecting system, wherein the fluorescence excitation collecting system is used for exciting a fluorescence signal on a sample; the fluorescence signal reflected by the sample returns through the same optical path, and the fluorescence beam emergent from the scanning galvanometer and the beam incident into the scanning galvanometer by the scanning system are on the same straight line; the emergent light beam after the scanning system is removed expands, and is guided to the scanning galvanometer again to realize photon reset; and the imaging system receives the light beams after the rescanning treatment and respectively images different scanning positions of the sample. Because the real position of a transmitted photon is half of the distance between the excitation focus and the detection point, the system is provided with twice beam expansion in the de-scanning and re-scanning light paths, and each detected photon is reset to the corresponding position by an optical method, so that an image with higher detection efficiency and resolution is obtained.
Description
Technical Field
An all-optical super-resolution microscopic device based on a photon reset technology belongs to the field of optical microscopic measurement, and particularly relates to a microscopic imaging device which utilizes a photon redistribution principle and realizes high resolution by means of all-optical.
Background
Laser scanning confocal microscopy (CLSM) has been widely used in the biomedical detection field due to its optical sectioning capability and high imaging contrast. As a well-studied fluorescence microscope, the excellent imaging properties of CLSM are achieved by using a detector with a high dynamic range and setting a confocal detection pinhole. In common CLSM, the detected intensity value for each scan point is recorded by an integrating detector, such as a photomultiplier tube (PMT), avalanche photodiode, etc.; the pinhole is coaxially conjugated to the excitation focus position and the corresponding image is constructed by assigning the detected illumination intensity to the corresponding excitation scan position. Later, there have been studies showing that the use of off-axis pinholes can further improve resolution. Because the area array detector has a plurality of off-axis pixel points, and research shows that the actual position of the fluorescence emission light source on the sample is 1/2 between the focal point of laser and the detection point deviating from the optical axis, when the final image is synthesized, the pinhole plane image shot at each excitation scanning position and the position information thereof are combined. The resolution of the traditional microscope can be improved by means of the area array detector and the photon reset algorithm.
Following the above principle, some researchers have proposed an image scanning microscope, that is, replacing a photomultiplier tube (PMT) as a detector in a laser scanning confocal microscope with a CCD (charge coupled device) area array detector, based on the photon resetting principle, in subsequent computer image processing, shifting an image collected by off-axis pixel points of the detector, that is, shifting a detected image to a position where the distance between the focal point of laser and a detection point deviated from the optical axis is half, redistributing photons detected by each pixel point on the area array detector to an accurate position, and superimposing all shifted images to obtain an image with higher detection efficiency and resolution, which can be up to twice the original resolution. Besides the improvement of resolution, the equipment of the image scanning microscope is simple, is easy to operate and can be better compatible with the existing laser scanning confocal microscope.
Because in the original image scanning microscope, the image of each pixel point needs to be collected, and in addition, each collected image needs corresponding shift processing, so that the imaging speed of the image scanning microscope is very slow, experiments show that tens of minutes are consumed for imaging micron-sized small images, the applicability of the image scanning microscope is greatly limited by later-stage image reconstruction, and if the imaging speed of the image scanning microscope can be improved, the application of the image scanning microscope in the fields of biomedicine and the like can be greatly improved.
Disclosure of Invention
The invention solves the problem of how to finish the photon reset step while imaging by an image scanning microscope by means of optics, thereby saving the subsequent image processing time, greatly improving the imaging speed on one hand, eliminating the problem of inaccurate photon reset caused by artificial factors in a subsequent processing algorithm from the source on the other hand, and simultaneously still maintaining the advantage of improving the double resolution.
In order to solve the above problems, the present invention provides an all-optical super-resolution microscopy device based on photon reset technology, comprising an illumination system, a scanning system, a fluorescence excitation collection system, a descanning system, a rescanning system and an imaging system, wherein:
an illumination system for generating a multi-focus illumination beam;
furthermore, in this technical scheme, the illumination system includes the laser instrument and is located lens group, the microlens array on its emergent light path, and the laser instrument is as the light source for generate laser, and the lens group is used for expanding the laser beam, adjusts the light beam size, and the microlens array is used for receiving laser to generate a plurality of parallel illuminating beam under the spotlight effect of a plurality of microlenses, when follow-up scanning the sample, can promote the efficiency of scanning, promote micro-imaging device's imaging speed.
A scanning system: receiving a multi-focus illumination beam generated by an illumination system, and realizing the scanning of the sample through the deflection of a scanning galvanometer;
further, the scanning system is located after the illumination system, i.e., in the output optical path of the multi-focus illumination beam, and before the fluorescence excitation collection system.
Further, in the technical scheme, the scanning system includes a beam splitter, a scanning galvanometer and a lens group, the scanning galvanometer is used for receiving the multi-focus laser beam generated by the illumination system, the lens group is used for adjusting the size of the laser beam, the reflector is used for changing the direction of the light path, and the multi-focus laser beam sequentially scans the sample plane under the deflection action of the scanning galvanometer.
Fluorescence excitation collection system: scanning the sample by the multi-focus illumination beam transmitted by the scanning system to excite a fluorescence signal on the sample;
further, the fluorescence excitation collection system is located after the scanning system.
Further, in the technical scheme, the fluorescence excitation collection system includes a reflector, an optical filter, an objective lens, and a sample, where the multi-focus illumination laser beam is guided to the sample through the objective lens to excite fluorescence on the surface of the sample, the fluorescence signal is collected by the same objective lens after being reflected, the fluorescence collected by the objective lens exits from the objective lens and is guided to the scanning system after passing through the optical filter, where the optical filter is used to filter out stray light.
A descanning system: the fluorescence signal reflected by the sample returns through the same optical path and is scanned by the scanning galvanometer, so that the position of the light beam incident to the scanning galvanometer from the illumination system is consistent with the position of the light beam emitted by the fluorescence excitation system through the scanning galvanometer;
further, in this technical solution, the descan system is located behind the fluorescence excitation collection system and on an emergent light path of the fluorescence excitation collection system. The descan system and the scanning system comprise the same optical elements, and the traveling directions of the optical paths are opposite.
Furthermore, the system guides the fluorescence emitted by the fluorescence excitation collection system to the scanning galvanometer, and the scanning galvanometer has the same deflection angle when the light beam enters the scanning galvanometer and the fluorescence entrance scanning galvanometer, so that the emergent direction of the fluorescence light beam is the same as the direction of the laser entrance scanning galvanometer, namely after the scanning system is removed, the deflection of the scanning galvanometer does not influence the propagation direction of the light beam, and the scanning removal effect is realized.
Furthermore, an emergent light beam of the scanning removing system and a light beam incident on the scanning galvanometer are always kept on the same straight line, so that the deviation of the position of the light beam caused by the deflection of the scanning galvanometer is eliminated, and the imaging quality is influenced.
In the technical scheme, the scanning of the multifocal illuminating beam to different positions of a sample plane is realized through the deflection motion of the scanning galvanometer, and when a fluorescence signal excited by the sample scanning returns to the scanning galvanometer through the same path, because the scanning galvanometer still keeps the same deflection angle within a short time, the laser incident on the scanning galvanometer is the same as the position of the fluorescence emitted from the scanning galvanometer subsequently, so that the scanning deflection effect is realized, and the imaging effect is ensured.
Rescanning the system: the outgoing light beam processed by the descanning system is expanded twice and is guided to the scanning galvanometer again under the reflection action of the reflector, so that photon resetting is realized;
further, in this technical solution, the rescan system is located behind the descan system, and the rescan system includes a lens group and a mirror.
Further, the focal length ratio of the lens group is 1: 2;
furthermore, the light beam after being descanned exits from the scanning galvanometer and is guided to the reflecting mirror, and a pair of lens groups is arranged in the reflected light path to perform double beam expansion on the light beam. Due to the enlargement of the beam size, the size of the corresponding point spread function is reduced by half, namely the distance between the imaging points in the whole imaging plane is reduced to 1/2, and the requirement of shifting the detection image to the position 1/2 between the focal point of the laser and the detection point deviated from the optical axis in the photon resetting principle is met.
Furthermore, the optical realization of photon reset is realized by expanding the light path between the scanning removing system and the re-scanning system, namely, the operation of shifting the detected image is completed in the imaging process, the content of subsequent computer image processing in the traditional image scanning microscope is eliminated, and the imaging time is greatly reduced.
An imaging system: receiving the light beams after the rescanning treatment, and respectively imaging different scanning positions of the sample;
furthermore, in the technical scheme, the imaging system comprises a reflector, an emission filter, an imaging lens and an area array detector, wherein the reflector is used for changing the direction of a light path, the emission filter is used for filtering stray light except for fluorescence, and the imaging lens is used for focusing light beams on the area array detector for imaging.
In the technical scheme, a laser of the illumination system emits laser, the light beam is shaped by a first lens group and then is guided to a micro lens array to generate a plurality of parallel illumination light beams, the light beams are then directed to a beam splitter and then are deflected by the beam splitter and then are directed to a scanning galvanometer, an emergent light beam after the scanning galvanometer is expanded by a second lens group and is reflected by a first reflector, and the light beam is guided to an objective lens and scans a sample. The sample receives laser scanning to produce fluorescence, and this fluorescence is gathered by same objective to return along incident path after first light filter filtering stray light, realize going the scanning behind first speculum, second lens group, the scanning galvanometer in proper order, the light beam after going the scanning separates fluorescence and exciting light in beam splitter department, and the fluorescence after the separation is 1 through the focus ratio behind the second speculum: the lens group of 2 realizes twice beam expansion, the expanded light beam is guided to the same scanning system after being reflected by the third reflector to finish rescanning, the fourth reflector is utilized to change the propagation direction of a light path, stray light is filtered by the second optical filter, and finally the stray light is projected to the area array detector through the imaging lens to be imaged.
Drawings
FIG. 1 is a schematic block diagram of an all-optical super-resolution microscopy apparatus based on photon reset technology in an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of an all-optical super-resolution microscopy device based on photon reset technology in an embodiment of the present invention
FIG. 3 is a schematic diagram of photon reset principle of an all-optical super-resolution microscopy device based on photon reset technology in an embodiment of the present invention
Description of the reference numerals
1-a lighting system; 101-a laser; 102-a first lens group; 103-a microlens array; 2-a scanning system; 201-a beam splitter; 202-scanning galvanometer; 203-a second lens group; 3-a fluorescence excitation collection system; 301-a first mirror; 302-a first filter; 303-objective lens; 304-the sample; 4-go scanning system; 5-a rescan system; 501-a second mirror; 502-third lens group; 503-a third mirror; 6-an imaging system; 601-a fourth mirror; 602-a second filter; 603-an imaging lens; 604-area array detector;
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
It is noted that the terms first, second and the like in the description and in the claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein.
Referring to fig. 1 and 2, the present invention provides an all-optical super-resolution microscopy device based on photon reset technology, comprising:
an illumination system 1 for generating a multi-focus illumination beam;
the scanning system 2 receives the multi-focus illumination light beam generated by the illumination system and realizes the scanning of the sample through the deflection of the scanning galvanometer;
the fluorescence excitation collection system 3 scans the sample through the multifocal illuminating beam transmitted by the scanning system to excite the fluorescence signal on the sample;
the fluorescence signal reflected by the sample returns through the same optical path and is scanned by the scanning galvanometer, so that the positions of the light beam incident to the scanning galvanometer from the illumination system and the light beam emergent from the fluorescence excitation system through the scanning galvanometer are kept consistent;
the rescanning system 5 is used for expanding the outgoing beam processed by the descanning system twice and guiding the outgoing beam to the scanning galvanometer again under the reflection action of the reflector so as to realize photon resetting;
the imaging system 6 receives the light beams after the rescanning treatment and respectively images different scanning positions of the sample;
referring to fig. 2, the illumination system 1 in the present embodiment includes a laser 101, and a lens group 102 and a microlens array 103 located on an exit optical path thereof. The laser 101 is used as a light source and is used for generating laser, the lens group 102 is used for expanding laser beams and adjusting beam size, the micro lens array 103 is used for receiving the laser and generating a plurality of parallel illuminating beams under the condensation effect of a plurality of micro lenses, and when a sample 304 is scanned subsequently, the scanning efficiency can be improved, and the imaging speed of the micro imaging device is improved.
Referring to fig. 2, the scanning system 2 in this embodiment is located behind the illumination system 1, and includes a beam splitter 201, a scanning galvanometer 202, and a lens group 203, where the scanning galvanometer 202 is configured to receive a multi-focus laser beam generated by the illumination system 1, the lens group 203 is configured to adjust a beam size, and the multi-focus laser beam sequentially scans a plane of the sample 304 under a deflection action of the scanning galvanometer 202.
Referring to fig. 2, the fluorescence excitation collection system 3 in this embodiment includes a reflector 301, a filter 302, an objective 303, and a sample 304, where the multi-focus illumination laser beam is guided to the sample 304 through the objective 303 to excite fluorescence on the surface of the sample 304, the fluorescence signal is collected by the same objective 303 after being reflected, the fluorescence collected by the objective 303 exits, and is guided to the descan system 4 through the filter 302, where the filter 302 is used to filter out stray light.
Referring to fig. 2, the descan system 4 in this embodiment includes the same optical components as the scanning system 2, and the optical paths travel in opposite directions. The system guides the fluorescence emitted by the fluorescence excitation collection system 3 to the scanning galvanometer 202, and because the scanning galvanometer 202 has the same deflection angle when the light beam enters the scanning galvanometer 202 and the fluorescence enters the scanning galvanometer 202, the emergent direction of the fluorescence light beam is the same as the direction of the laser entering the scanning galvanometer 202, namely after the scanning system is removed, the deflection of the scanning galvanometer 202 does not influence the propagation direction of the light beam, and the scanning removal effect is realized.
Referring to fig. 2, the re-scanning system 5 in the present embodiment includes a mirror 501, a lens group 502, a mirror 503, and a scanning galvanometer 202. The light beam after being descanned exits from the scanning galvanometer 202 and is guided to a reflecting mirror 501, and a pair of lens groups 502 is arranged in the reflected light path to perform double beam expansion on the light beam. Due to the enlargement of the beam size, the size of the corresponding point spread function is reduced by half, namely the space between the imaging points in the whole imaging plane is reduced to 1/2, the requirement that the detection image is shifted to the position 1/2 between the focal point of the laser and the detection point deviated from the optical axis in the photon reset principle is met, and the photon reset is realized.
Referring to fig. 3, in the image scanning microscope, an area array detector is used for imaging, and in order to improve the resolution of the confocal laser scanning microscope, the image is shifted using different position information of each excitation scanning point, and more specifically, a true position of an emitted photon is a half of the distance between an excitation focal point and a detection point. If each detected photon is reset to its corresponding position, an image with higher detection efficiency and resolution can be obtained. In fig. 3, the upper diagram shows the relative positions of the emission beam of the sample plane and the excitation beam, and the lower diagram shows the relative positions of the emission beam of the image plane and the excitation beam. p denotes the position of the excitation beam and s denotes the actual position of the emission beam corresponding to the image plane. By de-scanning and subsequent re-scanning, the beam is expanded twice, the size of the point spread function is reduced by half and the corresponding position of the two beams is reduced by half, i.e., in the figure, s-p-1/2, thereby completing the content of photon redistribution by optical means during the imaging process.
Referring to fig. 2, the imaging system 6 in this embodiment includes a reflector 601, an emission filter 602, an imaging lens 603, and an area array detector 604, where the reflector 601 changes a light path direction to the emission filter 602, and after filtering stray light except for fluorescence, focuses a light beam onto the area array detector 604 through the imaging lens 603 for imaging.
Referring to fig. 1 and 2, in the present technical solution, a laser 101 in an illumination system 1 emits laser light, the light beam is shaped by a lens group 102 and then directed to a micro lens array 103 to generate a plurality of parallel illumination light beams, the light beams are then directed to a beam splitter 201, deflected by the beam splitter 201 and directed to a scanning galvanometer 202, an emergent light beam after passing through the scanning galvanometer 202 is expanded by a lens group 203, reflected by a mirror 301, and directed to an objective lens 303 under the filtering action of a filter 302 and scans a sample 304. The sample 304 is scanned by the laser to generate fluorescence, the fluorescence is collected by the same objective 303, returns along an incident path after being filtered by the filter 302 to remove stray light, sequentially passes through the reflector 301, the lens group 203 and the scanning galvanometer 202 to realize descanning, the descanned light beam separates the fluorescence from the excitation light at the beam splitter 201, and the separated fluorescence passes through the reflector 501 and then passes through a focal length ratio of 1: the lens group 502 of 2 realizes double beam expansion, the expanded light beam is guided to the same scanning system 2 after being reflected by the reflector 503 to complete rescanning, the reflector 601 is used for changing the propagation direction of the light path, stray light is filtered by the optical filter 602, and finally the stray light is projected to the area array detector 604 through the imaging lens 603 to be imaged.
Claims (8)
1. An all-optical super-resolution microscopic device based on photon reset technology is characterized by comprising:
an illumination system (1) for generating a multi-focus illumination beam;
the scanning system (2) receives the multi-focus illumination light beam generated by the illumination system and realizes the scanning of the sample through the deflection of the scanning galvanometer;
a fluorescence excitation collection system (3) for scanning the sample by means of a multi-focus illumination beam delivered by said scanning system to excite a fluorescence signal on the sample;
the fluorescence signal reflected by the sample returns through the same optical path and is scanned by the scanning galvanometer, so that the position of a light beam incident to the scanning galvanometer from the illumination system is consistent with the position of a light beam emitted by the fluorescence excitation system through the scanning galvanometer;
the re-scanning system (5) expands the emitted light beams processed by the de-scanning system, guides the expanded light beams to the scanning galvanometer again under the reflection action of the reflector, changes the size of the light spots, and equivalently changes the relative distance between the detected light spots, so that photons are repositioned at the position with the optimal imaging resolution to realize photon resetting;
and the imaging system (6) receives the light beams after the rescanning treatment and respectively images different scanning positions of the sample.
2. The all-optical super-resolution microscopy device based on the photon reset technology is characterized in that the illumination system (1) generates a plurality of parallel illumination light beams, the light beams are then directed to the beam splitter, deflected by the beam splitter and directed to the scanning galvanometer, an emergent light beam after passing through the scanning galvanometer is expanded through the second lens group, reflected by the first reflector, directed to the objective lens and scanned on a sample; the sample is scanned by laser to generate fluorescence, the fluorescence is collected by the same objective lens, the fluorescence returns along an incident path after stray light is filtered by a first optical filter, the fluorescence is removed by sequentially passing through a first reflector, a second lens group and a scanning galvanometer, the fluorescence and exciting light are separated by a light beam after the fluorescence is removed, the separated fluorescence is expanded by passing through the second reflector and the lens group, the light beam after the expansion is guided to the same scanning system after being reflected by a third reflector to complete rescanning, the propagation direction of a light path is changed by using a fourth reflector, the stray light is filtered by a second optical filter, and finally the fluorescence is projected to an area array detector through an imaging lens to form an image.
3. The all-optical super-resolution microscopy device based on the photon reset technology as claimed in claim 1, wherein a micro lens array is arranged on an emergent light path of laser in the illumination system (1), and a single laser beam is divided into a plurality of parallel laser beams after passing through the micro lens array, and can scan and image a sample simultaneously.
4. All-optical super-resolution microscopy device based on photonic reset technology as claimed in claim 1, characterized in that said descan system (4) comprises the same optical elements as said scanning system (2) with opposite direction of travel of the optical path.
5. The all-optical super-resolution microscopy device based on the photon resetting technology as claimed in claim 1, wherein in the descan system (4), when the light beam enters the scanning galvanometer and the fluorescence entering scanning galvanometer, the scanning galvanometers have the same deflection angle, so that the emitting direction of the fluorescence light beam and the direction of the laser entering the scanning galvanometer are on the same straight line.
6. All-optical super-resolution microscopy apparatus based on photon reset technology according to claim 2, characterized in that the focal length ratio of the third lens group in the rescanning system (5) is 1: and 2, realizing double beam expansion of the fluorescent light beam.
7. The all-optical super-resolution microscopy device based on the photon reset technology according to claim 2, wherein the size of the light beam in the re-scanning system (5) is doubled, so that the size of the corresponding point spread function is reduced by half, the operation of shifting the detection image is completed in the imaging process, and the photon reset principle is realized.
8. The all-optical super-resolution microscopy device based on the photon resetting technology as claimed in claim 2, wherein the fluorescence beam after beam expansion in the re-scanning system (5) is reflected by a reflector and then enters the scanning galvanometer again.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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