CN116256880A - Drift correction system based on interference scattering image and control system thereof - Google Patents
Drift correction system based on interference scattering image and control system thereof Download PDFInfo
- Publication number
- CN116256880A CN116256880A CN202310158216.XA CN202310158216A CN116256880A CN 116256880 A CN116256880 A CN 116256880A CN 202310158216 A CN202310158216 A CN 202310158216A CN 116256880 A CN116256880 A CN 116256880A
- Authority
- CN
- China
- Prior art keywords
- drift correction
- light
- laser
- drift
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Abstract
The embodiment of the application relates to the technical field of microscopic imaging, in particular to a drift correction system based on interference scattering images and a control system thereof. The system provided by the application is simple in structure, only one laser beam is used for realizing the drift correction in the XYZ three-dimensional direction, the system adopts focus locking to actively lock the Z-direction drift in real time, the complexity of sample Z-direction drift amount evaluation is reduced, the XY-direction drift is selected, the three-dimensional drift correction is realized based on the scheme of combining objective lens illumination with interference scattering imaging and then performing post-processing, the problems of vibration, displacement table compensation error and the like during switching equipment can be effectively avoided, and the drift correction effect is improved; the system is easy to build, can be assembled into the existing imaging system in the form of a plug-in, and has good compatibility.
Description
Technical Field
The embodiment of the application relates to the technical field of microscopic imaging, in particular to a drift correction system based on interference scattering images and a control system thereof.
Background
The microscopic imaging technology, especially the super-resolution microscopic imaging technology, is very sensitive to the position information of the sample, and the sample is inevitably drifted due to mechanical vibration, temperature change and other reasons in the data acquisition process, so that fuzzy artifacts appear in the reconstructed image, and the final imaging quality is obviously reduced.
Disclosure of Invention
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.
The main purpose of the disclosed embodiments is to provide a drift correction system based on interference scattering images and a control system thereof, which adopts focus locking to actively lock Z-direction drift in real time, reduce the complexity of sample Z-direction drift amount evaluation, and free XY-direction drift, realize three-dimensional drift correction based on objective illumination combined with interference scattering imaging and then post-processing scheme, so as to effectively avoid the problems of vibration introduction, displacement table compensation error and the like during switching equipment, and promote drift correction effect.
To achieve the above object, a first aspect of the embodiments of the present disclosure proposes a drift correction system based on interference scattering images, the system comprising:
the first laser module is used for generating first laser;
the first acquisition module is used for acquiring fluorescence reflected from the objective lens to obtain a first image; the fluorescence is generated by the first laser irradiated onto a sample of a sample slide through the objective lens and collected by the objective lens;
the second laser module is used for generating second laser;
an objective lens type illumination module for focusing the second laser light to a back focal plane of the objective lens in a set focal length, so that the second laser light irradiates the sample slide in an approximately parallel light manner to obtain reflected light and illumination light generated by the slide of the sample slide, first scattered light generated by the illumination light irradiating the surface of the slide, second scattered light generated by the illumination light irradiating the sample, and interference signals generated after the first scattered light and the second scattered light interfere;
a photoelectric position detector for detecting the reflected light and outputting a corresponding voltage according to the reflected light;
the axial nano displacement platform is sleeved on the objective lens and is used for locking the axial distance between the objective lens and the sample slide according to the voltage;
the second acquisition module is used for acquiring the first scattered light, the second scattered light and the interference signal to obtain a second image;
and the drift correction module is used for calculating the drift amount of the sample according to the first image and reconstructing a super-resolution image after drift correction according to the drift amount and the first image.
In some embodiments, the second laser module includes a laser for generating a linearly polarized laser light, a fiber coupler for coupling the linearly polarized laser light into the polarization maintaining fiber jumper, and the polarization maintaining fiber jumper generating the second laser light.
In some embodiments, the objective illumination module includes a first doublet lens, a polarizing beam splitter prism, and a rotatable quarter wave plate, and the second laser light is focused to a back focal plane of the objective lens sequentially through the first doublet lens, the polarizing beam splitter prism, and the rotatable quarter wave plate.
In some embodiments, the high-precision drift correction system based on interference scattering images further comprises a diaphragm stop and a second double-cemented lens, and the first scattered light, the second scattered light, and the interference signal are sequentially transmitted to the second acquisition module through the diaphragm stop and the second double-cemented lens.
In some embodiments, the first scattered light, the second scattered light, and the interference signal also pass through the rotatable quarter wave plate and the polarizing beam splitter prism before passing through the aperture stop.
In some embodiments, the interference scattering image based drift correction system further comprises a first dichroic mirror, a first mirror, a second dichroic mirror, a filter wheel, a microscope tube lens, and a plano-convex cylindrical lens, the fluorescence passing from the objective lens to the first acquisition module in sequence through the first dichroic mirror, the second dichroic mirror, the filter wheel, the microscope tube lens, and the plano-convex cylindrical lens.
In some embodiments, the first acquisition module and the second acquisition module are both cameras.
In some embodiments, the first acquisition module and the second acquisition module acquire images synchronously or in frequency division.
In some embodiments, the drift correction module calculates the amount of drift of the sample using a cross-correlation algorithm.
To achieve the above object, a second aspect of the embodiments of the present disclosure provides a Micro-FPGA based control system, in which the interference scattering image based drift correction system described in the first aspect is integrated, and the Micro-FPGA based control system controls the interference scattering image based drift correction system according to an EMU plug-in of a Micro-Manager of microscope control software.
The first aspect of the application provides a drift correction system based on interference scattering images, which has a simple structure, realizes drift correction in XYZ three-dimensional directions by using only one laser beam, is easy to build and can be assembled into an existing imaging system in a plug-in mode, and has good compatibility. In addition, the system adopts focus locking to actively lock Z-direction drift in real time, reduces the complexity of sample Z-direction drift amount estimation, enables XY-direction drift to be selected, realizes three-dimensional drift correction based on objective illumination combined with interference scattering imaging and then post-processing scheme, can effectively avoid the problems of vibration, displacement table compensation error and the like when switching equipment is avoided, and improves the drift correction effect.
The second aspect of the application provides a control system based on a Micro-FPGA, and the system can realize automatic data acquisition and automatic super-resolution image reconstruction.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required for the embodiments or the description of the related art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort to a person having ordinary skill in the art.
FIG. 1 is a schematic diagram of a drift correction system based on interference scattering images according to one embodiment of the present application;
FIG. 2 is a schematic diagram of interference between first and second scattered light provided by one embodiment of the present application;
FIG. 3 is an image captured by a second acquisition module provided in one embodiment of the present application;
FIG. 4 is a schematic diagram of an electronic unit Micro-FPGA provided in one embodiment of the present application;
reference numerals in fig. 1 describe:
101. a first acquisition module; 102. a second acquisition module; 103. a four-quadrant photodiode array; 104. an objective lens; 1041. a back focal plane; 105. a sample slide; 106. an axial nano-displacement table; 107. a first laser module; 108. 785nm laser; 109. an optical fiber coupler; 110. polarization maintaining optical fiber jumper wire; 201. a first dichroic mirror; 202. a second dichroic mirror; 203. a first mirror; 204. a second mirror; 205. a third mirror; 206. a first doublet lens; 207. a second double cemented lens; 301. a polarization beam splitter prism; 302. a quarter wave plate; 303. a stop; 304. a filter wheel; 305. a microscope tube lens; 306. a plano-convex cylindrical lens.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.
It should be noted that although functional block division is performed in a device diagram and a logic sequence is shown in a flowchart, in some cases, the steps shown or described may be performed in a different order than the block division in the device, or in the flowchart. The terms first, second and the like in the description and in the claims and in the above-described figures, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing embodiments of the present application only and is not intended to be limiting of the present application.
First, technical names and elements used in the present application will be described:
(1) Based on the objective lens type illumination method, the laser beam is firstly expanded by a beam expanding system with a certain multiple, then focused on the back focal plane of the high-power objective lens by a focusing lens with a proper focal length, and finally is hit on the sample in a mode similar to parallel light. A significant advantage of this illumination mode is that different illumination modes can be achieved by adjusting the "focus spot" to strike different positions of the objective lens, for example: total internal reflection illumination, high incidence angle illumination and epi-illumination, and these three illumination modes can be switched with each other easily according to the needs of experiments.
(2) Interference scattering imaging technology, illuminating light irradiates a sample, the illuminating light generates scattered light on the surface of a slide and the sample, the scattered light generated at the two positions can also generate interference phenomenon, and the collected interference and scattered signals are used for imaging application.
(3) An active coke locking system can be referred to as follows: TIRF Lock TM |Keeps the sample in the focal plane and maintains TIRF signal(madcitylabs.com)，Piezo Focus Motor Drives/Nano-focus Systems|Voice Coil,Stage&Controller|PIFOC。
(4) The cross-correlation image registration algorithm (cross-correlation algorithm) can be referred to as: efficient subpixel image registration by cross-corridation-File Exchange-MATLAB Central (mathworks. Cn).
(5) Micro-Manager, an open source microscope hardware control and automation software, can be seenhttps:// micro-manager.org/。
The microscopic imaging technology, especially the super-resolution microscopy, is a powerful imaging technology, the application takes a single-molecule positioning microscopic technology as an example for description, the single-molecule positioning microscopic technology is one of the super-resolution imaging technologies widely applied at present, the principle is that fluorescent signals which are sparsely scintillated in time and space are collected to carry out high-precision spatial positioning, then a multi-frame image which is collected for a long time is reconstructed into a super-resolution image, and the technology can break through the optical diffraction limit to observe subcellular structures under the nanoscale.
The single-molecule positioning microscopy is very sensitive to the position information of the sample, and the sample is inevitably drifted due to mechanical vibration, temperature change and other reasons in the data acquisition process, so that fuzzy artifacts appear in the reconstructed image, and the final imaging quality is obviously reduced.
Drift assessment methods can be broadly divided into two categories, the first category being marked assessment. Such methods are accomplished by introducing fiducial markers (e.g., gold nanoparticles, polystyrene microspheres, bright fluorophores, etc.) into the sample. They can be easily tracked during imaging thanks to the stability and brightness of the fiducial marks. However, the introduction of a marker in a sample not only results in a limitation of the imaging region, but also may reduce the positioning accuracy of nearby probe molecules due to the high brightness of the marker. The second category is a label-free assessment, which has the unique advantage over the first category of methods that no additional labels are required. It mainly uses the biological characteristics of the sample itself: such as bright field patterns, speckle patterns, diffraction patterns, differential phase contrast patterns, etc., and then calculate the amount of drift of the sample from images taken at different times by corresponding algorithms.
The drift compensation method can be roughly classified into two types as well. The first is real-time compensation, i.e. the drift amount is calculated by an algorithm while data is acquired, and then the nano-displacement table is moved in real time to compensate the drift amount, which obviously increases the complexity of the imaging system. The second is post-processing, this kind of scheme does not need to carry on the complex control to the optical system, in the imaging process, the free drift of the free sample, only compensate the drift amount calculated to the primitive single-molecule point data when rebuilding the super-resolution fluorescent image, thus get the more accurate fluorescent point coordinate, and then improve and rebuild the image resolution ratio.
The application utilizes the principle of a focusing locking technology to realize axial drift locking of a sample through one laser beam. For transverse movement, a fluorescence microscope based on objective illumination is combined with an interference scattering imaging technology, transverse drift assessment is completed through the same laser beam, and then XY direction drift compensation is performed through a post-processing mode by using a cross correlation algorithm. The three-dimensional drift correction device has wide applicability, and can realize the three-dimensional drift correction function by simply modifying a single-molecule positioning microscope commonly used in a laboratory.
Referring to fig. 1, in one embodiment of the present application, a drift correction system based on interference scatter images is provided, the system comprising:
a first laser module 107 for generating a first laser light. The role of the first laser is to obtain a fluorescence image, and the embodiment of the present application is not limited to the type of the laser and the specific model of the first laser module 107.
A first acquisition module 101 for acquiring fluorescence reflected from the objective lens 104 to obtain a first image; fluorescence is generated by the first laser light shining through the objective lens 104 onto the sample of the sample slide 105 and collected by the objective lens 104.
In some embodiments of the present application, the first acquisition module 101 is an sCMOS camera for capturing fluorescent images.
In some embodiments of the present application, the specific optical paths of the first laser and the fluorescence in the system include:
the first laser module 107 generates a first laser light, which is reflected and focused by the first dichroic mirror 201 to the back focal plane 1041 of the objective lens 104, and illuminates the imaging sample, which generates fluorescence, which is collected by the objective lens 104, and passes through the first dichroic mirror 201, the first reflecting mirror 203, the second dichroic mirror 202, the filter wheel 304 (for filtering stray light such as residual laser light), the microscope tube lens 305, and the plano-convex cylindrical lens 306 until captured by the sCMOS camera. It should be noted that the devices such as the first dichroic mirror 201 and the first reflecting mirror 203 may be disposed in the system, and these devices are common devices in the field, and will not be described in detail herein.
And the second laser module is used for generating second laser.
In some embodiments of the present application, the second laser module includes a 785nm laser 108, a fiber coupler 109, and a polarization maintaining fiber jumper 110, where the 785nm laser 108 is configured to generate linearly polarized laser light, the fiber coupler 109 is configured to couple the linearly polarized laser light into the polarization maintaining fiber jumper 110, the polarization maintaining fiber jumper 110 generates the second laser light, and the polarization maintaining fiber jumper 110 can be integrally rotated to adjust the linear polarization direction of the laser light, for example: polarization maintaining fiber jumper 110 is integrally mounted on a frame with adjustable pitch and yaw to control the position of the emitted laser in the optical path. It should be noted that this is only an example of the common model of 785nm laser, which is not intended to be limiting of the full type of laser options available for the present application.
And an objective 104 type illumination module for focusing the second laser beam to the back focal plane 1041 of the objective 104 at a set focal length, so that the second laser beam irradiates the sample slide 105 in an approximately parallel light manner to obtain reflected light and illumination light generated by the slide of the sample slide 105, first scattered light generated by the illumination light irradiating the surface of the slide, second scattered light generated by the illumination light irradiating the sample, and interference signals generated after the interference of the first scattered light and the second scattered light.
And the photoelectric position detector is used for detecting the reflected light and outputting corresponding voltage according to the reflected light.
In some embodiments of the present application, the photo position detector is exemplified by a four-quadrant photodiode array 103, with current-voltage amplifiers for the four-quadrant photodiode array 103 that can provide differential signals for the bottom minus the top and the left minus the right of the array. The four-quadrant photodiode array 103 also provides a sum signal of the four quadrant outputs of the array, the differential signal being a voltage analog signal of the difference in light intensity sensed by pairs of photodiode elements in the array.
The axial nano-displacement table 106 is sleeved on the objective lens 104, and the axial nano-displacement table 106 is used for locking the axial distance between the objective lens 104 and the sample slide 105 according to voltage.
In some embodiments of the present application, since the voltage output by the photoelectric position detector is related to the distance between the objective lens 104 and the slide, the distance between the objective lens 104 and the slide can be accurately positioned by using the voltage output by the photoelectric position detector, so that the axial nano-displacement table 106 can lock the distance between the objective lens 104 and the slide in real time, and avoid errors caused by mechanical vibration of the sample.
The second acquisition module 102 is configured to acquire the first scattered light, the second scattered light, and the interference signal, and obtain a second image.
In some embodiments of the present application, the optical path of the second laser in the system includes:
the second laser light is focused to the back focal plane 1041 of the objective lens 104 through the first double cemented lens 206, the third mirror 205, the polarization splitting prism 301, and the rotatable quarter wave plate 302. The first double-cemented lens 206 may be integrally mounted on a frame with adjustable pitch and yaw, and adjusts the linear polarization direction of the laser by integrally rotating in combination with the polarization maintaining fiber jumper 110. The polarization splitting prism 301 and the rotatable quarter wave plate 302 can improve 785 illumination light utilization. It should be noted that the first doublet 206, the third mirror 205, the polarization splitting prism 301, the rotatable quarter wave plate 302, and other devices may be disposed in the system, and these devices are common devices in the art and will not be described in detail herein.
In some embodiments of the present application, the optical path of the reflected light in the system includes:
the reflected light passes through the first mirror 203, the second dichroic mirror 202, the rotatable quarter wave plate 302, the polarization splitting prism 301, and the second mirror 204 to the photo position detector. A polarizing beam splitter prism 301 and a rotatable quarter wave plate 302 may be used to condition the reflected light.
In some embodiments of the present application, the optical paths of the first scattered light, the second scattered light, and the interference signal in the system include:
the scattered light and the interference light pass through the first reflecting mirror 203, the second dichroic mirror 202, the rotatable quarter wave plate 302, the polarization splitting prism 301, the stop block 303 (for filtering the reflected light and the stray light), and the second double cemented lens 207 to be captured by the second acquisition module 102.
And the drift correction module is used for calculating the drift amount of the sample according to the first image and reconstructing a super-resolution image after drift correction according to the drift amount and the first image. The drift correction module calculates the drift amount of the sample according to the multi-frame second image, meanwhile, the first image is used for drift evaluation (corresponding evaluation algorithm exists in the field), and finally, the drift correction module and the first image are compared to remove abnormal data, so that the two acquisition light paths are prevented from being asynchronous to the same sample acquisition in the acquisition process.
The system mainly realizes the following functions:
(1) The axial drift locking of the sample is realized by a beam of laser by adopting a focusing locking technology, and the realization process is as follows:
the second laser module generates a beam of second laser, and the objective illumination module focuses the second laser to the back focal plane 1041 of the objective 104 at a set focal length, so that the second laser irradiates the sample slide 105 in a manner similar to parallel light, and reflected light generated by the slide can be obtained, and the reflected light mainly serves to lock the axial direction (Z-axis direction).
According to the focusing locking principle, the reflected light beam of the glass slide is detected by the photoelectric position detector, the output voltage of the photoelectric position detector changes along with the change of the distance between the objective lens 104 and the glass slide, so that the distance between the objective lens 104 and the glass slide can be accurately positioned according to the output voltage of the photoelectric position detector, and the axial nano displacement table 106 sleeved on the objective lens 104 can accurately control and lock the axial distance between the objective lens 104 and the glass slide according to the output voltage of the photoelectric position detector. It should be noted that axial nano-displacement stage 106 (typically a Z-axis nano-displacement stage) is well known in the art and will not be discussed in detail herein.
(2) Through the same laser beam, the drift correction of the sample is realized by utilizing the illumination based on an objective lens and combining the interference scattering imaging technology, and the realization process is as follows:
the second laser light is focused to the back focal plane 1041 of the objective lens 104 at a set focal length, and is irradiated onto the sample slide 105 in an approximately parallel light manner to obtain first scattered light (scattered light generated by irradiation of illumination light to the surface of the slide) generated by the slide of the sample slide 105, second scattered light (scattered light generated by irradiation of illumination light to the sample), and an interference signal (signal generated after interference of the first scattered light and the second scattered light).
After the second acquisition module 102 acquires a second image containing the first scattered light, the second scattered light and the interference signal, the drift amount of the sample is calculated according to the second image, and then the drift amount of the sample is compensated into the original fluorescent single-molecular point data so as to reconstruct a super-resolution image after drift correction.
Compared with the prior art, the system realizes the axial (Z direction) drift locking through the focusing locking, completes the drift evaluation on the transverse movement by combining the objective-based illumination with the interference scattering imaging, and then performs the transverse (XY direction) drift compensation through a post-processing mode by utilizing a cross correlation algorithm. The system has the following advantages:
(1) The system has simple structure, realizes the drift correction in the XYZ three-dimensional direction by only one laser (second laser), is easy to build and can be assembled into the existing imaging system in the form of an insert, and has good compatibility.
(2) The system adopts the focusing locking to actively lock the Z-direction drift in real time, so that the complexity of sample Z-direction drift amount evaluation is reduced; in addition, the XY direction drifting is selected, three-dimensional drifting correction is achieved based on the scheme of combining objective lens type illumination with interference scattering imaging and then performing post-processing, the problems of vibration, displacement table compensation error and the like during switching equipment can be effectively avoided, and the drifting correction effect is improved.
(3) The system integrates software and hardware and subsequent data processing flow.
Referring to fig. 1 to 4, in one embodiment of the present application, a drift correction method corresponding to a drift correction system based on interference scattering images is provided:
(1) Collecting super-resolution fluorescent images;
the first laser module 107 generates a first laser light, which is reflected by the first dichroic mirror 201, then focused on the back focal plane 1041 of the objective lens 104, and finally illuminates the imaged sample. Fluorescence generated by the sample is collected by the objective 104, then the fluorescence is transmitted through the first dichroic mirror 201, reflected by the first reflecting mirror 203, transmitted through the second dichroic mirror 202, filtered by an emission filter mounted on the filter wheel 304, and finally captured by the first collecting module 101 (sCMOS camera) after being collected by the microscope tube lens 305. In addition, a quick detachable plano-convex cylindrical lens 306 is placed in front of the first acquisition module 101 for three-dimensional super-resolution imaging based on astigmatism.
(2) Control and locking of the axial distance between the objective 104 and the slide;
In this process, the second laser light passes through the polarization splitting prism 301, the quarter wave plate 302, the second dichroic mirror 202, the first reflecting mirror 203, and the first dichroic mirror 201 in this order, is then focused onto the back focal plane 1041 of the same objective lens 104 as described above, and finally irradiates the sample slide 105 in an approximately parallel light manner. At this time, the second laser beam generates reflected light and illumination light on the surface of the slide, the illumination light generates first scattered light on the surface of the slide, and the second scattered light is generated at the sample, and the scattered light generated at both positions also interferes, referring to fig. 2, to obtain an interference signal.
The reflected light of the slide is detected by the four-quadrant photodiode array 103 after passing through the polarization splitting prism 301 and the second reflecting mirror 204, the output voltage related to the position changes along with the change of the distance between the objective lens 104 and the slide, and the output voltage of the array is fed back to the axial nano displacement table 106 sleeved on the objective lens 104. The movement of the up and down relative position between the objective 104 and the slide will cause the reflected spot to move to a different position such as Z-1Z0 Z1, and eventually the position of the spot on the four-quadrant photodiode array 103 will also move. By virtue of the positioning capability of the axial nano-displacement stage 106, the axial distance between the objective lens 104 and the slide can be precisely controlled and locked.
(3) Correcting three-dimensional drift;
the combination of the polarization splitting prism 301 and the rotatable quarter wave plate 302 is used for adjusting the signal light (the first scattered light, the second scattered light, and the interference signal) and the reflected light, and for improving the illumination light utilization ratio (whether the linearly polarized light is transmitted or reflected in the polarization splitting prism 301 is related to whether the linear polarization state of the incident light is P-polarized or S-polarized, light of one polarization direction such as P-polarized becomes circularly polarized after passing through the quarter wave plate 302 once, and circularly polarized light returned from the sample end becomes linearly polarized after passing through the quarter wave plate 302 again, but is turned 90 degrees to become S-polarized with respect to the incident direction).
The first scattered light, the second scattered light and the interference signal pass through the rotatable quarter wave plate 302, the polarization splitting prism 301, the diaphragm stop 303 and the second double-cemented lens 207, and then are captured by the second acquisition module 102 (camera), referring to fig. 1. The stop 303 serves to filter out reflected light and stray light.
The drift correction module calculates the drift amount of the sample according to the multi-frame second image acquired by the first acquisition module 101, and then reconstructs a super-resolution image after drift correction according to the drift amount of the sample and the first image.
The system has the following advantages:
(1) The system has simple structure, realizes the drift correction in the XYZ three-dimensional direction by only one laser (second laser), is easy to build and can be assembled into the existing imaging system in the form of an insert, and has good compatibility.
(2) The system adopts the focusing locking to actively lock the Z-direction drift in real time, thereby reducing the complexity of locking the Z-direction of the sample; in addition, the XY direction drifting is selected, three-dimensional drifting correction is achieved based on the scheme of combining objective lens type illumination with interference scattering imaging and then performing post-processing, the problems of vibration, displacement table compensation error and the like during switching equipment can be effectively avoided, and the drifting correction effect is improved.
(3) The system integrates software and hardware and subsequent data processing flow.
In some embodiments of the present application, the present interferometric scatter image based drift correction system may be controlled by a custom EMU plug-in integrated into the microscope control software Micro-Manager. Referring to fig. 4, under the control of the electronic unit Micro-FPGA, the data may be automatically collected, and the first collection module 101 and the second collection module 102 (two cameras) may be used for performing operations such as exposure synchronization or frequency division, excitation light synchronization or sequence emission. In addition, the analog signals (e.g., voltages) generated by the device (e.g., the four-quadrant photodiode array 103) are converted to digital signals for computer display for real-time monitoring. Typically, the first acquisition module 101 obtains 50000-100000 frames of images, with an exposure time of 15ms, for super-resolution image reconstruction; the second acquisition module 102 obtains 12500-50000 frames of images for lateral drift assessment. Under the control of an electronic unit Micro-FPGA, through the second image which is synchronous with the first image or acquired by frequency division, the drift amount of a sample can be obtained by utilizing a cross-correlation algorithm, the calculated drift amount is compensated into the original fluorescent single-molecular-point data through SMAP software, and then the super-resolution image after drift correction is reconstructed, wherein the related content of the SMAP software can be seen in documents Ries and J. (2020). A module super-resolution microscopy analysis platform for SMLM data Nature Methods,17 (9) and 870-872 are adopted by SMAP.https://doi.org/10.1038/s41592-020-0938-1And will not be described in detail herein. .
While the preferred embodiments of the present application have been described in detail, the embodiments are not limited to the above-described embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the embodiments, and these equivalent modifications and substitutions are intended to be included in the scope of the embodiments of the present application as defined in the appended claims.
Claims (10)
1. A drift correction system based on interferometric scatter images, the high-precision drift correction system based on interferometric scatter images comprising:
the first laser module is used for generating first laser;
the first acquisition module is used for acquiring fluorescence reflected from the objective lens to obtain a first image; the fluorescence is generated by the first laser irradiated onto a sample of a sample slide through the objective lens and collected by the objective lens;
the second laser module is used for generating second laser;
an objective lens type illumination module for focusing the second laser light to a back focal plane of the objective lens in a set focal length, so that the second laser light irradiates the sample slide in an approximately parallel light manner to obtain reflected light and illumination light generated by the slide of the sample slide, first scattered light generated by the illumination light irradiating the surface of the slide, second scattered light generated by the illumination light irradiating the sample, and interference signals generated after the first scattered light and the second scattered light interfere;
a photoelectric position detector for detecting the reflected light and outputting a corresponding voltage according to the reflected light;
the axial nano displacement platform is sleeved on the objective lens and is used for locking the axial distance between the objective lens and the sample slide according to the voltage;
the second acquisition module is used for acquiring the first scattered light, the second scattered light and the interference signal to obtain a second image;
and the drift correction module is used for calculating the drift amount of the sample according to the second image and reconstructing a super-resolution image after drift correction according to the drift amount and the first image.
2. The interferometric scatter image based drift correction system of claim 1, wherein the second laser module comprises a laser for producing linearly polarized laser light, a fiber coupler for coupling the linearly polarized laser light into the polarization maintaining fiber jumper producing the second laser light.
3. The interference scattering image based drift correction system of claim 1, wherein the objective illumination module comprises a first doublet lens, a polarizing beamsplitter and a rotatable quarter wave plate, the second laser light being focused to a back focal plane of the objective lens sequentially through the first doublet lens, the polarizing beamsplitter and the rotatable quarter wave plate.
4. The interferometric scatter image based drift correction system of claim 3, further comprising a stop block and a second double cemented lens, the first scattered light, the second scattered light and the interference signal being transmitted to the second acquisition module sequentially through the stop block and the second double cemented lens.
5. The interferometric scatter image based drift correction system of claim 4, wherein the first scattered light, the second scattered light and the interference signal further pass through the rotatable quarter wave plate and the polarizing beam splitter prism before passing through the stop block.
6. The interferometric scatter image based drift correction system of claim 1, further comprising a first dichroic mirror, a first mirror, a second dichroic mirror, a filter wheel, a microscope tube lens, and a plano-convex cylindrical lens, wherein the fluorescence passes from the objective lens, in order, through the first dichroic mirror, the second dichroic mirror, the filter wheel, the microscope tube lens, and the plano-convex cylindrical lens to the first acquisition module.
7. The interferometric scatter image based drift correction system of claim 1, wherein the first acquisition module and the second acquisition module are each cameras.
8. The interferometric scatter image based drift correction system of claim 7, wherein the first acquisition module and the second acquisition module acquire images synchronously or in frequency division.
9. The interferometric scatter image based drift correction system of any of claims 1-8, wherein the drift correction module calculates the amount of drift of the sample using a cross-correlation algorithm.
10. A Micro-FPGA based control system, wherein the Micro-FPGA based control system has the interference scattering image based drift correction system according to any one of claims 1 to 9 integrated therein, and the Micro-FPGA based control system controls the interference scattering image based drift correction system according to an EMU plug-in of a Micro-Manager of microscope control software.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310158216.XA CN116256880A (en) | 2023-02-13 | 2023-02-13 | Drift correction system based on interference scattering image and control system thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310158216.XA CN116256880A (en) | 2023-02-13 | 2023-02-13 | Drift correction system based on interference scattering image and control system thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116256880A true CN116256880A (en) | 2023-06-13 |
Family
ID=86680516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310158216.XA Pending CN116256880A (en) | 2023-02-13 | 2023-02-13 | Drift correction system based on interference scattering image and control system thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116256880A (en) |
-
2023
- 2023-02-13 CN CN202310158216.XA patent/CN116256880A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108971747B (en) | Ultrafast laser micro-nano machining device with online monitoring function | |
| CN107490562B (en) | Ultra-high-speed three-dimensional refractive index image shooting and fluorescent structured light illuminating microscope system using wave surface shaper and using method thereof | |
| US9696686B2 (en) | Method and device for focussing a microscope automatically | |
| CN109712072B (en) | Fringe illumination Fourier domain iterative updating super-resolution microscopic imaging method based on total internal reflection | |
| CN106885796B (en) | Super-resolution fluorescence digital holographic tomography microscopic imaging system and method | |
| CN108982456B (en) | Three-dimensional living cell super-resolution microscopic imaging method and device based on evanescent wave illumination | |
| JP7150867B2 (en) | microscope system | |
| WO2011136382A1 (en) | Observation device | |
| JP7453981B2 (en) | Calibration of light field imaging systems | |
| US9791682B2 (en) | Quantitative nonlinear optical microscopy using a shaped beam | |
| JP2009264752A (en) | Three-dimensional image acquisition apparatus | |
| US20160313548A1 (en) | Method for capturing image of three-dimensional structure of specimen and microscopic device | |
| WO2019097587A1 (en) | Quantitative phase image generating method, quantitative phase image generating device, and program | |
| CN110849289A (en) | Double-camera parallel confocal differential microscopic 3D morphology measurement device and method | |
| CN110927945A (en) | Three-dimensional wide-field and high-resolution tomography method and device | |
| CN116183568B (en) | High-fidelity reconstruction method and device for three-dimensional structured light illumination super-resolution microscopic imaging | |
| US11947098B2 (en) | Multi-focal light-sheet structured illumination fluorescence microscopy system | |
| CN113218635A (en) | Non-contact vector polarization light field test system | |
| US11067510B2 (en) | System and method for estimating and compensating for sample drift during data acquisition in fluorescence microscopy | |
| US20140022373A1 (en) | Correlative drift correction | |
| CN113768472B (en) | Three-dimensional image acquisition device with fluorescent marker and method | |
| JP2009169420A (en) | Polariscopic phase microscope | |
| CN110824684B (en) | High-speed three-dimensional multi-modal imaging system and method | |
| CN109238131A (en) | A kind of optical coherence tomography method and system of transverse direction super-resolution | |
| WO2016126250A1 (en) | Particle tracking using spatiotemporal offset light beams |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |