CN117629958A - Three-dimensional single-molecule rapid tracking method and system - Google Patents
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Abstract
The invention relates to a three-dimensional single molecule rapid tracking method and a system, wherein the method comprises the following steps: acquiring fluorescence of the activated object to be detected, and tracking the fluorescence to obtain a symmetrical index point spread function image of molecules to be detected in the object to be detected; determining the transverse position of the molecule to be detected based on the position of the midpoint of the central connecting line of the symmetrical exponential point spread function sidelobe in the symmetrical exponential point spread function image on the imaging surface; determining the axial position of the molecule to be detected based on the distance and the direction of the center connecting line of the symmetrical index point spread function sidelobe in the symmetrical index point spread function image; and determining the space coordinate position of the molecule to be detected based on the transverse position and the axial position of the molecule to be detected, and drawing the three-dimensional motion trail of the moving molecule to be detected according to the change of the space coordinate position of the molecule to be detected. Compared with the prior art, the invention has the advantages of improving the depth of field of the rapid tracking of the polymolecules and the like.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a three-dimensional single molecule rapid tracking method and system.
Background
The rapid development of life sciences requires that one understand interactions between substances and life processes in real time, in situ, and in vivo from the single cell and single molecule level. Under complete living cells, the real-time tracking of the dynamic characteristics of intracellular molecules is important for researching key scientific problems such as virus infection mechanisms, interaction rules among nucleic acid macromolecules and the like, and is helpful for better understanding the occurrence mechanism of nervous system diseases and the intervention mechanism of medicines, so that the development progress of novel medicines is accelerated. However, the movement of the molecules in the cell is often a rapid three-dimensional process, and monitoring the real-time movement process of a plurality of molecules in the whole cell at the same time is helpful for more efficient and objective research on the dynamic characteristics of the molecules, which is an urgent requirement of life science and a great challenge to imaging science.
In the single molecule tracking method (SPT), a common means is to image the movement of fluorescent marker particles by using a wide-field fluorescent microscope with a high-speed camera, but the obtained information is often the movement of the particles in a two-dimensional plane, and the movement condition of the particles in the axial direction cannot be obtained. Laser confocal microscopy or multiphoton microscopy can be used to achieve optical sectioning of tissue, three-dimensional reconstruction, and qualitative and quantitative analysis of certain specific components in a sample. But this method requires point-by-point, line-by-line, and face-by-face scanning imaging, with low temporal resolution. Super-resolution microscopy, such as STED, STORM, PALM, has a resolution of tens of nanometers, but in order to achieve higher spatial resolution, it often takes several minutes or even tens of minutes to form an image, and the requirements of rapid or even real-time three-dimensional tracking of nanoparticles in biological samples still cannot be met.
Currently, three-dimensional real-time tracking of nanometer positioning accuracy for cells with diameters above 10 μm using single molecule tracking technology remains a challenging problem. First, conventional optical imaging techniques are limited to two-dimensional imaging, and if three-dimensional tracking is implemented, some axial positioning methods, such as defocusing imaging, astigmatism, z-stack, dual/multi-focal plane detection, fluorescence interferometry, and light field modulation, are required. Although the methods can realize dynamic tracking of a single molecule three-dimensional space, the imaging depth is mostly limited to be within 4 mu m, and the large depth of field tracking capability of molecules with close intracellular separation distance is insufficient.
Intracellular biomacromolecule dynamic tracking is to realize simultaneous tracking of multiple molecules in living cells, which requires that the tracking technology rapidly detect multiple target molecules within a field depth range of tens of micrometers with nanometer positioning accuracy in a three-dimensional space. Although the current SPT method has developed various axial positioning imaging methods such as a focal imaging method, an astigmatic method, a z-stack method, a double/multiple focal plane detection method, a fluorescence interferometry method, a light field regulation method and the like, three-dimensional nanometer positioning and tracking can be realized, the detection depth realized at present is only about 4 μm, and the thickness of a general whole cell is tens of micrometers, so that the current method still cannot meet the requirement of large depth of field for quickly tracking the intracellular polymolecules.
Disclosure of Invention
The invention aims to provide a three-dimensional single-molecule rapid tracking method and a three-dimensional single-molecule rapid tracking system for improving the depth of field of multi-molecule rapid tracking.
The aim of the invention can be achieved by the following technical scheme:
a three-dimensional single molecule rapid tracking method comprises the following steps:
acquiring fluorescence of the activated object to be detected, and tracking the fluorescence to obtain a symmetrical exponential point spread function image of molecules to be detected in the object to be detected, wherein the symmetrical exponential point spread function image consists of two side lobes which are symmetrical about a center and are positioned on a diagonal;
determining the transverse position of the molecule to be detected based on the position of the midpoint of the central connecting line of the symmetrical exponential point spread function sidelobe in the symmetrical exponential point spread function image on the imaging surface;
determining the axial position of the molecule to be detected based on the distance and the direction of the center connecting line of the symmetrical index point spread function sidelobe in the symmetrical index point spread function image;
and determining the space coordinate position of the molecule to be detected based on the transverse position and the axial position of the molecule to be detected, and drawing the three-dimensional motion trail of the moving molecule to be detected according to the change of the space coordinate position of the molecule to be detected.
Further, the specific steps of tracking fluorescence to obtain a symmetric index point spread function image of a molecule to be detected in the object to be detected are as follows:
modulating the point spread function by adopting the spliced index phase, optimizing the point spread function, generating an optimized symmetrical index point spread function, and tracking fluorescence based on the symmetrical index point spread function to obtain a symmetrical index point spread function image of molecules to be detected in the object to be detected.
Further, modulating and optimizing the point spread function by adopting the spliced index phase, and generating the optimized symmetrical index point spread function comprises the following specific steps:
combining two opposite index phases to obtain a double-index phase, rotating the double-index phase by 90 degrees to obtain an opposite double-index phase, combining the double-index phase with a positive defocusing phase to obtain a positive defocusing double-index phase, combining the opposite double-index phase with a negative defocusing phase to obtain a negative defocusing opposite double-index phase, and finally combining the positive defocusing double-index phase with the negative defocusing opposite double-index phase to obtain a final spliced index phase, wherein the transfer function of the spliced index phase is an initial symmetric index point diffusion function, and then optimizing the initial symmetric index point diffusion function through Fresnel approximate imaging inverse operation to obtain an optimized symmetric index point diffusion function.
Further, the exponential phase is:
wherein x, y are three-dimensional Cartesian coordinates of a focus area, and alpha and beta are modulation parameters.
Further, the positive defocus double index phase and the negative defocus double index phase are:
wherein,and->Is positive defocus double-index phase +.>And->Is a negative defocus double-index phase, and gamma is a defocus modulation parameter.
Further, the splice index phase is:
wherein,for the round down operator mod is the remainder, R represents the radius of the pupil, N is the pupil function design parameter, i.e. the fourier plane is divided into N parts, N representing the nth part of the fourier plane.
Further, the initial symmetric index point diffusion function is optimized through Fresnel approximate imaging inverse operation, and the optimized symmetric index point diffusion function is obtained through the following specific steps:
and carrying out Fresnel approximate imaging on the initial symmetric index point spread function, removing side lobes of the obtained imaging result, carrying out Fresnel approximate imaging inverse operation on the image after the side lobes are removed, and optimizing the symmetric index point spread function based on the image after the inverse operation.
The invention also provides a three-dimensional single-molecule rapid tracking system, which comprises an illumination subsystem and a detection subsystem, wherein the detection subsystem comprises a detector, a phase modulation optical component, a reflecting mirror, a detection lens, a dichroic mirror and an objective lens, the objective lens faces to an object to be detected,
the phase modulation optical component is a reflective phase modulation optical component or a transmissive phase modulation optical component, wherein the reflective phase modulation optical component comprises a first phase modulation module, the transmissive phase modulation optical component comprises a second phase modulation module, and the phase modulation module realizes the three-dimensional single-molecule rapid tracking method of any one of claims 1 to 7.
Further, the illumination subsystem comprises a laser, a beam expanding first lens, a beam expanding second lens, an illumination lens, a dichroic mirror and an objective lens which are sequentially arranged along the transmission direction of the optical path.
Further, the phase modulation optical assembly further comprises a first lens, a polarizing plate, a phase modulation module and a second lens which are sequentially arranged along the transmission direction of the optical path, wherein the phase modulation module is a first phase modulation module for the reflection type phase modulation optical assembly, and is a second phase modulation module for the transmission type phase modulation optical assembly.
Compared with the prior art, the invention has the following beneficial effects:
after the optical modulation module for realizing the tracking method of the invention is used for modulating, a symmetric index point spread function is generated, and the positioning accuracy is high and the depth of field is larger. In addition, the laser illumination light path is adopted to excite fluorescence, so that the astigmatic interference is avoided. The imaging depth of field of the invention can reach tens of micrometers, and the depth of field can be adjusted by changing the design parameters of the splicing index phase, so that the imaging depth of field can be used for dynamic functional images of a plurality of moving molecules with any depth in a living whole cell.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a graph of a mosaic exponential point spread function and a conventional point spread function at different depths, wherein (a) is a symmetric exponential point spread function at different depths according to the present invention, and (b) is a conventional point spread function at different depths;
FIG. 3 is a graph of the distance of a splice index point spread function sidelobe center line versus axial probe depth (Z-axis position);
FIG. 4 is a schematic block diagram of the optimization of a splice index point spread function;
fig. 5 is a schematic diagram of an initial splice index phase plate and an optimized splice index phase plate.
Wherein (a) is an initial splicing index phase plate schematic diagram, and (b) is an optimized splicing index phase plate schematic diagram;
FIG. 6 is an effect diagram of imaging using the phase plate of FIG. 5, wherein (a) is an effect diagram of imaging of a point spread function of an initial stitched index phase plate and (b) is an effect diagram of imaging of a point spread function of an optimized stitched index phase plate;
FIG. 7 is a schematic diagram of two phase modulating optical components in a system according to the present invention, wherein (a) is a structural diagram of a reflective phase modulating optical component, and (b) is a structural diagram of a transmissive phase modulating optical component;
FIG. 8 is a block diagram of an illumination subsystem in the system of the present invention;
FIG. 9 is a block diagram of a detection subsystem in a system employing the two phase modulating optical assemblies of FIG. 7, wherein (a) is a block diagram of a detection subsystem of a reflective phase modulating optical assembly and (b) is a block diagram of a detection subsystem of a transmissive phase modulating optical assembly;
FIG. 10 is a block diagram of a tracking system employing two detection subsystems of FIG. 9, wherein (a) is a block diagram of a tracking system corresponding to a detection subsystem of a reflective phase modulation optical assembly, and (b) is a block diagram of a tracking system corresponding to a detection subsystem of a transmissive phase modulation optical assembly;
in the figure, a first lens 201, a polarizing plate 202, a first phase modulation module 203, a second lens 204, a second phase modulation module 205, a laser 301, a beam expansion first lens 302, a beam expansion second lens 303, an illumination lens 304, a dichroic mirror 305, an objective lens 306, a detector 401, a phase modulation module 402, a mirror 403, and a detection lens 404.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
The invention provides a three-dimensional single-molecule rapid tracking method, and then an optical modulation module for realizing the symmetric index point spread function based on splicing index phase modulation by the tracking method is created, a three-dimensional single-molecule rapid tracking system is built, and after an imaging light path is modulated by the optical modulation module, the symmetric index point spread function is generated, so that the positioning accuracy is high and the depth of field is larger.
A flow chart of the method proposed by the present invention is shown in fig. 1. The method comprises the following steps:
acquiring fluorescence of the activated object to be detected, and tracking the fluorescence to obtain a symmetrical exponential point spread function image of molecules to be detected in the object to be detected, wherein the symmetrical exponential point spread function image consists of two side lobes which are symmetrical about a center and are positioned on a diagonal;
determining the transverse position of the molecule to be detected based on the position of the midpoint of the central connecting line of the symmetrical exponential point spread function sidelobe in the symmetrical exponential point spread function image on the imaging surface;
determining the axial position of the molecule to be detected based on the distance and the direction of the center connecting line of the symmetrical index point spread function sidelobe in the symmetrical index point spread function image;
and determining the space coordinate position of the molecule to be detected based on the transverse position and the axial position of the molecule to be detected, and drawing the three-dimensional motion trail of the moving molecule to be detected according to the change of the space coordinate position of the molecule to be detected.
The specific steps for obtaining the symmetrical index point spread function image of the molecules to be detected in the object to be detected are as follows:
modulating a point spread function by adopting a splicing index phase, optimizing the point spread function, generating an optimized symmetric index point spread function, tracking fluorescence based on the symmetric index point spread function, and obtaining a symmetric index point spread function image of molecules to be detected in the object to be detected.
Modulating and optimizing a point spread function by adopting a splicing index phase, and generating an optimized symmetrical index point spread function by the following specific steps:
combining two opposite index phases to obtain a double-index phase, rotating the double-index phase by 90 degrees to obtain an opposite double-index phase, combining the double-index phase with a positive defocusing phase to obtain a positive defocusing double-index phase, combining the opposite double-index phase with a negative defocusing phase to obtain a negative defocusing opposite double-index phase, and finally combining the positive defocusing double-index phase with the negative defocusing opposite double-index phase to obtain a final spliced index phase, wherein the transfer function of the spliced index phase is an initial symmetric index point diffusion function, and then optimizing the initial symmetric index point diffusion function through Fresnel approximate imaging inverse operation to obtain an optimized symmetric index point diffusion function.
Wherein, the index phase can be expressed as,
x and y are three-dimensional Cartesian coordinates of a focus area, and alpha and beta are modulation parameters;
the defocus index phase may be expressed as,
the four sets of defocus index phases may be represented separately as,
gamma is the defocus modulation parameter;
the annular splicing method is adopted to splice the defocusing index phases under four groups of different parameters together, so that the expression of the spliced index phase is obtained,
for the round down operator mod is the remainder, R represents the radius of the pupil, N is the pupil function design parameter, i.e. the fourier plane is divided into N parts, N representing the nth part of the fourier plane. The splice index phase can be obtained by using equation 4, and if the function is applied as an optical transfer function to an optical system, the point spread function of the optical system is modulated into a symmetric index point spread function, as shown in fig. 2. And optimizing the designed symmetric index point diffusion function through Fresnel approximate imaging inverse operation, wherein the optimization principle block diagram is shown in figure 4.
Optimizing an initial symmetric index point diffusion function through Fresnel approximate imaging inverse operation, and obtaining an optimized symmetric index point diffusion function comprises the following specific steps:
and carrying out Fresnel approximate imaging on the initial symmetric index point spread function, removing side lobes of the obtained imaging result, carrying out Fresnel approximate imaging inverse operation on the image after the side lobes are removed, and optimizing the symmetric index point spread function based on the image after the inverse operation.
Fresnel approximate imaging formula:
(u, v) represents the image plane coordinates, F is the Fourier transform operator, lambda is the incident light waveLength f 4f The focal length of the 4f system is set, and M is the magnification of the system;
ignoring the constant term, the fresnel approximation imaging inverse operates as,
a schematic of the initial splice index phase plate and the optimized splice index phase plate is shown in fig. 5. An effect diagram of imaging using the phase plate shown in fig. 5 is shown in fig. 6.
An optical system of symmetrical index point spread function is to add a specially designed optical module to the Fourier plane of a standard imaging system, the optical module makes its transmissivity function form symmetrical index point spread function in the focusing area of Fourier change, the optical module created by the above method has the characteristics, the imaged by the optical module is two side lobes which are located on the diagonal of the imaging plane and symmetrical about the center, and the distance between the two side lobes changes along with the change of the axial detection depth. When the symmetric index point spread function is used for three-dimensional positioning, the transverse position coordinates of the molecules are estimated through the middle point of the central connecting line of the two side lobes, the axial positions of the molecules are obtained according to the distance and the direction of the central connecting line of the two side lobes, the positioning precision is extremely high, and the relationship curve of the distance of the central connecting line of the two side lobes and the axial detection depth of the symmetric index point spread function shown in fig. 3 can be specifically referred to.
After the symmetrical index point spread function image is obtained, the transverse position of the molecule to be detected can be determined by the position of the midpoint of the central connecting line of the symmetrical index point spread function sidelobe in the obtained image on the imaging surface; the axial position of the molecule to be detected can be determined by the distance and the direction of the central connecting line of the side lobe of the symmetric index point spread function in the obtained image.
When the module is actually used, the system is calibrated in advance, the corresponding relation between the distribution position of the side lobe of the symmetric index point spread function and the spatial position of the molecule to be measured is established, and the information is prestored in a database and is called when the actual measurement is to be carried out. During actual measurement, the transverse space position of the molecule to be measured can be determined according to the position of the midpoint of the central connecting line of the symmetric index point spread function sidelobe in the calibration experiment on the imaging surface; and determining the axial space position of the molecule to be detected through the distance and the direction of the central connecting line of the side lobe of the symmetric index point spread function in the calibration experiment.
According to the optical transmission property, the optical module has two implementation modes, one is realized by a transmission modulation mode, such as optical modulation components of a phase plate, a transmission type digital micromirror device DMD, a transmission type spatial light modulator and the like; the other is realized by means of reflection modulation, such as a reflective spatial light modulator. The optical module has the advantages of large depth of field, high positioning precision and axial coding capability by utilizing the symmetric index point spread function, can be used for dynamic tracking of biological macromolecules in complete cells during imaging, and has important significance for understanding the relationship and rules of subcellular structure and cell function change at a higher level.
The invention also provides a three-dimensional single-molecule rapid tracking system, which comprises an illumination subsystem 501 and a detection subsystem 502, wherein the detection subsystem comprises a detector 401, a phase modulation optical component 402, a reflecting mirror 403, a detection lens 404, a dichroic mirror 305 and an objective lens 306, the objective lens 306 faces to an object to be detected,
the phase modulation optical component 402 is a reflective phase modulation optical component or a transmissive phase modulation optical component, where the reflective phase modulation optical component includes a first phase modulation module 203, and the transmissive phase modulation optical component includes a second phase modulation module 205, and the phase modulation module implements the three-dimensional single-molecule fast tracking method described above.
The phase modulation optical assembly mainly includes a first lens 201, a polarizing plate 202, a first/second phase modulation module 203/205, and a second lens 204, which are disposed in order along the optical path transmission direction. In the system, a first lens 201 collimates fluorescence emitted by a molecule to be detected and outputs the fluorescence to a polaroid 202, the polaroid 202 carries out polarization state modulation on the fluorescence, linear polarized light applicable to a first/second phase modulation module 203/205 is output, the first/second phase modulation module 203/205 outputs the collimated fluorescence, the first/second phase modulation module 203/205 converts the collimated fluorescence into an imaging light beam with a symmetric index point spread function property, then the imaging light beam is output by a second lens 204, the imaging light beam is focused on an image plane of a detector, and symmetric index point spread function imaging is realized on the detector. A schematic structure of two phase modulating optical components is shown in fig. 7. A block diagram of a detection subsystem in a system employing the two phase modulation optical assemblies of fig. 7 is shown in fig. 9.
The phase modulation module has two implementation manners, one is implemented by a reflection modulation manner, that is, the first phase modulation module 203, such as a reflection type spatial light modulator; the other is implemented by means of transmission modulation, i.e. a second phase modulation module 205, such as a phase plate, a transmission digital micromirror device DMD, a transmission spatial light modulator, or other optical modulation components.
In the detection subsystem, the dichroic mirror is shared with the objective and the dichroic mirror and the objective of the illumination subsystem. As an implementation, the phase modulation module 203/205 in the phase modulation optical component 402 may specifically be a phase plate, a spatial light modulator, a DMD, or other optical modulation device capable of loading a phase, for converting fluorescence into an imaging beam having symmetric exponential point spread function imaging properties.
In the detection subsystem, the object to be detected can emit fluorescence after being excited by the excitation light, a light beam containing the excitation light, the fluorescence and other stray light is received by the objective lens 306, the light beam filters the excitation light and the stray light after the light beam is filtered by the dichroic mirror 305, the fluorescence is focused by the detection lens 404, then the propagation direction of the light path of the fluorescence light beam is changed by the reflecting mirror 403, the light beam is subjected to phase modulation by the first/second phase modulation module 203/205, is converted into an imaging light beam with the symmetry index point diffusion function property, and finally is focused on the imaging surface of the detector 401 to form double-spiral image points on the imaging surface.
A block diagram of the illumination subsystem is shown in fig. 8. The illumination subsystem includes a laser 301, a beam expanding first lens 302, a beam expanding second lens 303, an illumination lens 304, a dichroic mirror 305, and an objective lens 306, which are sequentially arranged along the transmission direction of the optical path. The method of laser output illumination is adopted, so that the monochromaticity and the directivity are stronger, and the excitation of fluorescent dyed biomacromolecules in cells is more effective.
In the illumination subsystem, laser 301 emits laser light, the laser light is expanded by a beam expander first lens 302 and a beam expander second lens 303, focused by an illumination lens 304 and output, the focused laser light is reflected by a dichroic mirror 305 to a pupil surface of an objective lens 306, parallel light is output by the objective lens 306, and the parallel light irradiates an object to be measured, thereby exciting fluorescence of the object to be measured.
When the illumination subsystem 501 and the detection subsystem 502 work, laser 301 is emitted by a laser, the beam is expanded by the first beam expanding lens 302 and the second beam expanding lens 303, focused by the illumination lens 304 and output, reflected by the dichroic mirror 305 to the pupil surface of the objective lens 306, and parallel light is output by the objective lens 306 and irradiated to an object to be measured. The object to be measured can emit fluorescence after being excited by the excitation light, the light beam containing the excitation light, the fluorescence and other stray light is received by the objective 306, the excitation light and the stray light are filtered after the light beam is filtered by the dichroic mirror 305, the fluorescence is focused by the detection lens 404, the propagation direction of the light path is changed by the reflecting mirror 403, the light beam is phase modulated by the first/second phase modulation module 203/205, the light beam is converted into an imaging light beam with the symmetry index point spread function property, and finally the imaging light beam is focused on the imaging surface of the detector 401 to form double-spiral image points on the imaging surface.
A block diagram of a tracking system employing the two detection subsystems of fig. 9 is shown in fig. 10.
The large-depth-of-field nanometer positioning precision single-molecule rapid tracking system carries out large-depth-of-field real-time tracking through the optical component provided by the invention and the method based on the molecular tracking of the motion provided by the invention, and the high-precision axial positioning capability of the symmetric exponential point diffusion function with high positioning precision three-dimensional coding capability is utilized, so that the molecular rapid tracking method with ultra-large depth-of-field three-dimensional nanometer positioning precision is realized. The large depth-of-field three-dimensional nanometer positioning precision single-molecule rapid tracking system can also be built in cell imaging and other imaging equipment, so that the imaging equipment provided with the imaging system is also within the protection scope of the invention.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. A three-dimensional single-molecule rapid tracking method, which is characterized by comprising the following steps:
acquiring fluorescence of the activated object to be detected, and tracking the fluorescence to obtain a symmetrical exponential point spread function image of molecules to be detected in the object to be detected, wherein the symmetrical exponential point spread function image consists of two side lobes which are symmetrical about a center and are positioned on a diagonal;
determining the transverse position of the molecule to be detected based on the position of the midpoint of the central connecting line of the symmetrical exponential point spread function sidelobe in the symmetrical exponential point spread function image on the imaging surface;
determining the axial position of the molecule to be detected based on the distance and the direction of the center connecting line of the symmetrical index point spread function sidelobe in the symmetrical index point spread function image;
and determining the space coordinate position of the molecule to be detected based on the transverse position and the axial position of the molecule to be detected, and drawing the three-dimensional motion trail of the moving molecule to be detected according to the change of the space coordinate position of the molecule to be detected.
2. The method for quickly tracking three-dimensional single molecules according to claim 1, wherein the specific steps of tracking fluorescence to obtain a symmetric exponential point spread function image of the molecules to be detected in the object to be detected are as follows:
modulating the point spread function by adopting the spliced index phase, optimizing the point spread function, generating an optimized symmetrical index point spread function, and tracking fluorescence based on the symmetrical index point spread function to obtain a symmetrical index point spread function image of molecules to be detected in the object to be detected.
3. The method for quickly tracking three-dimensional single molecules according to claim 2, wherein the method for modulating and optimizing the point spread function by adopting the splicing index phase is characterized by comprising the following specific steps of:
combining two opposite index phases to obtain a double-index phase, rotating the double-index phase by 90 degrees to obtain an opposite double-index phase, combining the double-index phase with a positive defocusing phase to obtain a positive defocusing double-index phase, combining the opposite double-index phase with a negative defocusing phase to obtain a negative defocusing opposite double-index phase, and finally combining the positive defocusing double-index phase with the negative defocusing opposite double-index phase to obtain a final spliced index phase, wherein the transfer function of the spliced index phase is an initial symmetric index point diffusion function, and then optimizing the initial symmetric index point diffusion function through Fresnel approximate imaging inverse operation to obtain an optimized symmetric index point diffusion function.
4. A method for three-dimensional single-molecule rapid tracking according to claim 3, wherein the exponential phase is:
wherein x, y are three-dimensional Cartesian coordinates of a focus area, and alpha and beta are modulation parameters.
5. The method for three-dimensional single-molecule rapid tracking according to claim 4, wherein the positive defocus double-index phase and the negative defocus double-index phase are:
wherein,and->Is positive defocus double-index phase +.>And->Is a negative defocus double-index phase, and gamma is a defocus modulation parameter.
6. The method for three-dimensional single-molecule rapid tracking according to claim 5, wherein the splice index phase is:
wherein,for the round down operator mod is the remainder, R represents the radius of the pupil, N is the pupil function design parameter, i.e. the fourier plane is divided into N parts, N representing the nth part of the fourier plane.
7. The method for three-dimensional single-molecule rapid tracking according to claim 3, wherein the initial symmetric exponential point diffusion function is optimized by fresnel approximate imaging inverse operation, and the specific steps of obtaining the optimized symmetric exponential point diffusion function are as follows:
and carrying out Fresnel approximate imaging on the initial symmetric index point spread function, removing side lobes of the obtained imaging result, carrying out Fresnel approximate imaging inverse operation on the image after the side lobes are removed, and optimizing the symmetric index point spread function based on the image after the inverse operation.
8. A three-dimensional single-molecule rapid tracking system is characterized in that the system comprises an illumination subsystem (501) and a detection subsystem (502), wherein the detection subsystem (502) comprises a detector (401), a phase modulation optical component (402), a reflecting mirror (403), a detection lens (404), a dichroic mirror (305) and an objective lens (306), the objective lens (306) faces to an object to be detected,
the phase modulation optical assembly (402) is a reflective phase modulation optical assembly or a transmissive phase modulation optical assembly, wherein the reflective phase modulation optical assembly comprises a first phase modulation module (203) and the transmissive phase modulation optical assembly comprises a second phase modulation module (205), the phase modulation module implementing the three-dimensional single molecule rapid tracking method of any one of claims 1 to 7.
9. The three-dimensional single-molecule rapid tracking system according to claim 8, wherein the illumination subsystem (501) comprises a laser (301), a beam-expanding first lens (302), a beam-expanding second lens (303), an illumination lens (304), a dichroic mirror (305) and an objective lens (306) which are arranged in order along the transmission direction of the optical path.
10. The three-dimensional single-molecule rapid tracking system according to claim 8, wherein the phase modulation optical assembly further comprises a first lens (201), a polarizer (202), a phase modulation module and a second lens (204) sequentially arranged along the transmission direction of the optical path, the phase modulation module being a first phase modulation module (203) for the reflective phase modulation optical assembly and a second phase modulation module (205) for the transmissive phase modulation optical assembly.
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