Three-dimensional active feedback tracing imaging device and method

Abstract

The invention relates to the technical field of optical microscopic imaging, in particular to a three-dimensional active feedback tracing imaging device and method. According to the scheme, after the light source emits the laser, the laser is modulated through the laser focus modulation assembly to generate the illumination area, the laser focus scans at each scanning position of the illumination area, the fluorescence signal is generated after the laser irradiates the to-be-tracked object, and the fluorescence signal is collected through the fluorescence signal collection assembly; the signal is transmitted to the control and processing assembly, the control and processing assembly calculates the position deviation between the position of the object to be tracked and the reference position in the illumination area according to the position of the laser focus and the electric signal, and the spatial position of the illumination area is adjusted according to the position deviation, so that the object to be tracked is always located at the reference position in the illumination area. According to the invention, active feedback tracing of the to-be-tracked object is realized by controlling the movement of the illumination area, the imaging precision and the imaging speed are obviously improved, and the three-dimensional active feedback tracing function of the high-speed moving target is realized.

Description

Technical Field

[0001] The invention relates to the technical field of optical microscopic imaging, and in particular to a three-dimensional active feedback tracing imaging device and method. Background Art

[0002] In the field of life sciences, understanding dynamic biological processes requires not only studying the fine structure and state of biological systems such as cells and proteins, but also focusing on the dynamic processes of interactions between various biological molecules. A large number of dynamic biological processes, such as DNA transcription, protein folding, ribozyme catalysis, and receptor protein recognition, occur at the nanoscale spatial scale and millisecond or even microsecond time scale. Therefore, improving the spatial and temporal resolution of optical microscopes and achieving high-resolution spatial and temporal tracking imaging of single biological molecules has always been an important development goal in this field.

[0003] The common method for tracing and imaging the dynamic process of single molecules or single particles is the single particle tracking (SPT) technology based on traditional optical microscopy. This technology first requires the target molecules or particles to be photographed frame by frame in two or three dimensions, and then the images are superimposed in a time series to finally generate a dynamic video or motion trajectory. Although this technology has been widely used in various research fields of biology and has achieved many results. For example, through SPT tracing of various dynamic interactions between viruses and cells, the binding mechanism of viruses and cell surfaces has been revealed, the internalization and transport pathways of viruses have been distinguished, the location of fusion of viruses with cell membranes has been determined, and the transport characteristics of viruses on the cytoskeleton have been characterized. However, despite this, due to the limitations of exposure time and frame rate, this technology cannot track targets that move faster, and it is even more impossible to accurately achieve rapid three-dimensional tracking. Summary of the invention

[0004] The present invention provides a three-dimensional active feedback tracing imaging device and method, which effectively solves the problem that the existing tracing imaging system cannot perform three-dimensional active feedback tracing on a high-speed moving target.

[0005] According to the first aspect, an embodiment provides a three-dimensional active feedback tracing imaging device, comprising:

[0006] A light source, used to emit laser light, wherein the laser light is used to excite the object to be tracked;

[0007] A laser focus modulation component is arranged on the laser optical path, and is used to modulate the laser in three-dimensional space so that the focus of the laser after being emitted from the objective lens scans at various scanning positions in a preset scanning space area;

[0008] A three-dimensional dynamic focusing component, arranged on the laser light path emitted by the laser focus modulation component, for adjusting the spatial position of the preset scanning space area;

[0009] An imaging component is arranged on the optical path of the three-dimensional dynamic focusing component, and the imaging component includes an objective lens. After the laser emitted by the three-dimensional dynamic focusing component passes through the objective lens, the laser excites the object to be tracked in the sample so that the object to be tracked generates a fluorescent signal;

[0010] A fluorescence signal collection component, used to collect the fluorescence signal and process it into a corresponding electrical signal;

[0011] A control and processing component is used to control the laser focus modulation component to make the laser focus scan in a preset scanning space area, and calculate the position deviation between the object to be tracked and the reference position in the preset scanning space area according to the position of the laser focus and the electrical signal, and control the three-dimensional dynamic focusing component to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at the reference position in the preset scanning space area.

[0012] The solution of this embodiment can achieve nanometer-level tracing accuracy and millisecond-level tracing time resolution. In addition, the economy and compatibility of this device enable it to be easily implemented in most laboratories and compatible with existing optical systems, and has broad application prospects.

[0013] In one feasible implementation, the control and processing component is also used to record the spatial position of the preset scanning space area at different times, and obtain the three-dimensional movement trajectory of the object to be tracked according to the spatial position of the preset scanning space area at different times, so as to realize high-precision and high-speed real-time three-dimensional active feedback tracking imaging of the target.

[0014] In an achievable embodiment, a collimating and beam expanding component is also arranged on the optical path between the light source and the laser focus modulation component, and the collimating and beam expanding component includes a first lens, a first optical pinhole and a second lens; the first lens, the first optical pinhole and the second lens are arranged in sequence along the laser optical path.

[0015] In an achievable implementation, a Glan prism and a first half-wave plate are sequentially arranged on the optical path between the collimating beam expansion component and the laser focus modulation component;

[0016] The Glan prism is used to purify the polarization state of the laser;

[0017] The first half-wave plate is used to adjust the polarization angle of the laser light emitted from the Glan prism.

[0018] In an achievable implementation, the three-dimensional space includes a three-dimensional space formed by three directions of X, Y, and Z; the laser focus modulation component includes a first electro-optical deflector, a second half-wave plate, a second electro-optical deflector, and a tunable acoustic gradient lens;

[0019] The first electro-optical deflector is used to deflect the incident laser in the X direction, the second half-wave plate is used to change the polarization direction of the laser emitted by the first electro-optical deflector, the second electro-optical deflector is used to deflect the laser emitted by the second half-wave plate in the Y direction, and the tunable acoustic gradient lens is used to adjust the focal length of the laser emitted by the second electro-optical deflector in the Z-axis direction.

[0020] In an achievable implementation, a third lens and a fourth lens are further provided between the second electro-optical deflector and the tunable acoustic gradient lens;

[0021] The third lens and the fourth lens form a beam expansion component, and the beam expansion component is used to expand the laser emitted by the second electro-optical deflector.

[0022] In an achievable embodiment, the device further comprises a light spot modulation component; the light spot modulation component is arranged on the laser light path emitted by the laser focus modulation component, and the light spot modulation component comprises a fifth lens, a sixth lens and a dove prism;

[0023] The fifth lens and the sixth lens form a beam reduction assembly, and the beam reduction assembly is used to reduce the spot of the laser emitted by the laser focus modulation assembly, and the reduction ratio of the spot does not exceed a preset threshold;

[0024] The dove prism is used to rotate the spot angle of the laser light emitted by the laser focus modulation component.

[0025] In an achievable implementation, the device further comprises an optical path setting component; the optical path setting component is a dichroic mirror, and the dichroic mirror is arranged on the laser optical path emitted by the light spot modulation component;

[0026] The dichroic mirror is used to reflect the laser emitted by the spot modulation component to the three-dimensional dynamic focusing component so that the light path can propagate along a first direction, and to transmit the fluorescent signal so that the fluorescent signal is transmitted to the fluorescent signal collection component along a second direction; the first direction is the direction of the light path reflected by the dichroic mirror, and the second direction is the direction of the light path transmitted by the dichroic mirror.

[0027] In an achievable implementation, the three-dimensional dynamic focusing assembly includes a diverging lens, a relay lens, a first galvanometer and a second galvanometer, which are sequentially arranged along a first direction; the first galvanometer and the second galvanometer are orthogonal.

[0028] In an achievable implementation, the relay lens includes a first plano-convex lens and a second plano-convex lens, and the convex surface of the first plano-convex lens and the convex surface of the second plano-convex lens are arranged opposite to each other;

[0029] The diverging lens is a plano-concave lens.

[0030] In one feasible embodiment, the first galvanometer and the second galvanometer are both one-dimensional galvanometers; the control and processing component controls the deflection of the first galvanometer to move the focus of the laser in the X-axis direction, and the control and processing component controls the deflection of the second galvanometer to move the focus of the laser in the Y-axis direction; the control and processing component controls the movement of the plano-concave lens to move the focus of the laser in the Z-axis direction.

[0031] In one feasible embodiment, the imaging component also includes a laser scanning tube lens, which is arranged on the optical path between the objective lens and the three-dimensional dynamic focusing component, and the laser scanning tube lens is located at the focal length conjugate point of the three-dimensional dynamic focusing component, and the focal length of the objective lens is conjugate with the focal length of the laser scanning tube lens.

[0032] In an achievable implementation, the imaging assembly further includes a stage and a controller;

[0033] The stage is used to place samples, and the stage adopts a piezoelectric nano-platform;

[0034] The controller is used to control the movement of the piezoelectric nano-platform; the controller is electrically connected to the control and processing component.

[0035] In one feasible embodiment, the fluorescence signal collection component is arranged on the optical path in the second direction. After being collected by the objective lens, the fluorescence signal passes through the three-dimensional dynamic focusing component in reverse to the optical path setting component. The optical path setting component is also used to transmit the fluorescence signal along the second direction to the fluorescence signal collection component.

[0036] In an achievable embodiment, the fluorescence signal collection component includes an avalanche photodiode; the device also includes a bandpass filter and a spatial filter component;

[0037] The bandpass filter is arranged on the fluorescent light path emitted by the light path setting component along the second direction;

[0038] The spatial filtering component includes a seventh lens, a second optical pinhole and an eighth lens. The fluorescent signal passing through the bandpass filter sequentially passes through the seventh lens, the second optical pinhole and the eighth lens before entering the avalanche photodiode.

[0039] In an achievable implementation, a ninth lens is further provided on the optical path between the spatial filter component and the avalanche photodiode, and the ninth lens is used to focus the fluorescent signal emitted by the spatial filter component and transmit it to the avalanche photodiode.

[0040] In an achievable implementation, the control and processing component controls the laser focus modulation component to make the laser focus scan in a preset scanning space area, including:

[0041] Controlling the laser focus modulation component to perform grid scanning in the form of a "knight's tour" on the XY plane; this scanning method can ensure more uniform sampling on the two-dimensional plane;

[0042] The laser focus modulation component is controlled to perform sinusoidal scanning in the Z direction; the position of the laser focus in the Z direction at each moment can be obtained through this scanning method.

[0043] In an achievable implementation, the control and processing component is further used to calibrate the three-dimensional dynamic focusing component; the calibrating the three-dimensional dynamic focusing component includes:

[0044] focusing the laser light onto a sample;

[0045] Sending instructions to the controller to control the piezoelectric nano-platform to control the sample to move a preset distance in the X, Y and Z directions respectively, so that the sample leaves the preset scanning space area;

[0046] The three-dimensional dynamic focusing component is controlled to move a preset distance in three directions of X, Y and Z so that the sample is relocated in the preset scanning space area.

[0047] By correcting the 3D dynamic focusing component, the applied voltage of the 3D dynamic focusing component and the displacement in three directions can be highly linearly matched. This linear matching improves the accuracy of the 3D dynamic focusing component in controlling the laser position and avoids further data processing.

[0048] According to the second aspect, an embodiment provides a three-dimensional active feedback tracing imaging method, using the above-mentioned three-dimensional active feedback tracing imaging device, the method comprising:

[0049] Controlling the light source to emit laser light; the laser light is used to excite the object to be tracked;

[0050] Controlling the laser focus modulation component to modulate the laser in three-dimensional space so that the focus of the laser after being emitted from the objective lens scans at various scanning positions in a preset scanning space area;

[0051] Controlling the fluorescence signal collection component to collect the fluorescence signal generated after the laser excites the object to be tracked; the fluorescence signal collection component generates an electrical signal according to the fluorescence signal;

[0052] Calculating a position deviation between the object to be tracked and a reference position in the preset scanning space area according to the electrical signal and the position of the laser focus;

[0053] The three-dimensional dynamic focusing component is controlled to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at a reference position within the preset scanning space area.

[0054] Through the tracking imaging method of this embodiment, it can be ensured that the object to be tracked is always actively tracked and is located at the center of the illumination area.

[0055] According to a three-dimensional active feedback tracking imaging device / method of the above embodiment, after the light source emits laser, the laser is modulated by the laser focus modulation component to generate a three-dimensional illumination area, so that the laser focus scans at each scanning position of the illumination area, and when the laser is irradiated to the object to be tracked, a fluorescent signal is generated, and the fluorescent signal is collected by the fluorescent signal collection component, and the generated electrical signal is transmitted to the control and processing component, and the control and processing component calculates the position deviation between the object to be tracked and the reference position in the illumination area according to the position of the laser focus and the electrical signal, and controls the three-dimensional dynamic focusing component to adjust the spatial position of the illumination area according to the position deviation, so that the object to be tracked is always located at the reference position in the illumination area. The above scheme of the present application realizes active feedback tracking of the object to be tracked by controlling the movement of the illumination area, without mechanical movement of the sample, significantly improving the imaging accuracy and imaging speed, and can realize the three-dimensional active feedback tracking function of high-speed moving targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 A schematic diagram of the structure of a three-dimensional active feedback tracing imaging device provided in this embodiment;

[0057] Figure 2 Schematic diagram of the three-dimensional active feedback tracking principle in this embodiment, wherein (a) is a schematic diagram of the coordinate positioning principle of the object to be tracked, and (b) is a three-dimensional motion trajectory diagram of the object to be tracked;

[0058] Figure 3 A schematic diagram of the lighting area generation principle of this embodiment;

[0059] Figure 4 Schematic diagram of the scanning method of the laser focus modulation assembly of this embodiment, wherein (a) is a schematic diagram of the scanning method in the XY plane, and (b) is a schematic diagram of the scanning method in the Z direction;

[0060] Figure 5 is a working principle diagram of the three-dimensional dynamic focusing component of this embodiment;

[0061] Figure 6 It is a schematic diagram of the movement of the laser focus above the objective lens when the three-dimensional dynamic focusing assembly of this embodiment is working;

[0062] Figure 7 3D active feedback tracking process principle diagram of this embodiment, wherein (a), (b), (c), and (d) respectively represent the process schematic diagrams of the illumination area following the movement of the object to be tracked when the object to be tracked moves;

[0063] Figure 8 is a flow chart of the three-dimensional active feedback tracing imaging method of this embodiment;

[0064] Fig. 9 The three-dimensional tracing imaging diagram of the 200 nm fluorescent microspheres in the present embodiment placed in a glycerol solution with a volume concentration of 70% is shown; wherein (a) is the three-dimensional motion trajectory of the fluorescent microspheres in the glycerol solution over time, (b) is a trend diagram of the light intensity change of the fluorescent microspheres during the tracing process, and (c1), (c2), and (c3) are curve diagrams of the X, Y, and Z axial positions of the fluorescent microspheres in the glycerol solution over time, respectively;

[0065] Fig.10 The three-dimensional tracing imaging diagram of the 200 nm fluorescent microspheres in the aqueous solution of this embodiment; wherein, (a) is the three-dimensional motion trajectory of the fluorescent microspheres in the aqueous solution changing with time, (b) is the light intensity change trend diagram of the fluorescent microspheres during the tracing process, and (c1), (c2), and (c3) are respectively the curve diagrams of the X, Y, and Z axial positions of the fluorescent microspheres in the aqueous solution changing with time.

[0066] Figure numerals: 10, light source; 20, laser focus modulation component; 30, three-dimensional dynamic focusing component; 40, imaging component; 50, fluorescence signal acquisition component; 60, control and processing component; 70, collimation and beam expansion component; 80, spot modulation component; 90, optical path setting component. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. Wherein similar elements in different embodiments adopt associated similar element numbers. In the following embodiments, many detailed descriptions are for making the present application better understood. However, those skilled in the art can easily recognize that some features can be omitted in different situations, or can be replaced by other elements, materials, methods. In some cases, some operations related to the present application are not shown or described in the specification, this is to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0068] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0069] The serial numbers of the components in this document, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning. The "connection" and "coupling" mentioned in this application, unless otherwise specified, include direct and indirect connections (couplings).

[0070] Optical microscopy technology has become an important scientific research tool in the fields of life sciences, medicine, and materials science because of its advantages such as high spatiotemporal resolution, dynamic imaging, and non-destructive imaging. In the field of life sciences, optical microscopes can be used to observe the structure and morphology of cells and study a variety of complex physiological processes of cells. In the medical field, optical microscopes are used to detect tissue specimens and for the analysis and diagnosis of diseases. The development of optical microscopy technology has been promoting the progress of life sciences and medicine and has become one of its important driving forces. This driving force is mainly reflected in several key performances of optical microscopes, including spatial resolution, temporal resolution, observation time, and imaging field of view. These performances determine the degree of sophistication, dynamic accuracy, continuous observation time, and observation range of microscopic biological research.

[0071] At present, there are some methods that can achieve three-dimensional single-molecule / single-particle tracing imaging, such as the single particle tracking (SPT) technology based on traditional optical microscope mentioned in the background technology. This technology requires first shooting the target molecule or particle frame by frame in three dimensions, and then superimposing the images in time series to finally generate a dynamic video or motion trajectory. This technology requires complete shooting of all pictures of the entire three-dimensional space during three-dimensional tracking, which seriously affects the temporal resolution of the tracing. In addition, the tracing accuracy of this method is limited by the three-dimensional resolution of the optical imaging system and is limited by the optical diffraction limit, making it difficult to achieve a positioning accuracy of less than 200nm.

[0072] In this regard, the present application proposes a three-dimensional active feedback tracing imaging device and a three-dimensional active feedback tracing imaging method. Different from the traditional SPT method, the solution of the present application no longer images the entire field of view, but only focuses on a smaller illumination area. In some embodiments, the smaller illumination area refers to no more than 1×1×2μm 3 The three-dimensional scanning space area. By narrowing the field of view, the time resolution is greatly improved (the time resolution can reach 1ms), so dynamic information of the target particles with higher temporal and spatial accuracy can be obtained in a limited illumination area. In addition, this solution uses a three-dimensional dynamic focusing component to control the movement of the laser illumination area, realizing the system's active feedback function for single molecules / single particles (objects to be tracked), thereby eliminating the need to physically move the sample. Therefore, this solution can perform rapid three-dimensional tracing imaging of single molecules or particles in three-dimensional space (the three-dimensional tracing speed can reach 4μm 2 / s) without mechanical movement of the sample.

[0073] In one embodiment, the present solution also has good optical compatibility, and can be compatible with other optical systems in the implementation mode of this embodiment, such as combining with a traditional wide-field illumination microscope, a single objective oblique illumination light sheet microscope, and a structured light illumination microscope, etc., to realize a multimodal imaging system. In addition, the device of this solution also has high economic efficiency, so that it can be easily implemented in most laboratories and has better marketability. Below, the three-dimensional active feedback tracing imaging device and the three-dimensional active feedback tracing imaging method of this application are described in detail respectively.

[0074] In one embodiment, reference Figure 1The present embodiment provides a three-dimensional active feedback tracing imaging device, which includes a light source 10, a laser focus modulation component 20, a three-dimensional dynamic focusing component 30, an imaging component 40, a fluorescence signal acquisition component 50, and a control and processing component 60. The light source 10 is used to emit laser light, which is used to excite the object to be tracked; the laser focus modulation component 20 is arranged on the laser light path, and the laser modulation component is used to modulate the laser light in three-dimensional space, so that the focus of the laser light after being emitted from the objective lens scans at various scanning positions in the preset scanning space area; the three-dimensional dynamic focusing component 30 is arranged on the laser light path emitted by the laser focus modulation component 20, and is used to adjust the spatial position of the preset scanning space area; the imaging component 40 is arranged on the light path emitted by the three-dimensional dynamic focusing component 30, and the imaging component 40 includes an objective lens. After the laser light emitted by the three-dimensional dynamic focusing component 30 passes through the objective lens, it excites the object to be tracked in the sample so that the object to be tracked generates a fluorescence signal; the fluorescence signal acquisition component 50 is used to collect the fluorescence signal and process it into a corresponding electrical signal; the control and processing component 60 is used to control the laser focus modulation component 20 to make the laser focus scan in the preset scanning space area, and calculate the position deviation between the object to be tracked and the reference position in the preset scanning space area according to the position of the laser focus and the electrical signal, and control the three-dimensional dynamic focusing component 30 to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at the reference position in the preset scanning space area.

[0075] In one embodiment, in actual application, a suitable laser wavelength is selected according to the research purpose, and the laser enters the laser focus modulation component 20 to generate a three-dimensional illumination area (i.e., a preset scanning space area), wherein the preset scanning space area is set according to the experimental requirements. When tracking a target with a fast movement speed, a larger scanning range needs to be set, and the scanning voltage of the laser focus modulation component 20 should be increased at this time; conversely, when the target to be tracked has a moderate movement speed, the scanning voltage of the laser focus modulation component 20 needs to be reduced for a more precise tracking result. Subsequently, after the laser passes through the three-dimensional dynamic focusing component 30 and the imaging component 40, it irradiates the object to be tracked to generate a fluorescent signal, and then the fluorescent signal is collected in real time by the fluorescent signal acquisition component 50 and an electrical signal is generated and transmitted to the control and processing component 60. Then, the control and processing component 60 obtains the scanning position information of the laser from the laser focus modulation component 20, and calculates the position deviation between the object to be tracked and the reference position in the preset scanning space area according to the electrical signal generated by the fluorescence signal acquisition component 50, and controls the three-dimensional dynamic focusing component 30 to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at the reference position in the preset scanning space area, thereby realizing feedback compensation movement. In general, the reference position in the preset scanning space area is the center position in the preset scanning space area (that is, the center position of the illumination area).

[0076] Specifically, the strongest photon signal can be stimulated only when the laser focus is irradiated to the object to be tracked. Therefore, by calculating the relationship between the number of photons and the position of the laser focus, the specific position of the object to be tracked in the illumination area can be obtained. When it deviates from the center of the illumination area, the control and processing component 60 calculates the offset and outputs a corresponding feedback signal to the three-dimensional dynamic focusing component 30. The three-dimensional dynamic focusing component 30 actively compensates for this offset by controlling the movement of the entire illumination area, always keeping the object to be tracked in the center of the illumination area, thereby realizing real-time three-dimensional active feedback tracking of the object to be tracked.

[0077] By adopting the three-dimensional active feedback tracing imaging device of this embodiment, nanometer-level tracing accuracy and millisecond-level tracing time resolution can be achieved, allowing the observation of millisecond-level rapid single-molecule three-dimensional dynamic biological events. In addition, the economy and compatibility of this device make it easy to implement in most laboratories and compatible with existing optical systems, and has a wide range of application prospects.

[0078] Among them, the control and processing component 60 of this embodiment uses a software program developed by LabVIEW to realize the signal control, data acquisition and processing of the system. The software runs on the computer side, and controls the scanning of the laser focus modulation component 20 through the FPGA module, thereby determining the scanning position of the laser focus in the preset scanning space area. The FPGA also controls the movement of the three-dimensional dynamic focusing component 30, and records and collects the electrical signals collected by the fluorescence signal collection component 50. The FPGA controls the movement of the three-dimensional dynamic focusing component 30 to realize the precise manipulation of the laser position and reads its position information at the same time.

[0079] Furthermore, the control and processing component 60 is also used to record the spatial position of the preset scanning space area at different times, and obtain the three-dimensional movement trajectory of the object to be tracked according to the spatial position of the preset scanning space area at different times.

[0080] In one embodiment, the entire process is written in a visual panel by LabVIEW, which can observe in real time the signal strength of the object to be tracked, the scanning position of the laser focus modulation component 20, the moving position of the three-dimensional dynamic focusing component 30, etc., and the final trajectory data can be drawn into a three-dimensional image by MATLAB.

[0081] Specifically, the control and processing component 60 records the movement of the illumination area (i.e., the preset scanning space area) in real time after controlling the three-dimensional dynamic focusing component 30 to move for active feedback each time, because the movement of the illumination area reflects the movement of the target in the three-dimensional space. The control and processing component 60 analyzes the recorded illumination area movement data through MATLAB and draws a three-dimensional trajectory image (e.g., Figure 2(b) shows), thus the real-time three-dimensional motion trajectory information of the object to be tracked is obtained, thereby realizing high-precision and high-speed real-time three-dimensional active feedback tracking imaging of the target.

[0082] As a further improvement of this embodiment, a collimating and expanding assembly 70 is also arranged on the optical path between the light source 10 and the laser focus modulation assembly 20. The collimating and expanding assembly 70 includes a first lens L1, a first optical aperture PH1 and a second lens L2; ​​the first lens L1, the first optical aperture PH1 and the second lens L2 are arranged in sequence along the laser optical path.

[0083] In one embodiment, the light source 10 can use a multi-wavelength laser as the system light source 10 to meet the testing requirements of different dye molecules or nanoparticles, and can achieve multi-wavelength excitation and multi-color imaging. The laser generated by the light source 10 first enters the collimating beam expansion component 70 composed of a pair of achromatic doublet lenses (i.e., the first lens L1, the second lens L2) and the optical pinhole (PH1) to perform beam filtering, improve the spot quality and adjust the spot diameter.

[0084] It should be noted that the size of the laser spot needs to be reasonably adjusted according to the requirements of the laser focus modulation component 20 for the incident spot size. In some embodiments, the electro-optical deflector EOD used in the present application requires the laser spot diameter to be less than 2.5 mm. Therefore, the focal length ratio of the first lens L1 and the second lens L2 is adjusted according to the spot size of the laser light to meet the optimal incident spot size requirements of the electro-optical deflector EOD. For example, when using a laser with a wavelength of 488nm, the light output diameter is about 0.7mm. At this time, we use a lens combination of L1=50mm and L2=100mm. This 2-fold relationship of the lens combination can expand the spot diameter by 2 times, which can better exert the performance of the EOD.

[0085] Furthermore, a Glan prism GP and a first half-wave plate HWP1 are sequentially arranged on the optical path between the collimating beam expander component 70 and the laser focus modulation component 20; the Glan prism GP is used to purify the polarization state of the laser; and the first half-wave plate HWP1 is used to adjust the polarization angle of the laser emitted by the Glan prism GP.

[0086] Specifically, after the laser is expanded to a suitable size, it first passes through a Glan prism GP to obtain purer linear polarized light. The Glan prism GP can purify the polarization state so that the extinction ratio of the linear polarized light reaches 100000:1. The highly pure linear polarized light can be better modulated by the electro-optic deflector EOD. Subsequently, the laser passes through an achromatic half-wave plate (i.e., the first half-wave plate HWP1), which can adjust the polarization angle of the laser to match the modulation direction of the electro-optic deflector EOD.

[0087] In one embodiment, since the laser wavelength can be changed according to the experimental requirements, the first half-wave plate HWP1 can achieve almost the same adjustment effect for lasers of different wavelengths. Whether the Glan prism GP or the first half-wave plate HWP1 adjusts the laser polarization to make the laser better adapt to the electro-optic deflector EOD, so that the electro-optic deflector EOD can perform at its best performance.

[0088] Furthermore, the three-dimensional space includes a three-dimensional space formed by three directions of X, Y, and Z; the laser focus modulation component 20 includes a first electro-optical deflector EOD1, a second half-wave plate HWP2, a second electro-optical deflector EOD2, and a tunable acoustic gradient lens TAG; the first electro-optical deflector EOD1 is used to deflect the incident laser in the X direction, the second half-wave plate HWP2 is used to change the polarization direction of the laser emitted by the first electro-optical deflector EOD1, the second electro-optical deflector EOD2 is used to deflect the laser emitted by the second half-wave plate HWP2 in the Y direction, and the tunable acoustic gradient lens TAG is used to adjust the focal length of the laser emitted by the second electro-optical deflector EOD2 in the Z-axis direction.

[0089] In one embodiment, the laser enters the first electro-optical deflector EOD1, and the first electro-optical deflector EOD1 can control the laser to be rapidly deflected in the direction perpendicular to its polarization, that is, the first electro-optical deflector EOD1 can control the laser to be deflected in the X direction. Subsequently, the laser passes through an achromatic half-wave plate (the second half-wave plate HWP2) again, the purpose of which is to rotate the polarization of the laser by 90° so that it can be rapidly deflected in the Y direction when passing through the second electro-optical deflector EOD2. In this way, the laser can be scanned in the XY plane by the EOD electro-optical deflectors whose two polarization modulation directions are placed perpendicular to each other. Afterwards, the laser enters the tunable acoustic gradient lens TAG, and the tunable acoustic gradient lens TAG can control the laser to be rapidly scanned in the Z-axis direction at a speed of up to 70kHz. Therefore, the two electro-optical deflectors EOD cooperate with a tunable acoustic gradient lens TAG to realize the rapid scanning of the three-dimensional space formed by the laser in the XYZ three-dimensional directions. This rapid scanning enables the laser to form a 1×1×2μm at the objective lens. 3 The three-dimensional preset scanning space area, or "illumination area", can be used to calculate the position of the target. The specific principle of the generation of the illumination area can be referred to Figure 3 .

[0090] Furthermore, a third lens L3 and a fourth lens L4 are arranged between the second electro-optic deflector EOD2 and the tunable acoustic gradient lens TAG; the third lens L3 and the fourth lens L4 form a beam expansion assembly, which is used to expand the laser emitted by the second electro-optic deflector EOD2.

[0091] After completing the XY plane scan, the laser passes through a pair of achromatic doublet lenses (i.e., the third lens L3 and the fourth lens L4), where L3 = 75 mm and L4 = 300 mm. Two lenses are used here to enlarge the light spot by 4 times. On the one hand, this amplification can enable the scanning range of the two electro-optical deflectors EOD to achieve a range of about 1 μm at the objective lens, which is more suitable for the tracking capability of the device; on the other hand, a larger light spot can better utilize the performance of the tunable acoustic gradient lens TAG, and the enlarged light spot can just fill the entire optical pupil surface of the tunable acoustic gradient lens TAG.

[0092] In one embodiment, as a further improvement, the device also includes a spot modulation component 80; the spot modulation component 80 is arranged on the laser light path emitted by the laser focus modulation component 20, and the spot modulation component 80 is used to scale and rotate the spot of the laser emitted by the laser focus modulation component 20.

[0093] Since the incident pupil diameter of the three-dimensional dynamic focusing component 30 is limited, the light spot needs to be smaller than 10 mm. Therefore, in order to match the incident pupil between the three-dimensional dynamic focusing, the light spot needs to be reduced again. In addition, after the laser is modulated by the electro-optical deflector EOD, a square scanning area is formed in the XY direction. At this time, the image of the light spot should be a square light spot, and the horizontal direction is the X scanning direction, and the vertical direction is the Y scanning direction. However, in actual applications, in order to save system space, a reflector M is provided on the optical path of the tunable acoustic gradient lens TAG. The reflector M will mirror the light spot. In particular, the three-dimensional dynamic focusing component 30 will also twist the direction of the square light spot during reflection, so that after entering the objective lens, it is no longer the shape when the electro-optical deflector EOD is emitted, but may form a diamond light spot. Therefore, it is also necessary to rotate the angle of the light spot through the light spot modulation component 80.

[0094] Furthermore, the spot modulation component 80 includes a fifth lens L5, a sixth lens L6 and a Dove prism DP; the fifth lens L5 and the sixth lens L6 form a beam reduction component for reducing the spot of the laser emitted by the laser focus modulation component 20, and the reduction ratio of the spot does not exceed a preset threshold; the Dove prism DP is used to rotate the spot angle of the laser emitted by the laser focus modulation component 20.

[0095] The focal planes of the two lenses L5 and L6 are conjugated, and the laser spot is reduced to match the optimal pupil diameter between the subsequent three-dimensional dynamic focusing. DP can rotate the direction of the spot. By adjusting the angle of DP, the scanning spot of EOD can be transmitted to the objective lens in the correct scanning direction. Specifically, the spot is reduced by using two achromatic doublet lenses (fifth lens L5 and sixth lens L6) again, where L5=250mm and L6=150mm. However, the smaller the spot is, the better. Excessive reduction of the spot will affect the size of the three-dimensional illumination area scanned by the electro-optic deflector EOD and the tunable acoustic gradient lens TAG, and will also reduce the imaging quality. Therefore, the scaling ratio here needs to be strictly designed to ensure that the reduction ratio of the spot does not exceed the preset threshold. In this embodiment, the spot reduction ratio of the fifth lens L5 and the sixth lens L6 is approximately 1.67 times to achieve a trade-off between various performances. In addition, a dove prism DP is added to rotate the angle of the light spot. By rotating the angle of the dove prism DP, the scanning light spot of the electro-optical deflector EOD can be restored to a standard square light spot in the horizontal X direction and the vertical Y direction.

[0096] Furthermore, the device also includes an optical path setting component 90; the optical path setting component 90 is set on the laser optical path emitted by the spot modulation component 80, and the optical path setting component 90 is used to enable the optical path to propagate along a first direction. Specifically, the optical path setting component 90 uses a dichroic mirror, and the dichroic mirror is used to reflect the laser emitted by the spot modulation component 80 to the three-dimensional dynamic focusing component 30, and transmit the fluorescent signal so that the fluorescent signal is transmitted to the fluorescent signal collection component 50 along a second direction; the first direction is the direction of the optical path reflected by the dichroic mirror, and the second direction is the direction of the optical path transmitted by the dichroic mirror.

[0097] Since the dichroic mirror DC can have different transmission and reflection properties for lasers of different wavelengths, the optical path setting component 90 of this embodiment adopts the dichroic mirror DC. The laser emitted by the light spot modulation component 80 is reflected by the dichroic mirror DC to the three-dimensional dynamic focusing component 30; and the fluorescence signal generated after the laser irradiates the object to be tracked is transmitted to the fluorescence signal collection component 50 after passing through the three-dimensional dynamic focusing component 30 in the reverse direction, so as to realize the de-scanning processing of the fluorescence signal.

[0098] Furthermore, the three-dimensional dynamic focusing assembly 30 includes a diverging lens, a relay lens, a first galvanometer MX, and a second galvanometer MY arranged in sequence along a first direction; the first galvanometer MX and the second galvanometer MY are orthogonal. Specifically, the relay lens includes a first plano-convex lens L10 and a second plano-convex lens L11, and the convex surface of the first plano-convex lens L10 and the convex surface of the second plano-convex lens L11 are arranged oppositely; the diverging lens is a plano-concave lens LZ.

[0099] In one embodiment, the laser enters the three-dimensional dynamic focusing assembly 30 after being reflected by the dichroic mirror DC. The three-dimensional dynamic focusing assembly 30 includes two orthogonal one-dimensional galvanometers (a first galvanometer MX and a second galvanometer MY), two opposite plano-convex lenses (a first plano-convex lens L10 and a second plano-convex lens L11), and a plano-concave lens LZ mounted on a linear motor. The scanning of the laser in the X and Y directions is controlled by changing the angles of the two one-dimensional galvanometers (MX, MY), and the scanning of the laser focus in the Z direction is controlled by changing the position of the plano-concave lens LZ.

[0100] Specifically, the laser first enters the plano-concave lens LZ, which acts as a diverging lens to diverge the laser, so a pair of relay lenses (L10 and L11) are placed behind it. The relay lens receives the laser emitted by the plano-concave lens LZ and transmits it to the first galvanometer MX and the second galvanometer MY to ensure that the laser spot on MX and MY will not become too large and will not exceed the range of the mirror during scanning.

[0101] Furthermore, in one embodiment, in addition to manipulating the movement of the laser focus in three dimensions, the three-dimensional dynamic focusing component 30 can also descan the fluorescent signal generated by the object to be tracked, so that the fluorescent signal passes through the three-dimensional dynamic focusing component 30 in the reverse direction, so as to ensure that the fluorescent signal is focused to the center position of the avalanche photodiode APD and does not change with the scanning of the three-dimensional dynamic focusing component 30, so that a more sensitive and economical avalanche photodiode APD can be used to receive the signal.

[0102] Furthermore, the first galvanometer MX and the second galvanometer MY are both one-dimensional galvanometers; the control and processing component 60 controls the deflection of the first galvanometer MX to move the focus of the laser in the X-axis direction, and the control and processing component 60 controls the deflection of the second galvanometer MY to move the focus of the laser in the Y-axis direction; the control and processing component 60 controls the movement of the plano-concave lens LZ to move the focus of the laser in the Z-axis direction.

[0103] In one embodiment, specifically, the first galvanometer MX and the second galvanometer MY are respectively connected to independent high-speed motors, and the high-speed motors of the first galvanometer MX and the second galvanometer MY are driven by the control and processing component 60 to control the deflection of the first galvanometer MX to move the focus of the laser in the X-axis direction, and control the deflection of the second galvanometer MY to move the focus of the laser in the Y-axis direction. In addition, the plano-concave lens LZ is placed on the driving motor, and its position is controlled by the control and processing component 60 to control the focus position movement of the laser in the Z direction.

[0104] In one embodiment, if Figure 5As shown, it shows a simplified diagram of the three-dimensional dynamic focusing component 30 controlling the laser, Xmotor is the first galvanometer motor, Y motor is the second galvanometer motor, and Z motor is the plano-concave lens motor. This figure more clearly depicts the working principle of the three-dimensional dynamic focusing component 30. When a parallel laser beam enters the component, it first enters LZ, which controls the focus and divergence of the laser, thereby controlling the movement of the laser focus in the Z direction. It is then transmitted to the two galvanometers MX and MY by the relay lens composed of L10 and L11. The two orthogonal galvanometers MX and MY control the movement of the laser focus in the XY directions. Figure 6 The movement of the laser focus above the objective lens OL is reflected in the figure, wherein the XY direction movement controlled by MX and MY can make the laser focus move in a two-dimensional plane. The Z-axis direction movement controlled by LZ can control the laser focus to move in the Z direction. The above control method can achieve precise manipulation of the laser position. Specifically, the three-dimensional dynamic focusing component 30 is used to control the laser focus to perform XY two-dimensional slow scanning on the focal plane to find the object to be tracked that may appear in the area. Here, the first galvanometer MX and the second galvanometer MY are controlled to scan within the range of ±10μm, and the light intensity signal during the scanning process is detected in real time. When the target does not appear during the search and scanning process, no fluorescence is generated, and the light intensity is always at a low level. Once the search scan finds the target, the fluorescence signal is detected by the APD, and the light intensity increases sharply. Therefore, the light intensity is used as the tracking threshold here. When the light intensity reaches the set threshold, it indicates that the object to be tracked appears in the illumination area, the active feedback tracking program is started, the real-time calculation of the target position begins, and the three-dimensional dynamic focusing component 30 is used to control the movement of the laser focus to follow the target position to achieve feedback tracking.

[0105] When EOD and TAG are working, the laser focus above the objective lens will no longer be a simple focusing point, but a three-dimensional illumination area will be formed due to their rapid scanning modulation. Figure 2 (a) and Figure 7In (a), (b), (c), and (d), according to the above-mentioned tracking principle, when the object to be tracked is in the illumination area, the photon signal provided by APD and the laser focus position information provided by EOD and TAG can be used to calculate the precise position of the object to be tracked in the illumination area. Therefore, the first step of tracking is to calculate the position of the target (x1, y1, z1). Then, based on this coordinate position information, the three-dimensional focusing component is controlled to perform active feedback compensation movement, and the illumination area is moved to follow the target. When the object to be tracked moves again, the position of the target (x2, y2, z2) is calculated again. Finally, according to the new coordinate position, the three-dimensional dynamic focusing component 30 is controlled to move the illumination area to follow the target. The cycle is repeated to achieve real-time three-dimensional tracking of the object to be tracked. Finally, the moving position of the three-dimensional dynamic focusing component 30 is reconstructed, which is the three-dimensional motion trajectory of the object to be tracked.

[0106] In one embodiment, further, the imaging component 40 also includes a laser scanning tube lens TL, which is arranged on the optical path between the objective lens OL and the three-dimensional dynamic focusing component 30, and the laser scanning tube lens TL is located at the focal length conjugate point of the three-dimensional dynamic focusing component 30, and the focal length of the objective lens OL is conjugate with the focal length of the laser scanning tube lens TL.

[0107] In one embodiment, after being emitted from the three-dimensional dynamic focusing component 30, the laser enters the laser scanning tube lens TL, which is strictly placed at the focal conjugate point of the three-dimensional dynamic focusing component 30 and is conjugate with the focal length of the objective lens OL, thereby minimizing aberrations and achieving flat-field scanning.

[0108] In one embodiment, further, the imaging component 40 also includes a stage and a controller; the stage is used to place the sample, and the stage adopts a piezoelectric nano-platform; the controller is used to control the movement of the piezoelectric nano-platform; the controller is electrically connected to the control and processing component 60.

[0109] In one embodiment, specifically, the piezoelectric nano-platform includes NXY and NZ, NXY means that the sample can be controlled to move in the X direction and the Y direction, and NZ means that the sample can be controlled to move in the Z direction. The precise movement of NXY and NZ is controlled by the controller respectively. In this embodiment, the controller and the field programmable gate array FPGA are connected in communication. When in use, the controller can send the movement information of the X, Y, and Z directions to the field programmable gate array FPGA. When necessary, the sample is controlled to move or scan with nanometer precision by the piezoelectric nano-platform.

[0110] In one embodiment, further, the fluorescence signal collection component 50 is arranged on the optical path in the second direction. After being collected by the objective lens OL, the fluorescence signal passes through the three-dimensional dynamic focusing component 30 in reverse to the optical path setting component 90. The optical path setting component 90 is also used to transmit the fluorescence signal to the fluorescence signal collection component 50 along the second direction.

[0111] In one embodiment, specifically, the sample fluorescence signal is collected by the objective lens OL, and is descanned through the three-dimensional dynamic focusing component 30. Subsequently, the fluorescence signal passes through the dichroic mirror DC and is transmitted to the fluorescence signal collection component 50 along the second direction. The fluorescence signal is collected by the fluorescence signal collection component 50, and the fluorescence signal is converted into an electrical signal and transmitted to the control and processing component 60 for further processing. The descanning process specifically refers to passing the sample fluorescence signal collected by the objective lens OL through the three-dimensional dynamic focusing component 30 again in the reverse direction to achieve the purpose of descanning, so as to ensure that the fluorescence signal is focused on the center position of the fluorescence signal collection component 50 and does not change with the scanning of the three-dimensional dynamic focusing component 30, so as to ensure the stability of the fluorescence signal, thereby improving the imaging quality.

[0112] In one embodiment, further, the fluorescence signal collection component 50 includes an avalanche photodiode APD; the device also includes a bandpass filter F and a spatial filtering component; the bandpass filter F is arranged on the fluorescence light path along the second direction emitted by the optical path setting component 90; the spatial filtering component includes a seventh lens L7, a second optical aperture PH2 and an eighth lens L8, and the fluorescence signal passing through the bandpass filter F passes through the seventh lens L7, the second optical aperture PH2 and the eighth lens L8 in sequence and then enters the avalanche photodiode APD.

[0113] In this embodiment, the fluorescence signal collection component 50 uses an avalanche photodiode APD. After the fluorescence signal passes through the dichroic mirror DC and the bandpass filter F, it can block the laser purification signal. The purified fluorescence signal is relayed and spatially filtered through the seventh lens L7, the second optical aperture PH2 and the eighth lens L8 in sequence, and is collected by the highly sensitive avalanche photodiode APD. The spatial filtering component composed of the seventh lens L7, the second optical aperture PH2 and the eighth lens L8 can reduce the defocus signal and improve the imaging resolution.

[0114] In one embodiment, further, a ninth lens L9 is provided on the optical path between the spatial filter component and the avalanche photodiode APD, and the ninth lens L9 is used to focus the fluorescent signal emitted by the spatial filter component and transmit it to the avalanche photodiode APD. Since the photosensitive element area of ​​the avalanche photodiode APD is small, the ninth lens L9 is added in this embodiment, and the fluorescent signal is focused by the ninth lens L9 to enter the avalanche photodiode APD.

[0115] For further reference, Figure 3 and Figure 4 The control and processing component 60 controls the laser focus modulation component 20 to make the laser focus scan in the preset scanning space area, specifically including:

[0116] Combination Figure 4 As shown in (a), the laser focus modulation component 20 is controlled to perform grid scanning in the form of "Knight's Parade" in the XY plane;

[0117] Combination Figure 4 As shown in (b), the laser focus modulation component 20 is controlled to perform sinusoidal scanning in the Z direction.

[0118] The focal spot modulation of the illumination area requires not only high-speed scanning to construct a preset scanning space area, but also accurate knowledge of the specific information of each scanning position. This includes the position of the scanning point in the three-dimensional space at each moment and the number of photons collected by the APD at this moment. Specifically, this embodiment uses two electro-optical deflectors (EOD1, EOD2) and a tunable acoustic gradient lens TAG to modulate the laser focus at high speed, so that it can quickly scan in the three-dimensional space to form a dynamic illumination area, such as Figure 3 As shown in FIG. 1 , EOD1 and EOD2 control the focus spot to perform grid scanning in the form of a “Knight’s tour” on the XY plane. The scanning sequence is as follows: Figure 4 As shown in (a), this scanning form can ensure more uniform sampling on the two-dimensional plane. The entire scanning range is 1×1μm 2 The dwell time of each grid point is 20μs. The EOD is controlled in real time through the field-compilable logic gate array FPGA, and the specific scanning position of the laser focus is recorded. TAG can control the focus spot to perform sinusoidal scanning in the Z direction, with a scanning range of about 2μm and a scanning frequency of 70kHz. Figure 4 Similarly, by reading the three output phases of the TAG controller through FPGA, interpolating and fitting these three phases to the sine function, the position of the laser focal spot in the Z direction at each moment can be obtained. Therefore, through EOD and TAG, the focal spot can be 1×1×2μm in three-dimensional space. 3 Dynamic scanning can be performed, and the specific position of the laser focus in the scanning illumination area can be recorded in real time.

[0119] Subsequently, an avalanche photodiode APD is used to detect the fluorescence signal of the object to be tracked, and the detected photon signal is transmitted to the FPGA. The strongest photon signal can only be stimulated when the laser focus happens to illuminate the object to be tracked. Therefore, by calculating the relationship between the number of photons and the position of the laser focus, the specific position of the object to be tracked in the illumination area can be obtained. When it deviates from the center of the illumination area, the FPGA calculates the offset and outputs a corresponding feedback signal to the three-dimensional dynamic focusing component 30. The three-dimensional dynamic focusing component 30 actively compensates for this offset by controlling the movement of the entire illumination area, and always keeps the object to be tracked in the center of the illumination area, thereby realizing real-time three-dimensional active feedback tracking of the target particles.

[0120] As a further improvement of this embodiment, the control and processing component 60 is also used to calibrate the three-dimensional dynamic focusing component 30; wherein, the calibration of the three-dimensional dynamic focusing component 30 specifically includes:

[0121] Focus the laser on the sample;

[0122] Sending instructions to the controller to control the piezoelectric nano-stage to control the sample to move a preset distance in the X, Y and Z directions respectively, so that the sample leaves the preset scanning space area;

[0123] The three-dimensional dynamic focusing assembly 30 is controlled to move a preset distance in the three directions of X, Y and Z so that the sample is relocated in the preset scanning space area.

[0124] The three-dimensional dynamic focusing component 30 is the key to realize the active feedback function in this solution, and it is crucial to calibrate the relationship between the applied voltage of the three axes and the laser focus position. This can obtain a more accurate scanning position, which is conducive to improving imaging quality and accuracy.

[0125] Specifically, before calibration, the entire optical system is finely collimated and conjugated. The position of the plano-concave lens LZ in the three-dimensional dynamic focusing assembly 30 is adjusted to z=0, and the laser scanning tube lens TL is strictly conjugated with the three-dimensional dynamic focusing assembly 30 to ensure that the laser remains parallel after being emitted from the scanning tube lens TL. Similarly, the back focal plane of the objective lens OL is also conjugated with the laser scanning tube lens TL.

[0126] This embodiment uses a piezoelectric nano-platform as a calibration standard to calibrate various parameters of the three-dimensional dynamic focusing component 30, including precise positioning, step size and scanning range. The specific calibration process is as follows: First, focus the laser on a 200nm fluorescent bead and scan and image it. Then use the piezoelectric nano-platform to control the sample to move a certain distance in the X, Y and Z directions respectively, so that the fluorescent bead leaves the laser focus. Subsequently, voltage is applied to the motors of the X, Y and Z axes of the three-dimensional dynamic focusing component 30 to control the laser to refocus on the fluorescent bead. By carefully comparing the relationship between the voltage and the moving distance of the piezoelectric nano-platform, the calibration relationship between the laser focus change and the voltage applied by the three-dimensional dynamic focusing component 30 in the X, Y and Z directions can be obtained.

[0127] After calibration, the applied voltage of the three-dimensional dynamic focusing assembly 30 and the displacement in three directions will be highly linearly matched. This linear matching ensures the accuracy of the three-dimensional dynamic focusing assembly 30 in controlling the laser position and avoids further data processing.

[0128] refer to Figure 8 An embodiment provides a three-dimensional active feedback tracing imaging method, using the above three-dimensional active feedback tracing imaging device, the method comprises the following steps:

[0129] Step 100: Control the light source 10 to emit laser light; the laser light is used to excite the object to be tracked;

[0130] Step 200: Control the laser focus modulation component 20 to modulate the laser in three directions of X, Y, and Z, so that the focus of the laser after being emitted from the objective lens scans at various scanning positions within a preset scanning space area;

[0131] Step 300: Control the fluorescence signal collection component 50 to collect the fluorescence signal generated after the laser excites the object to be tracked; the fluorescence signal collection component 50 generates an electrical signal according to the fluorescence signal;

[0132] Step 400: Calculate the position deviation of the object to be tracked and the reference position in the preset scanning space area according to the electrical signal and the position of the laser focus;

[0133] Step 500: Control the three-dimensional dynamic focusing component 30 to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at the reference position in the preset scanning space area.

[0134] The three-dimensional active feedback tracing imaging device of this embodiment, and its imaging method are specifically as follows:

[0135] The control and processing component 60 controls the light source 10 to emit a laser of the target wavelength, and the laser focus modulation component 20 composed of the first electro-optical deflector EOD1, the second electro-optical deflector EOD2 and a tunable acoustic gradient lens TAG controls the laser to scan rapidly in the three directions of X, Y and Z. The scanning of the laser can form a 1×1×2μm 3 Three-dimensional illumination area. It can be understood that the laser focus modulation component 20 modulates the ordinary laser focus originally at the objective lens into a three-dimensional laser area (that is, the preset scanning space area). Since only the object to be tracked in the three-dimensional illumination area can be excited by the laser, a fluorescent signal is generated and collected by the APD. The control and processing component 60 obtains the electrical signal generated by the APD and the scanning position of the laser focus of the laser focus modulation component 20, and uses the single-molecule positioning algorithm to analyze and calculate the scanning position and the signal collected by the APD in real time. It can determine the specific position of the object to be tracked in the illumination area, and calculate its distance from the center of the illumination area, thereby obtaining a coordinate (x, y, z). The coordinate takes the center of the illumination area as the origin and is referenced to the center of the illumination area. Figure 2 (a). Therefore, the coordinate information is the position of the object to be tracked that deviates from the center of the illumination area (i.e., the reference position in the preset scanning space area). The above solution of the present application can achieve a three-dimensional positioning accuracy of 100nm.

[0136] The distance of the object to be tracked from the center of the illumination area can be obtained based on the calculated coordinate information. At this time, the three-dimensional dynamic focusing component 30 is used to control the movement of the illumination area, feedback compensates for the deviation, and always keeps the target in the center of the illumination area. When the target moves again and deviates from the center of the illumination area, the above steps are repeated to calculate the deviation of the target in real time and actively feedback the deviation to compensate for the deviation.

[0137] Through the above tracking imaging method, it can be ensured that the object to be tracked is always actively tracked and in the center of the lighting area. The movement of the lighting area is recorded in real time by the control and processing module, because the movement of the lighting area reflects the movement of the target in three-dimensional space. The recorded lighting area movement data is analyzed by MATLAB and a three-dimensional trajectory image is drawn. At this point, the real-time three-dimensional motion trajectory information of the target is obtained, thereby realizing high-precision and high-speed real-time three-dimensional active feedback tracking imaging of the target.

[0138] In addition, in order to better verify the feasibility of the solution of the present application, the inventors performed real-time three-dimensional tracing imaging on polystyrene fluorescent microspheres with a diameter of 200nm. Among them, polystyrene fluorescent microspheres are standard samples made of polystyrene wrapped with dyes, which are often used for performance testing of fluorescence microscopes. Here, we configure 200nm polystyrene fluorescent microspheres in a 70% volume concentration glycerol solution (the solvent is water and the solute is glycerol). Glycerol can provide higher viscosity for the entire sample and is used to simulate areas with higher viscosity under physiological conditions. Fig. 9 As shown, the device and method of the present application successfully performed three-dimensional tracing on 200nm polystyrene fluorescent microspheres. Fig. 9 (a) shows the three-dimensional motion trajectory of the fluorescent microspheres. The trajectory lines are marked with grayscale to indicate the time change. In order to more clearly show the changes of the fluorescent microspheres in the three axes, Fig. 9 (c1), (c2), and (c3) show the curves of the X, Y, and Z axis positions changing with time. Within the tracing time of about 20 seconds, the position information of the fluorescent microspheres in three-dimensional space is accurately recorded. In addition, the light intensity changes of the target can also be recorded in real time, such as Fig. 9 (b) The light intensity of the fluorescent microspheres was weakened during the tracing process, which may be caused by the fluorescence of the microspheres being bleached.

[0139] Subsequently, the fluorescent microspheres were placed in a pure aqueous solution, and the three-dimensional motion was tracked in real time using the device and method of the present application. Fig.10 As shown. Among them, Fig.10 (a) shows the three-dimensional motion trajectory of the fluorescent microspheres in the aqueous solution. The trajectory lines are marked with grayscale to indicate the time change. In order to more clearly show the changes of the fluorescent microspheres in the three axes, Fig.10 (c1), (c2), and (c3) show the curves of the X, Y, and Z axis positions changing with time. During the tracing time of about 35 seconds, the position information of the fluorescent microspheres in three-dimensional space was accurately recorded. Similarly, the changes in the light intensity of the target were also recorded, such as Fig.10 (b). The light intensity of the fluorescent microspheres during the tracing process showed an overall weakening trend, which may be caused by the bleaching of the fluorescence of the microspheres. It is worth noting that the movement speed of the fluorescent microspheres in the aqueous solution is faster and more difficult to capture. However, based on the tracking speed and accuracy advantages of the present application, it is entirely possible to achieve accurate tracking of targets in aqueous solutions.

[0140] Through experiments, it can be found that the implementation of the present application scheme can achieve various indicators that are superior to the existing technology. Specifically, for relevant imaging parameters: the time resolution of three-dimensional dynamic tracing imaging can reach 1ms, and the three-dimensional tracing speed can reach 4μm 2 / s, and the three-dimensional positioning accuracy reaches 100nm. In addition, although only 200nm fluorescent microspheres were used for testing in this experiment, this implementation case can fully perform real-time three-dimensional tracing imaging of single fluorescent molecules. The tracing target is not limited to fluorescent particles, and tracing imaging can be performed for single fluorescent molecules or single nanoparticles that do not fluoresce but scatter light.

[0141] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A three-dimensional active feedback tracing imaging device, characterized in that: include: A light source, used to emit laser light, wherein the laser light is used to excite the object to be tracked; A laser focus modulation component is arranged on the laser optical path, and is used to modulate the laser in three-dimensional space so that the focus of the laser after being emitted from the objective lens scans at various scanning positions in a preset scanning space area; A three-dimensional dynamic focusing component, arranged on the laser light path emitted by the laser focus modulation component, for adjusting the spatial position of the preset scanning space area; An imaging component is arranged on the optical path of the three-dimensional dynamic focusing component, and the imaging component includes an objective lens. After the laser emitted by the three-dimensional dynamic focusing component passes through the objective lens, the laser excites the object to be tracked in the sample so that the object to be tracked generates a fluorescent signal; A fluorescence signal collection component, used to collect the fluorescence signal and process it into a corresponding electrical signal; A control and processing component is used to control the laser focus modulation component to make the laser focus scan in a preset scanning space area, and calculate the position deviation between the object to be tracked and the reference position in the preset scanning space area according to the position of the laser focus and the electrical signal, and control the three-dimensional dynamic focusing component to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at the reference position in the preset scanning space area.

2. The three-dimensional active feedback tracing imaging device according to claim 1, characterized in that: The control and processing component is also used to record the spatial position of the preset scanning space area at different times, and obtain the three-dimensional movement trajectory of the object to be tracked according to the spatial position of the preset scanning space area at different times.

3. The three-dimensional active feedback tracing imaging device according to claim 1, characterized in that: A collimating beam expansion component is also arranged on the optical path between the light source and the laser focus modulation component, and the collimating beam expansion component includes a first lens, a first optical pinhole and a second lens; the first lens, the first optical pinhole and the second lens are arranged in sequence along the laser optical path.

4. The three-dimensional active feedback tracing imaging device according to claim 3, characterized in that: A Glan prism and a first half-wave plate are sequentially arranged on the optical path between the collimating beam expansion component and the laser focus modulation component; The Glan prism is used to purify the polarization state of the laser; The first half-wave plate is used to adjust the polarization angle of the laser light emitted from the Glan prism.

5. The three-dimensional active feedback tracing imaging device according to claim 1, characterized in that: The three-dimensional space includes a three-dimensional space formed by three directions of X, Y, and Z; the laser focus modulation component includes a first electro-optical deflector, a second half-wave plate, a second electro-optical deflector, and a tunable acoustic gradient lens; The first electro-optical deflector is used to deflect the incident laser in the X direction, the second half-wave plate is used to change the polarization direction of the laser emitted by the first electro-optical deflector, the second electro-optical deflector is used to deflect the laser emitted by the second half-wave plate in the Y direction, and the tunable acoustic gradient lens is used to adjust the focal length of the laser emitted by the second electro-optical deflector in the Z-axis direction.

6. The three-dimensional active feedback tracing imaging device according to claim 5, characterized in that: A third lens and a fourth lens are further arranged between the second electro-optical deflector and the tunable acoustic gradient lens; The third lens and the fourth lens form a beam expansion component, and the beam expansion component is used to expand the laser emitted by the second electro-optical deflector.

7. The three-dimensional active feedback tracing imaging device according to claim 1, characterized in that: It also includes a spot modulation component, which is arranged on the laser light path emitted by the laser focus modulation component, and the spot modulation component includes a fifth lens, a sixth lens and a dove prism; The fifth lens and the sixth lens form a beam reduction assembly, and the beam reduction assembly is used to reduce the spot of the laser emitted by the laser focus modulation assembly, and the reduction ratio of the spot does not exceed a preset threshold; The dove prism is used to rotate the spot angle of the laser light emitted by the laser focus modulation component.

8. The three-dimensional active feedback tracing imaging device according to claim 7, characterized in that: It also includes an optical path setting component; the optical path setting component is a dichroic mirror, and the dichroic mirror is arranged on the laser light path emitted by the light spot modulation component; The dichroic mirror is used to reflect the laser emitted by the spot modulation component to the three-dimensional dynamic focusing component so that the light path can propagate along a first direction, and to transmit the fluorescent signal so that the fluorescent signal is transmitted to the fluorescent signal collection component along a second direction; the first direction is the direction of the light path reflected by the dichroic mirror, and the second direction is the direction of the light path transmitted by the dichroic mirror.

9. The three-dimensional active feedback tracing imaging device according to claim 8, characterized in that: The three-dimensional dynamic focusing component includes a diverging lens, a relay lens, a first galvanometer and a second galvanometer, which are sequentially arranged along a first direction; the first galvanometer and the second galvanometer are orthogonal.

10. The three-dimensional active feedback tracing imaging device according to claim 9, characterized in that: The relay lens comprises a first plano-convex lens and a second plano-convex lens, wherein the convex surface of the first plano-convex lens and the convex surface of the second plano-convex lens are arranged opposite to each other; The diverging lens is a plano-concave lens.

11. The three-dimensional active feedback tracing imaging device according to claim 9, characterized in that: The first galvanometer and the second galvanometer are both one-dimensional galvanometers; the control and processing component controls the deflection of the first galvanometer to move the focus of the laser in the X-axis direction, and the control and processing component controls the deflection of the second galvanometer to move the focus of the laser in the Y-axis direction; the control and processing component controls the movement of the plano-concave lens to move the focus of the laser in the Z-axis direction.

12. The three-dimensional active feedback tracing imaging device according to claim 1, characterized in that: The imaging component also includes a laser scanning tube lens, which is arranged on the optical path between the objective lens and the three-dimensional dynamic focusing component, and the laser scanning tube lens is located at the focal length conjugate point of the three-dimensional dynamic focusing component, and the focal length of the objective lens is conjugate with the focal length of the laser scanning tube lens.

13. The three-dimensional active feedback tracing imaging device according to claim 1, characterized in that: The imaging assembly also includes a stage and a controller; The stage is used to place samples, and the stage adopts a piezoelectric nano-platform; The controller is used to control the movement of the piezoelectric nano-platform; the controller is electrically connected to the control and processing component.

14. The three-dimensional active feedback tracing imaging device according to claim 8, characterized in that: The fluorescence signal collection component is arranged on the optical path in the second direction. After being collected by the objective lens, the fluorescence signal passes through the three-dimensional dynamic focusing component in reverse to the optical path setting component. The optical path setting component is also used to transmit the fluorescence signal along the second direction to the fluorescence signal collection component.

15. The three-dimensional active feedback tracing imaging device according to claim 14, characterized in that: The fluorescence signal collection component includes an avalanche photodiode; the device also includes a bandpass filter and a spatial filtering component; The bandpass filter is arranged on the fluorescent light path emitted by the light path setting component along the second direction; The spatial filtering component includes a seventh lens, a second optical pinhole and an eighth lens. The fluorescent signal passing through the bandpass filter sequentially passes through the seventh lens, the second optical pinhole and the eighth lens before entering the avalanche photodiode.

16. The three-dimensional active feedback tracing imaging device according to claim 5, characterized in that: The control and processing component controls the laser focus modulation component to make the laser focus scan in a preset scanning space area, including: Controlling the laser focus modulation component to perform grid scanning in the form of "Knight's Parade" on the XY plane; The laser focus modulation component is controlled to perform sinusoidal scanning in the Z direction.

17. The three-dimensional active feedback tracing imaging device according to claim 13, characterized in that: The control and processing component is also used to calibrate the three-dimensional dynamic focusing component; the calibration of the three-dimensional dynamic focusing component includes: focusing the laser light onto a sample; Sending instructions to the controller to control the piezoelectric nano-platform to control the sample to move a preset distance in the X, Y and Z directions respectively, so that the sample leaves the preset scanning space area; The three-dimensional dynamic focusing component is controlled to move a preset distance in the three directions of X, Y and Z so that the sample is relocated in the preset scanning space area.

18. A three-dimensional active feedback tracing imaging method, characterized in that: Using the three-dimensional active feedback tracing imaging device described in any one of claims 1 to 17, the method comprises: Controlling the light source to emit laser light; the laser light is used to excite the object to be tracked; Controlling the laser focus modulation component to modulate the laser in three-dimensional space so that the focus of the laser after being emitted from the objective lens scans at various scanning positions in a preset scanning space area; Controlling the fluorescence signal collection component to collect the fluorescence signal generated after the laser excites the object to be tracked; the fluorescence signal collection component generates an electrical signal according to the fluorescence signal; Calculating a position deviation between the object to be tracked and a reference position in the preset scanning space area according to the electrical signal and the position of the laser focus; The three-dimensional dynamic focusing component is controlled to adjust the spatial position of the preset scanning space area according to the position deviation, so that the object to be tracked is always located at a reference position within the preset scanning space area.

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