CN114689558B

CN114689558B - Background-free wide-field and low-loss super-resolution dual-purpose imaging device and imaging method

Info

Publication number: CN114689558B
Application number: CN202210602811.3A
Authority: CN
Inventors: 张琪; 燕一皓; 殷俊; 石发展; 杜江峰
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-10-28
Anticipated expiration: 2042-05-31
Also published as: CN114689558A

Abstract

The invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device and an imaging method, wherein the device comprises: a first pump light unit for generating a first wavelength beam; a second pump light unit for generating a second wavelength beam; the device comprises a sample unit, a signal processing unit and a signal processing unit, wherein the sample unit is used for placing a sample to be detected comprising a fluorescent beacon and is used for receiving a first wavelength light beam, initializing the charge state of the fluorescent beacon, receiving a second wavelength light beam, modulating the charge state of the fluorescent beacon and outputting at least one of a first wavelength light beam signal, a second wavelength light beam signal and a fluorescent photon signal corresponding to the fluorescent beacon with different charge states, which are emitted by the fluorescent beacon; the collecting unit is used for outputting the target photon signal obtained through filtering processing; and the control unit is used for receiving the target photon signal and generating a background-free wide-field image or a low-loss super-resolution image according to the target photon signal.

Description

Background-free wide-field and low-loss super-resolution dual-purpose imaging device and imaging method

Technical Field

The invention relates to the field of biological microscopic imaging, in particular to a background-free wide-field and low-loss super-resolution dual-purpose imaging device and an imaging method.

Background

The development of imaging technology has enabled fluorescence microscopy to study substances of interest and kinetics within cells, even at the single molecule level. However, sensitivity and resolution of in vivo fluorescence imaging are often limited by autofluorescence and other background noise due to overlap between the fluorescent probe and the background emission spectrum.

Disclosure of Invention

Accordingly, the present invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device and an imaging method.

One aspect of the present invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device, comprising: a first pump light unit for generating a first wavelength beam; a second pump light unit for generating a second wavelength light beam including a second wavelength gaussian light beam or a second wavelength hollow light beam; a sample unit, on which a sample to be tested including a fluorescent beacon is placed, the sample unit being configured to receive the first wavelength light beam, initialize a charge state of the fluorescent beacon, receive the second wavelength light beam, modulate the charge state of the fluorescent beacon, and output at least one of a first wavelength light beam signal, a second wavelength light beam signal, and a fluorescent photon signal corresponding to the fluorescent beacon in a different charge state emitted via the fluorescent beacon; the collecting unit is used for receiving at least one of the first wavelength light beam signal, the second wavelength light beam signal and the fluorescence photon signal and outputting a target photon signal obtained through filtering processing; and the control unit is used for receiving the target photon signal and generating a background-free wide-field image or a low-loss super-resolution image according to the target photon signal.

Optionally, the first pump light unit includes: a first light source for generating the first wavelength light beam; a first pulse generator for receiving a first sequence of pulses transmitted by the control unit; a first modulator for controlling the first wavelength beam in accordance with the first sequence of pulses to produce a sequenced first wavelength beam; a first fiber coupled-collimating system for receiving the first wavelength beam and outputting a first collimated beam.

Optionally, the second pump light unit includes: a second light source for generating the second wavelength gaussian beam; a second pulse generator for receiving a second train of pulses transmitted by the control unit; a second modulator for controlling the second wavelength beam in accordance with the second sequence of pulses to produce a sequenced second wavelength beam; and the second fiber coupling-collimating system is used for receiving the second wavelength light beam and outputting a second collimated light beam.

Optionally, the first pump light unit may further include at least one of: the optical filter is used for adjusting the power of the first wavelength light beam; a mirror for adjusting the direction of the first wavelength beam; the first adjustable beam expander is used for adjusting the diameter of the first wavelength light beam; a first quarter wave plate for converting the linearly polarized first wavelength light beam into a circularly polarized first wavelength light beam.

Optionally, the second pump light unit may further include at least one of: the half-wave plate is used for adjusting the polarization direction of the second wavelength light beam; the polarization beam splitter is used for combining the half-wave plate and adjusting the power of the second wavelength light beam; the second adjustable beam expander is used for adjusting the diameter of the second wavelength light beam; the vortex phase plate is used for converting the second wavelength Gaussian beam into the second wavelength hollow beam; a second quarter wave plate for converting the linearly polarized second wavelength beam into a circularly polarized second wavelength beam; 1:1 non-polarizing beam splitter for splitting the second wavelength beam in a 1:1, splitting the beams in a power ratio to obtain a first split beam and a second split beam; an optical power meter for measuring the power of the first split beam or the second split beam and sending the measurement result to the control unit.

Optionally, the background-free wide-field and low-loss super-resolution dual-purpose imaging device further includes: and the beam combining unit is used for receiving the first wavelength light beam and the second wavelength light beam and outputting a combined light beam.

Optionally, the beam combining unit includes at least one of: the quick deflection mirror is used for adjusting the direction of the second wavelength light beam and adjusting the focus position of the second wavelength light beam after being focused by the microscope lens; the variable focal length lens is used for adjusting the focal plane position of the second wavelength light beam after being focused by the micro lens; a lens group for adjusting a position, a direction and a divergence angle of the second wavelength light beam; a short pass dichroic mirror for transmitting said first wavelength light beam and reflecting said second wavelength light beam, and receiving at least one of said first wavelength light beam signal, said second wavelength light beam signal and said fluorescence photon signal, filtering said first wavelength light beam, reflecting at least one of said second wavelength light beam signal and said fluorescence photon signal; and the long-pass dichroic mirror is used for transmitting the second wavelength light beam, receiving at least one of the second wavelength light beam signal and the fluorescence photon signal, filtering the second wavelength light beam, and reflecting the fluorescence photon signal.

Optionally, the sample unit comprises: the fluorescent beacon of the sample to be detected comprises nano diamond particles; the microscope lens is used for receiving the first wavelength light beam and/or the second wavelength light beam, focusing the first wavelength light beam and/or the second wavelength light beam on the sample to be detected, and collecting at least one of a first wavelength light beam signal, a second wavelength light beam signal and a fluorescence photon signal emitted by a fluorescence beacon in the sample to be detected; the piezoelectric ceramic displacement platform is used for moving the pixel point to be detected of the sample to be detected to a preset position with nanometer precision; and the temperature control box is used for maintaining the stability of the temperature and the humidity of the environment where the sample to be detected is located.

Optionally, the collection unit comprises: the filter plate group is used for receiving at least one of the first wavelength light beam signal, the second wavelength light beam signal and the fluorescence photon signal, and performing filtering processing to obtain the target photon signal; a CCD camera for collecting the target photon signal and sending the target photon signal to the control unit; and the single photon counter is used for collecting the target photon signal and sending the target photon signal to the control unit.

Another aspect of the present invention provides a low-loss super-resolution imaging method, including: for each pixel point to be detected in the sample to be detected, acquiring N first fluorescence intensities and N second fluorescence intensities based on a background-free wide-field and low-loss super-resolution dual-purpose imaging device, wherein the sample to be detected includes a plurality of pixel points to be detected, the first fluorescence intensity includes a fluorescence intensity under the condition that a first initialization fluorescence beacon is irradiated by the first wavelength light beam, the second fluorescence intensity includes a fluorescence intensity under the condition that a first enhanced fluorescence beacon is simultaneously irradiated by the first wavelength light beam and the second wavelength light beam, the first initialization fluorescence beacon is obtained by irradiating the fluorescence beacon in the sample to be detected based on the first wavelength light beam, the first enhanced fluorescence beacon is obtained by irradiating the first initialization fluorescence beacon based on the first wavelength light beam and the second wavelength light beam, and N is a positive integer; determining the target fluorescence intensity corresponding to the pixel point to be detected according to the average value of the N second fluorescence intensities; and determining a low-loss super-resolution image corresponding to the sample to be detected according to the fluorescence intensities of the targets corresponding to the pixel points to be detected.

Another aspect of the invention provides a background-free wide-field imaging method, comprising: acquiring a first image and a second image which are obtained by shooting aiming at the sample to be detected based on a background-free wide field and low-loss super-resolution dual-purpose imaging device, wherein the first image comprises an image obtained under the condition that the sample to be detected comprising a second initialization fluorescent beacon is irradiated by the first wavelength light beam, the second image comprises an image obtained under the condition that the image to be detected comprising a second enhancement fluorescent beacon is irradiated by the first wavelength light beam and the second wavelength light beam, the second initialization fluorescent beacon is obtained by irradiating the fluorescent beacon in the sample to be detected based on the first wavelength light beam, and the second enhancement fluorescent beacon is obtained by irradiating the second initialization fluorescent beacon based on the first wavelength light beam and the second wavelength light beam; and determining a background-free wide-field image corresponding to the sample to be detected according to the difference value of the second image and the first image.

According to the embodiment of the invention, the charge state of the fluorescent beacon in the sample to be detected is initialized by generating the first wavelength light beam by using the first pump light unit, and the charge state of the fluorescent beacon is modulated by generating the second wavelength light beam by using the second pump light unit. Because the fluorescent signals of the fluorescent beacons in different charge states are different, background-free imaging and super-resolution imaging can be performed on the sample to be detected according to the collected target photon signals. The technical scheme of the invention utilizes low-power continuous infrared light to modulate the number of the radiation photons of the fluorescent beacon, and has the advantages of no influence on the property of a sample to be detected, good biocompatibility, resistance to biological spontaneous background fluorescence, stable fluorescent signal, high spatial resolution, high signal-to-back ratio, simple structure of an optical system and an electronic system, and low operation cost and cost.

Drawings

In order to more clearly illustrate the embodiments or technical solutions of the present invention, the drawings used in the embodiments or technical solutions of the present invention will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 schematically illustrates a block diagram of a background-free wide-field and low-loss super-resolution dual-purpose imaging device according to an embodiment of the invention;

FIG. 2 schematically illustrates an optical path diagram of a background-free wide-field and low-loss super-resolution dual-purpose imaging device according to an embodiment of the invention;

FIG. 3 schematically illustrates a flow diagram of a low-loss super-resolution imaging method according to an embodiment of the invention;

FIG. 4 schematically illustrates a flow diagram of a background-free wide-field imaging method according to an embodiment of the invention;

FIG. 5 schematically illustrates a schematic diagram of low power continuous light assisted imaging according to an embodiment of the invention;

fig. 6 schematically illustrates a schematic view of a scene imaging nanodiamond particles according to an embodiment of the invention;

fig. 7 schematically shows a sequence diagram of control signals versus read signals when imaging nanodiamond particles according to an embodiment of the invention;

fig. 8A schematically illustrates a low-loss super-resolution imaging map of nano-diamond particles imaged using a conventional focused imaging method according to an embodiment of the invention;

FIG. 8B schematically shows a low loss super-resolution image of nanodiamond particles imaged using an apparatus of the invention, in accordance with an embodiment of the invention;

FIG. 8C is a graph schematically illustrating the result of extracting dark spot depressions using a deconvolution algorithm for the low loss super resolution imaging graph shown in FIG. 8B, in accordance with an embodiment of the present invention;

FIG. 9 schematically illustrates a graph of imaging resolution versus beam power density in accordance with an embodiment of the invention;

fig. 10 schematically illustrates a schematic of a scenario for imaging nematodes phagocytosing nanodiamond particles according to an embodiment of the invention;

fig. 11 schematically shows a sequence diagram of control signals versus read signals when imaging nematodes phagocytosing nanodiamond particles according to an embodiment of the invention;

fig. 12A schematically illustrates a background wide-field imaging plot of nematode-endocytosed nanodiamond particles imaged using a conventional wide-field imaging method, in accordance with an embodiment of the invention; and

fig. 12B schematically shows a background-free wide-field imaging plot of nematode endocytosed nanodiamond particles using the device of the invention, according to an embodiment of the invention.

Detailed Description

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It is to be understood that such description is merely illustrative and not intended to limit the scope of the present invention. In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the invention. It may be evident, however, that one or more embodiments may be practiced without these specific details. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms "comprises," "comprising," and the like, as used herein, specify the presence of stated features, steps, operations, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, or components.

All terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined. It is noted that the terms used herein should be interpreted as having a meaning that is consistent with the context of this specification and should not be interpreted in an idealized or overly formal sense.

Where a convention analogous to "A, B and at least one of C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include, but not be limited to, systems that have a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B, C together, etc.). Where a convention analogous to "A, B or at least one of C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B or C" would include, but not be limited to, systems that have a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B, C together, etc.).

Fluorescence is a common luminescence phenomenon in nature. Fluorescence lifetime imaging techniques that distinguish between signal and background based on fluorescence lifetime differences can be limited where the lifetime of the fluorochromes is similar.

The fluorescent nano-diamond is a fluorescent material which is bright, stable in light, good in cell absorption capacity and ultralow in cytotoxicity. A background-free wide-field imaging technology based on fluorescence nano-diamond spin state magnetic modulation is a method based on magnetic modulation or microwave modulation, a magnetic field or microwave is needed to operate a nitrogen-vacancy (NV) color center in a diamond, a complex system is needed, and the operation cost is high.

After absorbing energy, a fluorescent molecule can transit from a ground state with low energy to an excited state with high energy. The fluorescent molecule spontaneously transitions from an excited state back to a ground state and emits photons, a process known as autofluorescence radiation. The excited state fluorescent molecule generates radiation with the same frequency, the same phase and the same polarization as the external radiation under the external radiation, which is called an excited fluorescent radiation process. This process produces fluorescence that exceeds the emission of autofluorescence, competing for the suppression of the autofluorescence, a phenomenon known as STED (stimulated emission depletion). The traditional optical fluorescence microscope is limited by diffraction limit, the spatial resolution can only reach about 300nm, and the research requirement of modern science cannot be met. The super-resolution STED microscopy has the advantages that the energy required by the depletion of the stimulated radiation is large, generally about megawatts per square centimeter, the property of a target to be detected can be influenced by strong pump light, even a biological sample is damaged, and the application of the super-resolution STED microscopy in-situ imaging of a biological living body is greatly limited.

The invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device on one hand.

Fig. 1 schematically shows a block diagram of a background-free wide-field and low-loss super-resolution dual-purpose imaging device according to an embodiment of the invention.

As shown in fig. 1, the background-free wide-field and low-loss super-resolution dual-purpose imaging apparatus 100 may include a first pump light unit 110, a second pump light unit 120, a sample unit 130, a collection unit 140, and a control unit 150.

The first pump light unit 110 is configured to generate a first wavelength light beam.

And a second pump light unit 120 for generating a second wavelength beam. The second wavelength beam comprises a second wavelength gaussian beam or a second wavelength hollow beam.

And a sample unit 130 in which a sample to be tested including a fluorescent beacon is placed. The sample unit is configured to receive the first wavelength light beam, initialize a charge state of the fluorescent beacon, receive the second wavelength light beam, modulate the charge state of the fluorescent beacon, and output at least one of a first wavelength light beam signal, a second wavelength light beam signal, and a fluorescent photon signal corresponding to the fluorescent beacon of a different charge state emitted by the fluorescent beacon.

And the collecting unit 140 is configured to receive at least one of the first wavelength light beam signal, the second wavelength light beam signal, and the fluorescence photon signal, and output a target photon signal obtained through filtering processing.

And a control unit 150 for receiving the target photon signal and generating a background-free wide-field image or a low-loss super-resolution image according to the target photon signal.

According to an embodiment of the present invention, the first pump light unit 110 may generate a first wavelength beam with a tunable beam size. The method comprises the steps of utilizing a first wavelength light beam to irradiate a sample to be detected, enabling the charge state of a fluorescent beacon in a first position range which can be irradiated by the first wavelength light beam in the sample to be detected to be initialized, enabling the fluorescent beacon in the first position range to transit from a ground state of a current charge state to an excited state, then returning to the ground state from the excited state, and enabling the fluorescent beacon in the first position range to be excited to release energy in the form of photon radiation and emit a fluorescent signal.

Fluorescent beacons may include metal particles, nanopores, dielectric particles, fluorescent molecules, quantum dots, point defects, and the like, according to embodiments of the invention. Fluorescent beacons in embodiments of the invention may include NV (nitrogen-vacancy) color centers in diamond, and the charge state may include NV (nitrogen-vacancy) in a dark state where fluorescence is weaker ⁰ And strong bright NV ^- . Dark NV following illumination by a first wavelength beam and a second wavelength beam ⁰ And bright NV ^- May be varied.

According to an embodiment of the present invention, the second pump light unit 120 may generate a second wavelength beam with adjustable beam size, the power of the second wavelength beam may be different from that of the first wavelength beam, and the second wavelength beam may include a second wavelength gaussian beam or a second wavelength hollow beam. The sample to be detected is irradiated by the second wavelength light beam, so that the charge bright state layout degree of the fluorescent beacon in the second position range which can be irradiated by the second wavelength light beam in the sample to be detected is increased, and the fluorescence intensity is enhanced.

It should be noted that the first wavelength light beam and the second wavelength light beam may be laser light beams or any other light beams, such as infrared light, ultraviolet light, microwaves, X-rays, and the like. The first wavelength light beam and the second wavelength light beam can also be other energy, such as electric energy, chemical energy and the like. The embodiments of the present invention are not limited to these examples.

According to an embodiment of the present invention, the sample unit 130 may be placed with a sample to be measured. The sample to be tested may include any of a fluorescent material, a material modified with a fluorescent material, and the like. Materials modified with fluorescent materials may include, for example, nanodiamond-labeled cells. Fluorescent materials generally have different charge states, and the fluorescence spectra of different charge states are different, the fluorescence intensity is different, and the lifetime of the charge state is longer. Most molecules of the fluorescent material are in a ground state energy level having the lowest energy in a normal state, and the fluorescent material may absorb energy and make a transition upon receiving an external excitation. According to the energy level structures of the fluorescent material in different states, the energy to be absorbed for transition to a target energy level can be determined, and the molecules of the fluorescent material can be pumped to different quantum states by using electromagnetic waves with corresponding wavelengths.

For example, for a nitrogen-vacancy (NV) color center in diamond, the two charge states of NV are bright NV with strong fluorescence intensity ^- And NV in a dark state with weak fluorescence intensity ⁰ . The charge state of the NV molecule can be made to be in NV by pumping with a 1064 nm laser ^- And NV ⁰ And adjusting the NV charge state layout. The charge state of the NV molecule can be initialized to an equilibrium configuration using a 532nm laser.

It should be noted that the first wavelength light beam may correspond to a fluorescent beacon charge state layout. The power of the beam part and the power of the central part of the second wavelength hollow beam are different, and the power of the beam part and the power of the central part of the second wavelength hollow beam can correspond to different charge state population degrees of the fluorescent beacon, namely different fluorescent intensity.

According to an embodiment of the invention, the second wavelength hollow beam may be converted on the basis of a second wavelength gaussian beam in the low-loss super-resolution mode. For example, a beam having a gaussian intensity distribution can be converted into a hollow beam by modulating the phase of the beam differently according to the angular distribution on the incident surface using a vortex phase plate having a corresponding wavelength. The VPP-1a vortex phase plate can be used as the vortex phase plate, and the ratio of the central optical power density to the highest optical power density of the obtained hollow beam is 1. In the background-free wide-field mode, a gaussian beam of the second wavelength can be used directly.

According to an embodiment of the present invention, the first position range may be an irradiation range in which the first wavelength beam is focused on the sample. The second range of positions may be other irradiation positions than the central aperture when the hollow beam is focused on the sample. For example, when the beams are focused on a sample, the diameter of both the beams is 1cm, and the diameter of the central hole of the second wavelength hollow beam is 0.1cm, the first position range may be a circular region with a diameter of 1cm, and the second position range may be a circular region with an outer diameter of 1cm and an inner diameter of 0.1 cm.

The first position range may be the second position range, or may be a range in which the first wavelength light beam is focused on another region of the sample that does not include the second position range, or may be a range in which the second wavelength hollow light beam central hole is formed, or may be a range in which any specified light beam is irradiated on any one region of the sample. In the embodiment of the present invention, the first position range may be determined according to the needs of an experimenter or a user, and is not limited herein.

According to the embodiment of the present invention, in the case where imaging is required, the fluorescence of the fluorescent beacon at the intersection portion of the second position range and the first position range is strong, the fluorescence of the fluorescent beacon at the first position range outside the second position range is weak, and the fluorescent beacon outside the first position range does not radiate fluorescence.

According to the embodiment of the present invention, the collection unit 140 may collect the fluorescence photon signals emitted by the fluorescence beacon in the sample to be measured, and filter out photon signals with other wavelengths, for example, may obtain target photon signals. By transmitting the target photon signal to, for example, a computer control unit, it is possible to perform wide field imaging or scanning imaging, for example, based on the light intensity information of the fluorescence photon signal in combination with software. The software that can be incorporated can include, for example, MATLAB (Matrix Laboratory), labVIEW (a program development environment), and the like.

According to an embodiment of the present invention, the first pump light unit 110 may include: a first light source for generating a first wavelength light beam. A first pulse generator for receiving the first sequence of pulses transmitted by the control unit. A first modulator for controlling the first wavelength beam in accordance with a first sequence of pulses to produce a sequenced first wavelength beam. The first fiber coupling-collimation system is used for receiving the first wavelength light beam and outputting a first collimated light beam.

According to an embodiment of the present invention, the second pump light unit 120 may include: and the second light source is used for generating the Gaussian beam with the second wavelength. A second pulse generator for receiving a second train of pulses transmitted by the control unit. A second modulator for controlling the second wavelength beam in accordance with a second sequence of pulses to produce a sequenced second wavelength beam. And the second optical fiber coupling-collimating system is used for receiving the second wavelength light beam and outputting a second collimated light beam.

According to an embodiment of the present invention, the first light source and the second light source may include at least one of a laser light source and other light sources. The laser light source may include at least one of a semiconductor laser and other types of lasers, and the like. The laser light source has the advantages of high collimation, high brightness, good monochromaticity and the like, and can achieve better effect by adopting the laser light source.

It should be noted that the first light source and the second light source may be pump lights with different wavelengths emitted by the same machine, or may be two pump lights emitted by two different machines respectively. The two beams of pump light generated by the first light source and the second light source can irradiate the sample to be measured simultaneously, can also irradiate the sample to be measured in sequence, and can also irradiate the sample to be measured according to preset time. The predetermined time may include the exposure time that would be concluded from multiple experiments to be the most effective. For example, the first wavelength beam irradiation time may be 10 μ s, the second wavelength beam irradiation time may be 10 μ s, the first wavelength beam may be further irradiated as excitation light 1 ms, or the like.

According to an embodiment of the present invention, the first modulator and the second modulator may be respectively and correspondingly disposed at the focal points of the first wavelength light beam and the second wavelength light beam. And adjusting the three-dimensional positions and angles of the first modulator and the second modulator to enable the first wavelength light beam and the second wavelength light beam to be diffracted after passing through the first modulator and the second modulator correspondingly respectively, so that high-order diffracted light beams corresponding to the first wavelength light beam and the second wavelength light beam can be generated. The first modulator and the second modulator may be controlled by the control unit 150. For example, in the case where the first modulator receives the high voltage TTL signal of the control unit 150, the first modulator may convert the first wavelength beam into a higher order diffracted beam output. In the case where the first modulator accepts a low voltage TTL signal, the first modulator may not output any beam.

The first modulator and the second modulator can adopt acousto-optic modulators, the high-order diffraction light can be selected as first-order diffraction light, the diffraction efficiency can reach 80%, and the extinction ratio can be about 2000. The TTL signal can be generated by a PCI (Peripheral Component Interconnect) board, and can be controlled by a LabVIEW program.

According to the embodiment of the invention, the first fiber coupling-collimating system can obtain the first collimated light beam by coupling the first wavelength light beam of the free space into the polarization-maintaining fiber for exiting. The second fiber coupling-collimating system can obtain a second collimated light beam by coupling the second wavelength light beam in the free space into the polarization-maintaining fiber for emission.

According to an embodiment of the present invention, the first pump light unit may further include at least one of: and the optical filter is used for adjusting the power of the first wavelength light beam. And the reflecting mirror is used for adjusting the direction of the first wavelength light beam. And the first adjustable beam expander is used for adjusting the diameter of the first wavelength light beam. A first quarter wave plate for converting the linearly polarized first wavelength light beam into a circularly polarized first wavelength light beam.

It should be noted that the first quarter-wave plate may be a quarter-wave plate suitable for the first wavelength light beam. The polarization of the first wavelength light beam can be in any direction, and the best effect can be achieved by selecting circular polarization.

According to an embodiment of the present invention, the second pump light unit may further include at least one of: and the half-wave plate is used for adjusting the polarization direction of the second wavelength light beam. And the polarization beam splitter is used for combining the half-wave plate and adjusting the power of the second wavelength light beam. And the second adjustable beam expander is used for adjusting the diameter of the second wavelength light beam. And the vortex phase plate is used for converting the Gaussian beam with the second wavelength into the hollow beam with the second wavelength. A second quarter wave plate for converting the linearly polarized second wavelength light beam into a circularly polarized second wavelength light beam. 1:1 non-polarizing beamsplitter for splitting the second wavelength beam in a 1: the power proportion of 1 is used for splitting to obtain a first split beam and a second split beam. And an optical power meter for measuring the power of the first split light beam or the second split light beam and transmitting the measurement result to the control unit.

According to the embodiment of the invention, the vortex phase plate can be a vortex phase plate suitable for the second wavelength light beam, and the second wavelength Gaussian light beam can be converted into the second wavelength hollow light beam in the low-loss super-resolution mode. This element can be eliminated in the background-free wide-field mode, directly using a gaussian beam of the second wavelength. The second quarter-wave plate may be a quarter-wave plate suitable for the second wavelength light beam, the polarization of the second wavelength light beam may be in any direction, and the circular polarization may be selected for best results.

According to an embodiment of the present invention, the sample unit 130 may include: the sample to be tested, the fluorescent beacon, may comprise nanodiamond particles. The microscope lens is used for receiving the first wavelength light beam and/or the second wavelength light beam, focusing the first wavelength light beam and/or the second wavelength light beam on a sample to be detected, and collecting at least one of a first wavelength light beam signal, a second wavelength light beam signal and a fluorescence photon signal emitted by a fluorescence beacon in the sample to be detected. And the piezoelectric ceramic displacement platform is used for moving the pixel point to be detected of the sample to be detected to a preset position with nanometer precision. And the temperature control box is used for maintaining the stability of the temperature and the humidity of the environment where the sample to be detected is located.

According to the embodiment of the present invention, the sample to be tested can be obtained by introducing the fluorescent beacon into the biological sample by biological or chemical methods. The sample to be tested may be placed on a slide for imaging. The temperature control box can maintain the stability of the position of the sample to be measured and the light beam in the measuring process. For example, in the embodiment of the invention, the temperature can be stabilized within 10 mK, and the humidity can be stabilized within 0.5%.

According to the embodiment of the invention, the background-free wide-field and low-loss super-resolution dual-purpose imaging device can further comprise a beam combination unit. The beam combination unit can receive the first wavelength light beam and the second wavelength light beam and output a combined light beam.

According to an embodiment of the present invention, the beam combining unit may be configured to coincide optical axes of the first wavelength light beam and the second wavelength light beam, and adjust a position of a focal plane of the second wavelength light beam focused by the micro lens.

According to an embodiment of the present invention, the beam combining unit may include at least one of: and the quick deflection mirror is used for adjusting the direction of the second wavelength light beam and adjusting the focus position of the second wavelength light beam after being focused by the microscope lens. And the variable focal length lens is used for adjusting the position of a focal plane of the second wavelength light beam after being focused by the micro lens. And the lens group is used for adjusting the position, the direction and the divergence angle of the second wavelength light beam. The short-pass dichroic mirror is used for transmitting the first wavelength light beam, reflecting the second wavelength light beam, receiving at least one of the first wavelength light beam signal, the second wavelength light beam signal and the fluorescence photon signal, filtering the first wavelength light beam, and reflecting at least one of the second wavelength light beam signal and the fluorescence photon signal. And the long-pass dichroic mirror is used for transmitting the second wavelength light beam, receiving at least one of the second wavelength light beam signal and the fluorescence photon signal, filtering the second wavelength light beam and reflecting the fluorescence photon signal.

According to the embodiment of the invention, the lens group can adjust and shape the second wavelength light beam, so that the effect of the quick deflection mirror on the second wavelength light beam is equivalent to the rear focal plane of the microscope lens, and a better imaging effect is realized. For example, a focal plane of the second wavelength light beam can be overlapped with a focal plane of the first wavelength light beam by using a variable focus lens, and the size of the second wavelength light beam can be adjusted by using two lenses, so that the sizes of the light spots of the first wavelength light beam and the second wavelength light beam after focusing are consistent. Both the short-pass dichroic mirror and the long-pass dichroic mirror may have high transmittance. The high-transmittance short-pass dichroic mirror can collect fluorescent photon signals of the fluorescent beacon without affecting the first wavelength light beam. The high-transmittance long-pass dichroic mirror can collect fluorescent photon signals of the fluorescent beacon without affecting the second wavelength beam.

According to an embodiment of the present invention, the second wavelength light beam is transmitted through the long-pass dichroic mirror, reflected by the short-pass dichroic mirror, and may be incident into the microscope lens together with the first wavelength light beam transmitted through the short-pass dichroic mirror. The position, direction and divergence angle of the second wavelength light beam can be adjusted by utilizing the quick deflection mirror, the variable focal length lens and the lens group, and finally the first wavelength light beam is coincided with the second wavelength light beam, and the focus after being focused by the microscope lens is also coincided. The combined pump light can improve the utilization efficiency of light beams, obtain more fluorescent signals and is beneficial to imaging a sample to be detected.

It should be noted that, when the first wavelength light beam and the second wavelength light beam respectively irradiate the sample to be measured in sequence within the respective preset time, the beam combining unit may not be needed.

According to an embodiment of the present invention, the collection unit 140 may include: and the filter plate group is used for receiving at least one of the first wavelength light beam signal, the second wavelength light beam signal and the fluorescence photon signal, and performing filtering processing to obtain a target photon signal. A charge coupled device camera for collecting the target photon signal and transmitting the target photon signal to the control unit 150. A single photon counter for collecting the target photon signal and sending the target photon signal to the control unit 150.

According to the embodiment of the invention, the filter patch group can separate the fluorescence signal of the fluorescence beacon in the second charge state from other wavelength optical signals such as pump light and the like, and filter interference signals to obtain the target photon signal. The control unit 150 may perform wide-field imaging based on the target photon signal collected by the ccd camera to obtain a background-free wide-field image. The control unit 150 may perform scanning imaging based on the target photon signals collected by the single photon counter to obtain a low-loss super-resolution image.

According to an embodiment of the present invention, the collection unit 140 may further include at least one of: 1:1 non-polarizing beam splitter can be used to split fluorescence photon signals proportionally for background-free wide field imaging of CCD (charge coupled device) cameras and low-loss super-resolution imaging of single photon counters. And the achromatic lens group can be used for focusing the fluorescence photon signals on the CCD camera and the single photon counter and matching with the small hole to carry out spatial filtering. And the small hole can be used for carrying out spatial filtering of a confocal system on the fluorescence photon signal.

According to the embodiment of the invention, when imaging is required, since the pump light is irradiated on the sample to be measured, various objects in the sample area may reflect the pump light, such as the pump light which may include reflecting the first wavelength beam signal, the second wavelength beam signal, and the like. Inevitably, the pump light and the fluorescence photon signal are incident into the rear collection unit 140 together, and the light intensity of the fluorescence is weak and is difficult to distinguish after being mixed with the pump light, so a dichroic mirror is needed to separate photons emitted from the sample to be measured. In addition, since the light intensity difference between the pump light and the fluorescence is too large, the subsequent operations such as collection can be affected by only mixing a small amount of pump light into the fluorescence photon signal, so that the collected photons need to be further separated by a filter, the doped pump light photons are filtered, the fluorescence photons with higher purity are obtained, and a better imaging effect is obtained.

Fig. 2 schematically illustrates an optical path diagram of a background-free wide-field and low-loss super-resolution dual-purpose imaging device according to an embodiment of the invention.

As shown in fig. 2, the first

light source unit

110, 111 is a first light source composed of a first wavelength laser, and can continuously emit a first wavelength light beam. 115 is a circular continuously variable reflective neutral density filter that can modulate the power of a first wavelength beam. 116 is a mirror that can be used to change the beam direction of the first wavelength beam. 112 is a pulse generator and the acousto-optic modulator 113 can be controlled to produce a sequenced first wavelength beam. 114 is a fiber coupled-collimating system that can collimate and redirect the first wavelength beam. 117 is a tunable beam expander that can tune the diameter of the first wavelength beam. 118 is a quarter wave plate that changes the polarization state of the first wavelength beam. Through the operation of the first pump light unit 110, a first wavelength beam can be obtained.

As shown in fig. 2, the second light source formed of a second wavelength laser corresponding to the second pump

light unit

120, 121 may continuously emit a second wavelength light beam. An electrically controlled rotating half-wave plate 125 is used in conjunction with the polarization beam splitter 126 to adjust the power of the second wavelength beam. 123 is an acousto-optic modulator that can be controlled by the pulse generator 122 to produce a sequenced beam of light of the second wavelength. A fiber coupled-collimating system 124 collimates and redirects the second wavelength beam. 127 is a tunable beam expander. 128 is an m =2 vortex phase plate for generating a second wavelength hollow beam. 129 is a quarter wave plate. 1210 is a 1:1 non-polarizing beam splitter for splitting the first wavelength signal into two parts. 1211 is an optical power meter for measuring the power of a portion of the second wavelength optical beam. The second wavelength beam may be obtained through the operation of the second pump light unit 120.

As shown in fig. 2, the

sample unit

130, 131 is a sample to be measured. 132 is a high numerical aperture microscope lens that can be used to focus the combined beam onto the sample 131 to be measured. Reference numeral 133 denotes a piezo-ceramic displacement stage, which can be used to control the position of the sample 131 to be measured. 134 is a temperature control box, which can be used to maintain the temperature and humidity of the environment where the sample 131 to be measured is located stable.

As shown in fig. 2, the

beam combining units

160, 161 are fast deflection mirrors, and can change the direction of the second wavelength light beam. 162 is a variable focus lens, the divergence angle of the second wavelength beam can be adjusted. 163 is a lens group. 165 is a long pass dichroic mirror that can separate the fluorescence photon signal from the pump light. 164 is a short pass dichroic mirror that can separate the fluorescence photon signal, the pump light, and a combined beam signal, which can be a signal corresponding to the combined beam. Through the operation of the beam combining unit 160, the combined beam to be transmitted to the sample unit 130 can be obtained based on the short-pass dichroic mirror 164, and the target photon signal to be transmitted to the collection unit 140 can be obtained based on the long-pass dichroic mirror 165.

As shown in fig. 2, the

collection units

140 and 141 are filter sets, and the target photon beam can be subjected to back-end filtering. 144 is a 1:1 non-polarizing beam splitter that can split the target photon beam. 145. 146, 147, 148 are achromatic lenses that focus and collimate the target photon beam. 149 is a pinhole that can spatially filter the target photon beam. 143 is a single photon counter which can be used to realize scanning imaging. 142 are CCD cameras that can be used to achieve wide field imaging.

As shown in fig. 2, 150 is a computer control unit that can combine the target photon signal collected by the CCD camera 142, the target photon signal collected by the single photon counter 143, and the power of the second wavelength light beam measured by the optical power meter 1211 to generate a low-loss super-resolution image and/or a background-free wide-field image. The computer control unit 150 can also be used for controlling the electrically controlled rotating half-wave plate 125, the fast deflection mirror 161, the variable focal length lens 162, the piezoelectric ceramic displacement stage 133, the

pulse generators

112, 122 and the acousto-

optic modulators

113, 123, and processing the fluorescence signals sent back by the camera and the single photon counter.

The invention provides a low-loss super-resolution imaging method.

Fig. 3 schematically shows a flow chart of a low-loss super-resolution imaging method according to an embodiment of the invention.

As shown in FIG. 3, the method includes operations S310 to S330.

In operation S310, for each pixel point to be detected in the sample to be detected, based on the background-free wide-field and low-loss super-resolution dual-purpose imaging device, N first fluorescence intensities and N second fluorescence intensities are obtained. The sample to be tested comprises a plurality of pixel points to be tested. The first fluorescence intensity includes a fluorescence intensity in a case where the first initialization fluorescent beacon is irradiated with the first wavelength light beam, and the second fluorescence intensity includes a fluorescence intensity in a case where the first enhancement fluorescent beacon is simultaneously irradiated with the first wavelength light beam and the second wavelength light beam. The first initialized fluorescent beacon is obtained by irradiating the fluorescent beacon in the sample to be detected based on the first wavelength light beam, and the first enhanced fluorescent beacon is obtained by irradiating the first initialized fluorescent beacon based on the first wavelength light beam and the second wavelength light beam. N is a positive integer.

In operation S320, the target fluorescence intensity corresponding to the pixel point to be detected is determined according to the average value of the N second fluorescence intensities.

In operation S330, a low-loss super-resolution image corresponding to the sample to be measured is determined according to the target fluorescence intensities corresponding to the pixel points to be measured.

According to the embodiment of the invention, referring to fig. 2 in combination, in the low-loss super-resolution mode, the small focused beam can be obtained at the sample to be measured by adjusting the diameter and power of the first wavelength beam and the second wavelength beam. The fluorescence photons in the target photon signal are collected by the single photon detector 143, so that the light intensity variation information of the fluorescence photons can be collected, and the light beam related to the target photon signal can be focused on the probe of the single photon detector 143 by the achromatic lens 148. The single photon detector 143 can emit a TTL pulse signal each time a fluorescence photon is measured. The TTL pulse signals can be recorded by the data acquisition card of the control unit 150 to obtain a fluorescence intensity count. During imaging, the pixel points to be detected of the sample to be detected can be moved to the detection position through the position scanning device, the fluorescence photons radiated by each pixel point are collected, then the position of the pixel point is used as an x-y coordinate, and the fluorescence light intensity is used as the z value of the point for imaging. The position scanning device may be a piezo-ceramic displacement stage 133, and the piezo-ceramic displacement stage 133 may be selected from P-562.3CD (nano positioning system). For example, when imaging a target region with an area of 2 μm × 2 μm, the step size may be selected to be 10nm, the region is divided into 200 × 200 pixels, the sample is sequentially moved to 200 × 200 coordinate positions by using the piezoelectric ceramic displacement stage 133, the fluorescence intensity of each coordinate point is measured, and low-loss super-resolution imaging may be performed on the sample to be measured.

It should be noted that the control unit 150 is required to be used with a data acquisition card and a pulse signal generator. The data acquisition card can adopt PCIe6363 series, and the pulse signal generator can adopt PCI pulser. The control program can be compiled based on LabVIEW software, and can be used for controlling the on-off and the position movement of the light source, acquiring fluorescence intensity and position information, processing and imaging.

According to the embodiment of the invention, when each pixel point to be detected is imaged, each region or each pixel point to be detected needs to be cycled for multiple times to obtain sufficient fluorescence photon counting. That is, the beam switching control sequence needs to be cycled multiple times. By the method, enough photon counts can be obtained, signals with higher signal-to-noise ratio can be obtained, and the imaging quality can be improved.

According to the embodiments of the present invention, since the first wavelength light beam and the second wavelength light beam have extremely low power and are continuous light, the damage to the sample is extremely small, and low-loss super-resolution imaging can be effectively realized.

Fig. 4 schematically shows a flow chart of a background-free wide-field imaging method according to an embodiment of the invention.

As shown in FIG. 4, the method includes operations S410 to S420.

In operation S410, a first image and a second image captured for a sample to be measured are acquired based on a background-free wide-field and low-loss super-resolution dual-purpose imaging device. The first image includes an image obtained while illuminating the sample under test including the second initialization fluorescent beacon with the first wavelength light beam, and the second image includes an image obtained while illuminating the image under test including the second enhanced fluorescent beacon with the first wavelength light beam and the second wavelength light beam. The second initialized fluorescent beacon is obtained by irradiating the fluorescent beacon in the sample to be detected based on the first wavelength light beam, and the second enhanced fluorescent beacon is obtained by irradiating the second initialized fluorescent beacon based on the first wavelength light beam and the second wavelength light beam.

In operation S420, a background-free wide-field image corresponding to the sample to be measured is determined according to a difference between the second image and the first image.

In accordance with an embodiment of the present invention, and with reference to FIG. 2, in a background-free wide-field mode, fluorescence photons in a target photon signal can be collected using CCD camera 142, and a light beam associated with the target photon signal can be focused by achromatic lens 145 onto CCD camera 142. The CCD camera 142 may transmit the optical signal sensed by each pixel point back to the control unit 150, and the control unit 150 may perform subtraction on the image in the on/off state of the second wavelength light beam to obtain a background-free wide-field imaging image.

According to the embodiment of the invention, in the wide field mode, the

adjustable beam expanders

117 and 127 are adjusted to change the size and the divergence angle of the first wavelength light beam and the second wavelength light beam, so that the irradiation range after focusing can be enlarged. In addition, the power of the second wavelength beam can be varied by adjusting the angle of the electrically controlled rotating half-wave plate 125 so that the second range of positions is changed to the entire first range of positions with the central hole removed. The CCD camera is used for collecting light intensity change information, and biological spontaneous background fluorescence is not influenced by the light beam with the second wavelength, so background signals can be filtered, and a background-free wide-field image with a high signal-to-back ratio can be obtained.

It should be noted that, in the embodiment of the present invention, specific implementation processes of the background-free wide-field imaging method and the low-loss super-resolution imaging method may refer to specific implementations of each functional module of the background-free wide-field and low-loss super-resolution dual-purpose imaging apparatus in the above embodiments, and details are not described herein again.

FIG. 5 schematically shows a schematic diagram of low power continuous light assisted imaging according to an embodiment of the invention.

According to an embodiment of the invention, NV has two charge states, a less fluorescent dark state NV ⁰ And strong bright NV ^- . In imaging, a 1064 nm laser can switch NV between two charge states under illumination by a 532nm laser, as shown in FIG. 5, with different charge state layouts for balancing under different powers 1064 nm light. Because the fluorescence intensities of the two charge states are different, the optical power of 1064 nm is different, and the fluorescence intensity of the NV equilibrium state is different. NV (non-volatile memory) ^- Fluorescent wavelength ratio NV of radiation ⁰ Longer, 600 nm long pass filter can be added into filter set to filter NV ⁰ Fluorescence to detect NV alone ^- The fluorescent photons of the radiation.

For better understanding of the principle and idea of the embodiment of the present invention, please refer to fig. 6 to 12, which will be described below with reference to several specific application scenarios to describe the principle of using the background-free wide-field and low-loss super-resolution dual-purpose imaging device of the embodiment of the present invention to image the nano-diamond particles and the nematodes phagocytosing the nano-diamond particles, respectively.

Fig. 6 schematically illustrates a schematic view of a scene imaging nanodiamond particles according to an embodiment of the invention.

Fig. 7 schematically shows a sequence diagram of a control signal and a read signal when imaging the nanodiamond particles according to an embodiment of the invention.

As shown in fig. 6, the sample to be measured is the nano-diamond particles 620 on the glass sheet 610, and the imaging mode is a low-loss super-resolution imaging mode. Referring to fig. 7, when a low-loss super-resolution imaging needs to be performed on the nano-diamond particles, when a pixel point to be measured is measured, 532nm gaussian beam with power of 20 μ W may be used to irradiate 200 μ s, so as to initialize NV charge state layout in the nano-diamond particles. 10 ms can then be irradiated with a 532nm Gaussian beam at 20 μ W and the fluorescence intensity recorded with a single photon counter. Next, the NV in the nanodiamond outside the beam center can be irradiated for 200 μ s with a 532nm Gaussian beam with power of 20 μ W and a 1064 nm hollow beam with power of 10 mW together ^- The ratio increases and reaches equilibrium, resulting in enhanced fluorescence counts. Thereafter, 10 ms can be co-irradiated with a 532nm gaussian beam with power 20 μ W and a 1064 nm hollow beam with power 10 mW. The "initialize-count-enhance-count" sequence may be cycled a number of times, such as 10 times. Then, the average of the count enhancement values can be taken as the signal value of the pixel. Under the condition that the signal of the next pixel point to be measured needs to be measured, the sample can be controlled to move through the piezoelectric ceramic displacement platform. NV due to beam center ^- Less, the dark-spot depression in the image may represent where the NV is located. Alternatively, dark spot depressions in the image can be extracted and converted into bright spots using a deconvolution algorithm.

Fig. 8A schematically illustrates a low-loss super-resolution imaging map obtained by imaging nanodiamond particles using a conventional focused imaging method according to an embodiment of the invention.

Fig. 8B schematically shows a low-loss super-resolution image obtained by imaging nanodiamond particles using the device of the invention according to an embodiment of the invention.

Fig. 8C schematically illustrates a graph of the results of extracting dark spot depressions using a deconvolution algorithm for the low-loss super-resolution imaging graph illustrated in fig. 8B, in accordance with an embodiment of the present invention.

In conventional focused imaging, the resolution of the nanodiamond particles was about 325 nm, see imaging results shown in fig. 8A. Based on the sequence shown in fig. 7, the results shown in fig. 8B can be obtained by specifically adjusting the fluorescence of the nanodiamond particles and imaging with structured light. Referring to fig. 8B, the nanodiamonds formed dark spots at the centers of the spots. Using the deconvolution algorithm, the result shown in fig. 8C can be obtained. Referring to fig. 8C, the resolution of the resulting nanodiamond particle imaging results was about 100 nm.

FIG. 9 schematically shows a graph of imaging resolution versus beam power density according to an embodiment of the invention.

According to an embodiment of the present invention, referring to fig. 9, by continuously increasing the power of the first wavelength beam and/or the second wavelength beam for irradiating the sample to be measured, the resolution of the nano-diamond particles can be increased to about 30 nm, which is far below the diffraction limit of the optical fluorescence microscope.

Fig. 10 schematically illustrates a schematic of a scenario for imaging nematodes phagocytosing nanodiamond particles according to an embodiment of the invention.

Fig. 11 schematically shows a sequence diagram of control signals versus read signals when imaging nematodes phagocytosing nanodiamond particles according to an embodiment of the invention.

As shown in fig. 10, the sample to be tested is the nano-diamond particles in the nematodes, and the imaging mode is a background-free wide-field imaging mode. Referring to fig. 11, in the case where wide field imaging of nematodes with strong fluorescent backgrounds is required, 100 ms can be first illuminated with 532nm gaussian beam at 100 μ W to initialize the population of NV charge states in the nanodiamond particles, taking a first image. Then, a 532nm Gaussian beam with a power of 100 μ W and a 1064 nm Gaussian beam with a power of 10 mW can be used together to irradiate 200 μ s in the regionNV in nanodiamonds ^- The ratio increases and reaches equilibrium, resulting in enhanced fluorescence counts. Thereafter, 100 ms can be co-illuminated with a 532nm gaussian beam with a power of 100 μ W and a 1064 nm gaussian beam with a power of 10 mW to capture a second image. Then, the difference between the second image and the first image can be taken as a frame of background-free wide-field image. Repeating the process to shoot a multi-frame image with a refresh time of, for example, 200.2 ms, bright spots in the image can represent the nano-diamond particles.

Fig. 12A schematically illustrates a background wide-field imaging profile obtained by imaging nematode-endocytosed nanodiamond particles using a conventional wide-field imaging method, in accordance with embodiments of the invention.

In conventional wide field imaging, the nanodiamond particles are submerged in the autofluorescence background, see imaging results illustrated in fig. 12A. Based on the sequence shown in fig. 11, specific modulation of the fluorescence of the nanodiamond particles in the nematodes and using structured light imaging, a background-free wide-field imaging result as shown in fig. 12B can be obtained. Referring to fig. 12B, it can be determined that the background-free wide-field imaging performed based on the apparatus and method provided by the present invention can remove the background well, improve the signal-to-back ratio, and easily achieve the extraction of the nano-diamond particles from the fluorescence background of the nematodes.

Through the embodiments of the invention, the invention provides a full-gloss background-free wide-field and low-loss super-resolution dual-purpose imaging device and an imaging method which are suitable for in-situ imaging of a biological living body sample. The related device and the method use the low-power continuous light to modulate the charge state of the fluorescent beacon so as to modulate the photon number of the fluorescent signal, not only can realize the background-free wide-field imaging of the sample to be detected, but also can realize the low-loss super-resolution imaging, and does not influence the property of the sample to be detected and has high signal-to-back ratio. Compared with the prior art, the charge state has long service life and can reach the second order, so the charge state of the fluorescent beacon can be controlled by the pump light with lower power, the property of a sample to be detected is not influenced, the sample is less damaged, and the biocompatibility is good. In addition, the embodiment of the invention adopts an all-optical method, has simple system structure and operation, is convenient to use and assemble, saves the operation cost and the cost, and improves the use experience of users.

It should be noted that the elements and algorithm steps of the various examples described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the implementation. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.

The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. A software module may reside in Random Access Memory (RAM), memory, read Only Memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.

The background-free wide-field and low-loss super-resolution dual-purpose imaging system and method provided by the invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it can make several improvements and modifications to the present invention, and those improvements and modifications also fall into the protection scope of the claims of the present invention.

Claims

1. A background-free wide-field and low-loss super-resolution dual-purpose imaging device comprises:

a first pump light unit for generating a first wavelength beam;

the second pump light unit is used for generating a second wavelength light beam which comprises a second wavelength Gaussian light beam or a second wavelength hollow light beam;

the sample unit is used for receiving the first wavelength light beam, initializing the charge state of the fluorescent beacon, receiving the second wavelength light beam and modulating the charge state of the fluorescent beacon, wherein the second wavelength light beam is low-power continuous infrared light and modulates the number of radiation photons of the fluorescent beacon, and the charge state of NV molecules in NV molecules is modulated in the modulation process ^- And NV ⁰ Switching between, adjusting the NV charge state layout, and outputting at least one of a first wavelength beam signal, a second wavelength beam signal, and a fluorescent photon signal corresponding to a different charge state fluorescent beacon emitted via the fluorescent beacon;

the collecting unit is used for receiving at least one of the first wavelength light beam signal, the second wavelength light beam signal and the fluorescence photon signal and outputting a target photon signal obtained through filtering processing;

the control unit is used for receiving the target photon signal and generating a background-free wide-field image or a low-loss super-resolution image according to the target photon signal;

under the low-loss super-resolution image, the second wavelength hollow beam is obtained by conversion on the basis of the second wavelength Gaussian beam, and under the background-free wide-field image, the second wavelength Gaussian beam is directly used;

the imaging method of the background-free wide-field image comprises the following steps: acquiring a first image and a second image which are obtained by shooting aiming at a sample to be detected, wherein the first image comprises an image obtained under the condition that the sample to be detected comprising a second initialization fluorescent beacon is irradiated by the first wavelength light beam, the second image comprises an image obtained under the condition that the image to be detected comprising a second enhancement fluorescent beacon is irradiated by the first wavelength light beam and the second wavelength light beam, the second initialization fluorescent beacon is obtained by irradiating the fluorescent beacon in the sample to be detected based on the first wavelength light beam, the second enhancement fluorescent beacon is obtained by irradiating the second initialization fluorescent beacon based on the first wavelength light beam and the second wavelength light beam, and the fluorescent beacon is a diamond; determining a background-free wide field image corresponding to the sample to be detected according to the difference value of the second image and the first image;

the imaging method of the low-loss super-resolution image comprises the following steps: acquiring N first fluorescence intensities and N second fluorescence intensities for each pixel point to be detected in a sample to be detected, wherein the sample to be detected comprises a plurality of pixel points to be detected, the first fluorescence intensity comprises fluorescence intensity under the condition that a first initialization fluorescence beacon is irradiated by the first wavelength light beam, the second fluorescence intensity comprises fluorescence intensity under the condition that a first enhancement fluorescence beacon is simultaneously irradiated by the first wavelength light beam and the second wavelength light beam, the first initialization fluorescence beacon is obtained by irradiating the fluorescence beacon in the sample to be detected based on the first wavelength light beam, the first enhancement fluorescence beacon is obtained by irradiating the first initialization fluorescence beacon based on the first wavelength light beam and the second wavelength light beam, and N is a positive integer; determining the target fluorescence intensity corresponding to the pixel point to be detected according to the average value of the N second fluorescence intensities; and determining a low-loss super-resolution image corresponding to the sample to be detected according to the fluorescence intensities of the targets corresponding to the pixel points to be detected.

2. The apparatus of claim 1, wherein,

the first pump light unit includes:

a first light source for generating the first wavelength light beam;

a first pulse generator for receiving a first sequence of pulses transmitted by the control unit;

a first modulator for controlling the first wavelength beam in accordance with the first sequence of pulses to produce a sequenced first wavelength beam;

a first fiber coupled-collimating system for receiving the first wavelength beam and outputting a first collimated beam;

the second pump light unit includes:

a second light source for generating the second wavelength gaussian beam;

a second pulse generator for receiving a second train of pulses transmitted by the control unit;

a second modulator for controlling the second wavelength beam in accordance with the second sequence of pulses to produce a sequenced second wavelength beam;

and the second fiber coupling-collimating system is used for receiving the second wavelength light beam and outputting a second collimated light beam.

3. The apparatus of claim 2, wherein the first pump light unit further comprises at least one of:

the optical filter is used for adjusting the power of the first wavelength light beam;

a mirror for adjusting the direction of the first wavelength beam;

the first adjustable beam expander is used for adjusting the diameter of the first wavelength light beam;

a first quarter wave plate for converting the linearly polarized first wavelength light beam into a circularly polarized first wavelength light beam.

4. The apparatus of claim 2, wherein the second pump light unit further comprises at least one of:

the half-wave plate is used for adjusting the polarization direction of the second wavelength light beam;

the polarization beam splitter is used for combining the half-wave plate and adjusting the power of the second wavelength light beam;

the second adjustable beam expander is used for adjusting the diameter of the second wavelength light beam;

the vortex phase plate is used for converting the second wavelength Gaussian beam into the second wavelength hollow beam;

a second quarter wave plate for converting the linearly polarized second wavelength beam into a circularly polarized second wavelength beam;

1:1 non-polarizing beam splitter for splitting the second wavelength beam in a 1:1, splitting the beams in a power ratio to obtain a first split beam and a second split beam;

an optical power meter for measuring the power of the first split beam or the second split beam and sending the measurement result to the control unit.

5. The apparatus of claim 1, further comprising:

and the beam combining unit is used for receiving the first wavelength light beam and the second wavelength light beam and outputting a combined light beam.

6. The apparatus of claim 5, wherein the beam combining unit comprises at least one of:

the quick deflection mirror is used for adjusting the direction of the second wavelength light beam and adjusting the focus position of the second wavelength light beam after being focused by the microscope lens;

the variable focal length lens is used for adjusting the focal plane position of the second wavelength light beam after being focused by the microscope lens;

a lens group for adjusting a position, a direction and a divergence angle of the second wavelength light beam;

a short pass dichroic mirror for transmitting the first wavelength beam and reflecting the second wavelength beam, and receiving at least one of the first wavelength beam signal, the second wavelength beam signal, and the fluorescence photon signal, filtering the first wavelength beam, and reflecting at least one of the second wavelength beam signal and the fluorescence photon signal;

and the long-pass dichroic mirror is used for transmitting the second wavelength light beam, receiving at least one of the second wavelength light beam signal and the fluorescence photon signal, filtering the second wavelength light beam, and reflecting the fluorescence photon signal.

7. The apparatus of claim 1, wherein the sample unit comprises:

the fluorescent beacon of the sample to be detected comprises nano diamond particles;

the microscope lens is used for receiving the first wavelength light beam and/or the second wavelength light beam, focusing the first wavelength light beam and/or the second wavelength light beam on the sample to be detected, and collecting at least one of a first wavelength light beam signal, a second wavelength light beam signal and a fluorescence photon signal emitted by a fluorescence beacon in the sample to be detected;

the piezoelectric ceramic displacement platform is used for moving the pixel point to be detected of the sample to be detected to a preset position with nanometer precision;

and the temperature control box is used for maintaining the stability of the temperature and the humidity of the environment where the sample to be detected is located.

8. The apparatus of claim 7, wherein the collection unit comprises:

the filter plate group is used for receiving at least one of the first wavelength light beam signal, the second wavelength light beam signal and the fluorescence photon signal, and performing filtering processing to obtain the target photon signal;

a charge coupled device camera to collect the target photon signal and to send the target photon signal to the control unit;

and the single photon counter is used for collecting the target photon signal and sending the target photon signal to the control unit.