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

Abstract

The invention provides a dual-purpose imaging device and imaging method for wide-field without background and low-loss super-resolution. The device includes: a first pump light unit for generating a first wavelength beam; a second pump light unit for for generating a second wavelength light beam; the sample unit is placed with a sample to be tested including a fluorescent beacon, the sample unit is used to receive the first wavelength light beam, initialize the charge state of the fluorescent beacon, receive the second wavelength light beam, and measure the charge of the fluorescent beacon. modulate the state of the fluorescent beacon, and output at least one of the first wavelength beam signal, the second wavelength beam signal and the fluorescent photon signal corresponding to the fluorescent beacon of different charge states emitted by the fluorescent beacon; the collecting unit is used for outputting the filter processing The target photon signal; 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, and more specifically, to a dual-purpose imaging device and imaging method for no-background wide-field and low-loss super-resolution.

Background technique

Advances in imaging techniques have enabled fluorescence microscopy to study substances of interest and dynamics within cells, and even study biological processes at the single-molecule level. However, the sensitivity and resolution of intravital fluorescence imaging are often limited by autofluorescence and other background noise due to overlap between the emission spectra of fluorescent probes and background.

Contents of the invention

In view of this, the present invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device and imaging method.

One aspect of the present invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device, including: a first pump light unit, used to generate a beam of the first wavelength; a second pump light unit, used to generate The second wavelength light beam, the second wavelength light beam includes a second wavelength Gaussian light beam or a second wavelength hollow light beam; a sample unit is placed with a sample to be measured including a fluorescent beacon, and the sample unit is used to receive the first wavelength light beam , initialize the charge state of the fluorescent beacon, receive the second wavelength beam, modulate the charge state of the fluorescent beacon, and output the first wavelength beam signal and the second wavelength beam signal emitted by the fluorescent beacon At least one of the beam signal and the fluorescent photon signal corresponding to the fluorescent beacon of a different charge state; a collection unit for receiving at least one of the first wavelength beam signal, the second wavelength beam signal and the fluorescent photon signal , and output the target photon signal obtained through filtering; the control unit is configured to receive the target photon signal, and generate 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, configured to generate the first wavelength light beam; a first pulse generator, configured to receive a first sequence of pulses sent by the control unit; A modulator, used to control the first wavelength beam according to the first sequence of pulses, to generate a serialized first wavelength beam; a first fiber coupling-collimation system, used to receive the first wavelength beam, and Output a first collimated beam.

Optionally, the second pump light unit includes: a second light source, configured to generate a Gaussian beam of the second wavelength; a second pulse generator, configured to receive a second sequence of pulses sent by the control unit; A second modulator, configured to control the second wavelength beam according to the second sequence of pulses, to generate a serialized second wavelength beam; a second fiber coupling-collimation system, configured to receive the second wavelength beam, and outputting a second collimated light beam.

Optionally, the first pump light unit may further include at least one of the following: an optical filter, used to adjust the power of the first wavelength beam; a reflector, used to adjust the power of the first wavelength beam direction; the first adjustable beam expander is used to adjust the diameter of the first wavelength beam; the first quarter-wave plate is used to convert the linearly polarized first wavelength beam into a circularly polarized first wavelength beam.

Optionally, the second pump light unit may further include at least one of the following: a half-wave plate, used to adjust the polarization direction of the second wavelength beam; a polarization beam splitter, used to combine the half-wave a wave plate for adjusting the power of the second wavelength beam; a second adjustable beam expander for adjusting the diameter of the second wavelength beam; a vortex phase plate for converting the second wavelength Gaussian beam It is the second wavelength hollow beam; the second quarter-wave plate is used to convert the linearly polarized second wavelength beam into a circularly polarized second wavelength beam; a 1:1 non-polarizing beam splitter is used to convert The second wavelength beam is split according to a power ratio of 1:1 to obtain a first split beam and a second split beam; an optical power meter is used to measure the first split beam or the second split beam power and send the measurement to the control unit.

Optionally, the background-free wide-field and low-loss super-resolution dual-purpose imaging device further includes: a beam combining unit, configured to receive the first wavelength beam and the second wavelength beam, and output a combined beam.

Optionally, the beam combining unit includes at least one of the following: a fast deflection mirror, used to adjust the direction of the second wavelength beam, and adjust the focus position of the second wavelength beam after being focused by the microscope lens ; variable focal length lens, used to adjust the focal plane position of the second wavelength beam after being focused by the microscope lens; lens group, used to adjust the position, direction and divergence angle of the second wavelength beam; short-pass dichroic mirror, used to transmit the first wavelength beam and reflect the second wavelength beam, and receive at least one of the first wavelength beam signal, the second wavelength beam signal and the fluorescent photon signal, and filter out The first wavelength beam reflects at least one of the second wavelength beam signal and the fluorescent photon signal; a long-pass dichroic mirror is used to transmit the second wavelength beam and receive the second wavelength At least one of the light beam signal and the fluorescent photon signal filters out the second wavelength light beam and reflects the fluorescent photon signal.

Optionally, the sample unit includes: the sample to be tested, the fluorescent beacon includes nano-diamond particles; a microscope lens, configured to receive the first wavelength light beam and/or the second wavelength light beam, and Focusing the first wavelength beam and/or the second wavelength beam onto the sample to be tested, and collecting the first wavelength beam signal, the second wavelength beam signal and the fluorescent light emitted by the fluorescent beacon in the sample to be tested At least one of the photon signals; a piezoelectric ceramic displacement stage, used to move the pixel points of the sample to be measured to a preset position with nanometer precision; a temperature control box, used to maintain the environment of the sample to be measured stable temperature and humidity.

Optionally, the collection unit includes: a filter set, configured to receive at least one of the first wavelength beam signal, the second wavelength beam signal, and the fluorescent photon signal, and perform filtering processing to obtain the A target photon signal; a charge-coupled device camera, used to collect the target photon signal, and send the target photon signal to the control unit; a single photon counter, used to collect the target photon signal, and send the target photon signal The photon signal is sent to the control unit.

Another aspect of the present invention provides a low-loss super-resolution imaging method, including: for each pixel to be measured in the sample to be tested, based on a background-free wide-field and low-loss super-resolution dual-purpose imaging device, obtain N first fluorescence intensities and N second fluorescence intensities, wherein the sample to be tested includes a plurality of pixel points to be measured, and the first fluorescence intensities include the first initialization fluorescence Fluorescence intensity in the case of a beacon, the second fluorescence intensity comprising the fluorescence intensity in the case of simultaneously illuminating a first enhanced fluorescent beacon with the first wavelength light beam and the second wavelength light beam, the first initialization fluorescence intensity The beacon is obtained based on the first wavelength beam irradiating the fluorescent beacon in the sample to be tested, and the first enhanced fluorescent beacon is based on the first wavelength beam and the second wavelength beam irradiating the The first initialization fluorescent beacon is irradiated, and N is a positive integer; according to the average value of the N second fluorescence intensities, determine the target fluorescence intensity corresponding to the pixel to be measured; A plurality of target fluorescence intensities corresponding to the pixel points are used to determine a low-loss super-resolution image corresponding to the sample to be tested.

Another aspect of the present invention provides a background-free wide-field imaging method, including: based on a background-free wide-field and low-loss super-resolution dual-purpose imaging device, acquiring the first image and the second image taken for the sample to be tested images, wherein the first image comprises an image obtained when a sample to be tested comprising a second initialized fluorescent beacon is irradiated with a light beam of the first wavelength, and the second image comprises an image obtained when a light beam of the first wavelength is used to and the image obtained under the condition that the second wavelength beam irradiates the image to be measured comprising a second enhanced fluorescent beacon, and the second initialized fluorescent beacon is based on the effect of the first wavelength beam on the fluorescence in the sample to be measured Obtained by beacon irradiation, the second enhanced fluorescent beacon is obtained by irradiating the second initialization fluorescent beacon based on the first wavelength light beam and the second wavelength light beam; according to the second image and the first A difference value of an image to determine a background-free wide-field image corresponding to the sample to be tested.

According to an embodiment of the present invention, by using the first pump light unit to generate the first wavelength beam, the charge state of the fluorescent beacon in the sample to be tested is initialized, and the second pump light unit is used to generate the second wavelength beam, and the charge state of the fluorescent beacon is initialized. The charge state is modulated. Since the fluorescent signals of fluorescent beacons with different charge states are different, background-free imaging and super-resolution imaging can be performed on the sample to be tested according to the collected target photon signals. The technical scheme of the present invention uses low-power continuous infrared light to modulate the number of radiation photons of the fluorescent beacon, which has the characteristics of not affecting the properties of the sample to be tested, good biocompatibility, resistance to biological spontaneous background fluorescence, stable fluorescence signals, high spatial resolution, and high signal-to-background ratio , the optical system and the electronic system have the advantages of simple structure, low operating cost and low cost.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only For some embodiments of the present invention, those skilled in the art can also obtain other drawings based on these drawings without creative work.

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

Fig. 2 schematically shows 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 present invention;

FIG. 3 schematically shows a flowchart of a low-loss super-resolution imaging method according to an embodiment of the present invention;

FIG. 4 schematically shows a flowchart of a background-free wide-field imaging method according to an embodiment of the present invention;

Fig. 5 schematically shows a principle diagram of low-power continuous light-assisted imaging according to an embodiment of the present invention;

Fig. 6 schematically shows a schematic diagram of a scene of imaging nanodiamond particles according to an embodiment of the present invention;

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

Fig. 8A schematically shows a low-loss super-resolution imaging image obtained by imaging nano-diamond particles using a traditional focusing imaging method according to an embodiment of the present invention;

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

FIG. 8C schematically shows the results of extracting dark spot depressions using a deconvolution algorithm for the low-loss super-resolution imaging image shown in FIG. 8B according to an embodiment of the present invention;

Fig. 9 schematically shows a graph of imaging resolution-beam power density according to an embodiment of the present invention;

Fig. 10 schematically shows a schematic diagram of a scene of imaging a nematode that has swallowed nanodiamond particles according to an embodiment of the present invention;

Fig. 11 schematically shows a sequence diagram of control signals and reading signals when imaging nematodes that have swallowed nanodiamond particles according to an embodiment of the present invention;

Fig. 12A schematically shows a wide-field imaging image with background obtained by imaging nanodiamond particles endocytosed by nematodes using a traditional wide-field imaging method according to an embodiment of the present invention; and

Fig. 12B schematically shows a background-free wide-field imaging image obtained by using the device of the present invention to image nanodiamond particles endocytosed by nematodes according to an embodiment of the present invention.

Detailed ways

Hereinafter, embodiments of the present invention will be described with reference to the drawings. It should be understood, however, that these descriptions are exemplary only and are 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. Also, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concept 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 "comprising", "comprising", etc. used herein indicate the presence of stated features, steps, operations and/or components, but do not exclude 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 meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted to have a meaning consistent with the context of this specification, and not be interpreted in an idealized or overly rigid manner.

Where expressions such as "at least one of A, B, and C, etc." are used, they should generally be interpreted as those skilled in the art would normally understand the expression (for example, "having A, B, and C A system of at least one of "shall include, but not be limited to, systems with A alone, B alone, C alone, A and B, A and C, B and C, and/or A, B, C, etc. ). Where an expression such as "at least one of A, B, or C, etc." is used, it should generally be interpreted in accordance with the meaning that those skilled in the art would normally understand the expression (for example, "having A, B, or C A system of at least one of "shall include, but not be limited to, systems with A alone, B alone, C alone, A and B, A and C, B and C, and/or A, B, C, etc. ).

Fluorescence is a common light-emitting phenomenon in nature. Fluorescence lifetime imaging, which distinguishes signal from background based on differences in fluorescence lifetimes, is limited when fluorophores have similar lifetimes.

Fluorescent nanodiamond is a bright, light-stable fluorescent material with good cell absorption capacity and ultra-low cytotoxicity. The background-free wide-field imaging technology based on magnetic modulation of fluorescent nano-diamond spin state is based on magnetic modulation or microwave modulation. Manipulation requires a more complex system and high operating costs.

Fluorescent molecules can transition from a low-energy ground state to a high-energy excited state after absorbing energy. Fluorescent molecules spontaneously transition from the excited state back to the ground state and emit photons, which is called autofluorescence radiation process. Under external radiation, excited fluorescent molecules produce radiation with the same frequency, phase and polarization as the external radiation, which is called the process of stimulated fluorescence radiation. When the fluorescence generated by this process exceeds the autofluorescent radiation, it will compete to suppress the spontaneously emitted fluorescence. This phenomenon is called STED (stimulated emission depletion, stimulated emission depletion). The traditional optical fluorescence microscope is limited by the diffraction limit, and the spatial resolution can only reach about 300nm, which cannot meet the research needs of modern science. Super-resolution STED microscopy requires a large amount of energy required to deplete stimulated radiation, usually around megawatts per square centimeter. Strong pump light will affect the properties of the target to be measured and even damage biological samples, which greatly limits its Applications in in situ imaging of living organisms.

One aspect of the present invention provides a background-free wide-field and low-loss super-resolution dual-purpose imaging device.

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

As shown in FIG. 1 , a background-free wide-field and low-loss super-resolution dual-purpose imaging device 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 light beams with a first wavelength.

The second pump light unit 120 is configured to generate light beams with a second wavelength. The second wavelength beam includes a second wavelength Gaussian beam or a second wavelength hollow core beam.

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

The collection unit 140 is configured to receive at least one of the first wavelength beam signal, the second wavelength beam signal and the fluorescence photon signal, and output the target photon signal obtained through filtering.

The control unit 150 is configured to receive the target photon signal, and generate 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 can generate a first wavelength beam with an adjustable beam size. Utilizing the first wavelength beam to irradiate the sample to be measured can initialize the charge state of the fluorescent beacons in the first position range that can be irradiated by the first wavelength beam in the sample to be tested, including making the fluorescent beacons in the first position range from The ground state of the current charge state transitions to the excited state, and then returns to the ground state from the excited state, and can excite the fluorescent beacon within the first position to release energy in the form of photon radiation and emit a fluorescent signal.

According to embodiments of the present invention, fluorescent beacons may include metal particles, nanopores, dielectric particles, fluorescent molecules, quantum dots, point defects, and the like. The fluorescent beacon in the embodiment of the present invention may include an NV (nitrogen-vacancy, nitrogen-vacancy) color center in diamond, and the charge state may include a dark state NV ⁰ with weak fluorescence and a bright state NV ⁻ with strong fluorescence. With the irradiation of the first wavelength light beam and the second wavelength light beam, the layout ratio of the dark state NV ⁰ and the bright state NV ⁻ can change.

According to an embodiment of the present invention, the second pump light unit 120 can generate a second wavelength beam with an adjustable beam size, the power of the second wavelength beam and the first wavelength beam can be different, and the second wavelength beam can include the second wavelength Gaussian beam. beam or a second wavelength hollow beam. Irradiating the sample to be tested with the light beam of the second wavelength can increase the charge bright state layout degree of the fluorescent beacons within the second position range that can be irradiated by the light beam of the second wavelength in the sample to be tested, and enhance the fluorescence intensity.

It should be noted that, the first wavelength beam and the second wavelength beam may be laser beams or any other light beams, such as infrared light, ultraviolet light, microwave, X-ray and so on. The first wavelength light beam and the second wavelength light beam can also be other energies, such as electrical energy, chemical energy, etc. The embodiment of the present invention does not make any limitation thereto.

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

For example, for a nitrogen-vacancy (nitrogen vacancy, NV) color center in diamond, the two charge states of NV are the bright state NV with stronger fluorescence intensity ^- and the dark state NV ⁰ with weaker fluorescence intensity. Pumping with a 1064 nm laser can switch the charge state of NV molecules between NV ^- and NV ⁰ and adjust the layout of the NV charge state. The charge state of the NV molecules can be initialized to a balanced layout using a 532 nm laser.

It should be noted that the light beam of the first wavelength may correspond to a degree of layout of the charge state of the fluorescent beacon. The power of the beam part of the second wavelength hollow beam is different from the power of the central part, which can correspond to different charge state populations of the fluorescent beacons, that is, different fluorescence intensities.

According to an embodiment of the present invention, in the low-loss super-resolution mode, the second-wavelength hollow beam can be converted on the basis of the second-wavelength Gaussian beam. For example, for a beam whose intensity distribution is Gaussian, use a vortex phase plate of the corresponding wavelength to modulate the beam phase differently according to the angular distribution on the incident surface, and the beam can be converted into a hollow beam. The vortex phase plate can be VPP-1a vortex phase plate, and the ratio of the central optical power density to the highest optical power density of the obtained hollow beam is 1:50. In background-free widefield mode, a second wavelength Gaussian beam can be used directly.

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

It should be noted that the first position range can be the second position range, or it can be the first wavelength beam focused on other areas of the sample that do not include the second position range, or it can be the area of the central hole of the second wavelength hollow beam The range can also be the range where any specified beam irradiates any area of the sample. In the embodiment of the present invention, the first position range may be determined according to the requirements of the experimenter or the user, which is not limited here.

According to an embodiment of the present invention, when imaging is required, the fluorescence of the fluorescent beacons at the intersection of the second position range and the first position range is stronger, and the fluorescence of the fluorescent beacons in the first position range outside the second position range is weaker , fluorescent beacons outside the range of the first location do not fluoresce.

According to an embodiment of the present invention, the collection unit 140 can collect the fluorescent photon signals emitted by the fluorescent beacons in the sample to be tested, and filter out photon signals of other wavelengths, for example, to obtain target photon signals. By transmitting the target photon signal to a computer control unit, for example, wide-field imaging or scanning imaging can be performed based on the light intensity information of the fluorescent photon signal combined with software. The software that can be combined may include, for example, MATLAB (Matrix Laboratory, 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, configured to generate a light beam with a first wavelength. A first pulse generator for receiving the first sequence of pulses sent by the control unit. The first modulator is configured to control the first wavelength light beam according to the first sequence of pulses to generate a sequenced first wavelength light beam. The first fiber coupling-collimation system is used for receiving the first wavelength light beam and outputting the first collimated light beam.

According to an embodiment of the present invention, the second pump light unit 120 may include: a second light source for generating a Gaussian beam of a second wavelength. a second pulse generator for receiving the second sequence of pulses sent by the control unit. The second modulator is configured to control the second wavelength light beam according to the second sequence of pulses to generate a sequenced second wavelength light beam. The second fiber coupling-collimation system is used for receiving the second wavelength light beam and outputting the 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 semiconductor lasers and other types of lasers. The laser light source has the advantages of high collimation, high brightness, and good monochromaticity. Using a laser light source can achieve better results.

It should be noted that the first light source and the second light source may be pump light of different wavelengths emitted by the same machine, or may be two beams of pump light emitted by two different machines. The two beams of pump light generated by the first light source and the second light source can irradiate the sample to be tested at the same time, sequentially, or according to a preset time. The preset time may include the irradiation time with the best effect summed up in multiple experiments. For example, the irradiation time of the first wavelength beam may be 10 μs, the second wavelength beam irradiation time may be 10 μs, and the first wavelength beam as excitation light may be irradiated for another 1 ms, etc.

According to an embodiment of the present invention, the first modulator and the second modulator may be placed at the focal points of the first wavelength light beam and the second wavelength light beam respectively. Adjust the three-dimensional position and angle of the first modulator and the second modulator, so that the first wavelength beam and the second wavelength beam respectively pass through the first modulator and the second modulator and then diffract, which can generate , High-order diffracted beams corresponding to the second wavelength beams. The first modulator and the second modulator can be controlled by the control unit 150 . For example, when the first modulator receives a high-voltage TTL signal from the control unit 150, the first modulator may convert the first wavelength beam into a high-order diffracted beam for output. In case the first modulator accepts a low voltage TTL signal, the first modulator may not output any light beam.

The first modulator and the second modulator can be acousto-optic modulators, the high-order diffracted light can be selected as first-order diffracted light, the diffraction efficiency can reach 80%, and the extinction ratio can be about 2000:1. The TTL signal can be generated by the PCI (Peripheral Component Interconnect, computer and its peripheral device interconnection standard) board, and can be controlled by the LabVIEW program.

According to an embodiment of the present invention, the first optical fiber coupling-collimation system can obtain the first collimated beam by coupling the first wavelength beam in free space into the polarization-maintaining fiber to emit. The second fiber coupling-collimation system can obtain the second collimated beam by coupling the second wavelength beam in the free space into the polarization-maintaining fiber and outputting it.

According to an embodiment of the present invention, the first pump light unit may further include at least one of the following: an optical filter, configured to adjust the power of the first wavelength light beam. The reflecting mirror is used to adjust the direction of the first wavelength light beam. The first adjustable beam expander is used to adjust the diameter of the first wavelength beam. The first quarter-wave plate is used to convert the linearly polarized first wavelength beam into a circularly polarized first wavelength beam.

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

According to an embodiment of the present invention, the second pump light unit may further include at least one of the following: a half-wave plate, configured to adjust the polarization direction of the light beam of the second wavelength. A polarizing beam splitter, used in conjunction with a half-wave plate, to adjust the power of the second wavelength beam. The second adjustable beam expander is used to adjust the diameter of the second wavelength beam. A vortex phase plate for converting a second wavelength Gaussian beam into a second wavelength hollow core beam. The second quarter-wave plate is used to convert the linearly polarized light beam of the second wavelength into a circularly polarized light beam of the second wavelength. The 1:1 non-polarizing beam splitter is used to split the second wavelength beam according to the power ratio of 1:1 to obtain the first split beam and the 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.

According to an embodiment of the present invention, the vortex phase plate can be a vortex phase plate suitable for the second wavelength beam, and can convert the second wavelength Gaussian beam into the second wavelength hollow core beam in the low-loss super-resolution mode. This component can be eliminated in the background-free widefield mode and the second wavelength Gaussian beam can be used directly. The second quarter-wave plate can be a quarter-wave plate suitable for the second wavelength light beam, the polarization of the second wavelength light beam can be in any direction, and circular polarization can be selected to achieve the best effect.

According to an embodiment of the present invention, the sample unit 130 may include: a sample to be tested, and the fluorescent beacon may include nano-diamond particles. The microscope lens is used to receive the first wavelength light beam and/or the second wavelength light beam, focus the first wavelength light beam and/or the second wavelength light beam on the sample to be measured, and collect the first wavelength light emitted by the fluorescent beacon in the sample to be measured At least one of the beam signal of the first wavelength, the beam signal of the second wavelength and the fluorescent photon signal. The piezoelectric ceramic stage is used to move the pixel points of the sample to be measured to the preset position with nanometer precision. The temperature control box is used to maintain the stability of the temperature and humidity of the environment where the sample to be tested is located.

According to the embodiments of the present invention, the fluorescent beacon can be introduced into the biological sample through biological or chemical methods to obtain the sample to be tested. The sample to be tested can be placed on a glass slide for imaging. The temperature control box can maintain the stability of the sample to be tested and the position of the beam during the measurement process. For example, in the embodiment of the present invention, the temperature can be stabilized within 10 mK, and the humidity can be stabilized within 0.5%.

According to an embodiment of the present invention, the dual-purpose imaging device for background-free wide-field and low-loss super-resolution may further include a beam combining unit. The beam combining unit can receive the beam of the first wavelength and the beam of the second wavelength, and output the beam of combining.

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

According to an embodiment of the present invention, the beam combining unit may include at least one of the following: a fast deflection mirror for adjusting the direction of the second wavelength beam, and adjusting the focus position of the second wavelength beam after being focused by the microscope lens. The variable focal length lens is used to adjust the focal plane position of the second wavelength light beam after being focused by the microscope lens. The lens group is used to adjust the position, direction and 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 fluorescent photon signal, and filtering the first wavelength beam, At least one of the light beam signal at the second wavelength and the fluorescent photon signal is reflected. 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 present invention, the lens group can adjust and shape the second wavelength beam, so that the effect of the fast deflecting mirror on the second wavelength beam is equivalent to the rear focal plane of the microscope lens, so as to achieve better imaging effect. For example, a variable focus lens can be used to coincide the focal plane of the second wavelength beam with the focal plane of the first wavelength beam, and two lenses can be used to adjust the size of the second wavelength beam so that the first wavelength beam and the second wavelength beam are focused and the spot Same size. Both short pass dichroic mirrors and long pass dichroic mirrors can have high transmission. The high-transmittance short-pass dichroic mirror can collect the fluorescent photon signal of the fluorescent beacon without affecting the first wavelength beam. The high-transmittance long-pass dichroic mirror can collect the fluorescent photon signal of the fluorescent beacon without affecting the second wavelength beam.

According to an embodiment of the present invention, the second wavelength beam is transmitted through the long-pass dichroic mirror, and after being reflected by the short-pass dichroic mirror, it can enter the microscope lens together with the first wavelength beam transmitted through the short-pass dichroic mirror . The position, direction and divergence angle of the second wavelength beam can be adjusted by using a fast deflection mirror, a variable focal length lens, and a lens group, and finally the first wavelength beam and the second wavelength beam coincide, and the focus after being focused by the microlens also coincides . The combined pump light can improve the beam utilization efficiency and obtain more fluorescent signals, which is beneficial for imaging the sample to be tested.

It should be noted that when the first wavelength light beam and the second wavelength light beam sequentially irradiate the sample to be tested within respective preset times, the beam combining unit may not be needed.

According to an embodiment of the present invention, the collection unit 140 may include: a filter set for receiving at least one of the first wavelength beam signal, the second wavelength beam signal, and the fluorescence photon signal, and performing filtering processing to obtain the target photon signal. The charge-coupled device camera is used to collect target photon signals and send the target photon signals to the control unit 150 . The single photon counter is used to collect the target photon signal and send the target photon signal to the control unit 150 .

According to an embodiment of the present invention, the filter set can separate the fluorescent signal of the fluorescent beacon in the second charge state from the optical signal of other wavelengths such as pumping light, and filter out the interference signal to obtain the target photon signal. The control unit 150 can perform wide-field imaging based on the target photon signal collected by the charge-coupled device camera to obtain a background-free wide-field image. The control unit 150 can perform scanning imaging based on the target photon signal 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 the following: a 1:1 non-polarizing beam splitter, which may be used to split the fluorescent photon signal in proportion to be used in a CCD (Charge Coupled Device) Camera for background-free wide-field imaging and single-photon counter for low-loss super-resolution imaging. The achromatic lens group can be used to focus the fluorescence photon signal on the CCD camera and single photon counter, and cooperate with the pinhole for spatial filtering. A small hole that can be used for spatial filtering of the fluorescent photon signal in a confocal system.

According to an embodiment of the present invention, when imaging is required, since the pump light is irradiated on the sample to be measured, various objects in the sample area will reflect the pump light, such as reflecting the first wavelength beam signal, the second wavelength beam signal, etc. Wait for the pump light. Inevitably, the pump light and the fluorescence photon signal will enter the back-end collection unit 140 together, and the light intensity of the fluorescence is weak, and it is difficult to distinguish after mixing with the pump light, so it is necessary to use a dichroic mirror to collect The emitted photons are separated. In addition, due to the large difference between the light intensity of the pump light and the fluorescence, as long as a small amount of pump light is mixed into the fluorescence photon signal, it will affect the subsequent operations such as collection, so it is necessary to further filter the collected photons. Separation, filter out the doped pump light photons, obtain higher purity fluorescent photons, and obtain better imaging effects.

Fig. 2 schematically shows 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 present invention.

As shown in FIG. 2 , corresponding to the first pumping light units 110 and 111 being a first light source composed of a first-wavelength laser, they can continuously emit light beams of the first wavelength. 115 is a circular continuously variable reflective neutral density filter, which can adjust the power of the first wavelength light beam. 116 is a reflector, which can be used to change the beam direction of the first wavelength beam. 112 is a pulse generator, which can control the acousto-optic modulator 113 to generate a serialized light beam of the first wavelength. 114 is a fiber coupling-collimation system, which can collimate and adjust the direction of the first wavelength beam. 117 is an adjustable beam expander, which can adjust the diameter of the first wavelength beam. 118 is a quarter wave plate, which can change the polarization state of the first wavelength light beam. Through the operation of the first pump light unit 110, a light beam of the first wavelength can be obtained.

As shown in FIG. 2 , corresponding to the second pump light unit 120 , 121 being a second light source composed of a second-wavelength laser, it can continuously emit light beams of the second wavelength. 125 is an electronically controlled rotating half-wave plate, which cooperates with the polarization beam splitter 126 to adjust the power of the second wavelength beam. 123 is an acousto-optic modulator, which can be controlled by the pulse generator 122 to generate serialized light beams of the second wavelength. 124 is a fiber coupling-collimation system, which can collimate and adjust the direction of the second wavelength beam. 127 is an adjustable beam expander. 128 is an m=2 vortex phase plate, which is used to generate a hollow beam of the second wavelength. 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, which is used to measure the power of a part of the light beam with the second wavelength. Through the operation of the second pump light unit 120, a light beam of the second wavelength can be obtained.

As shown in FIG. 2 , corresponding to the sample units 130 and 131 are samples to be tested. 132 is a high numerical aperture microscope lens, which can be used to focus the combined beam onto the sample 131 to be measured. 133 is a piezoelectric ceramic displacement stage, which can be used to control the position of the sample 131 to be tested. 134 is a temperature control box, which can be used to maintain the stable temperature and humidity of the environment where the sample 131 to be tested is located.

As shown in FIG. 2 , corresponding to the beam combining units 160 and 161 are fast deflection mirrors, which can change the direction of the second wavelength beam. 162 is a variable focal length lens, which can adjust the divergence angle of the light beam of the second wavelength. 163 is a lens group. 165 is a long pass dichroic mirror, which can separate fluorescent photon signal and pump light. 164 is a short-pass dichroic mirror, which can separate the fluorescent photon signal, the pump light, and the beam combining signal, and the beam combining signal can be a signal corresponding to the beam combining beam. Through the work of the beam combining unit 160 , the combined beam to be sent to the sample unit 130 can be obtained based on the short-pass dichroic mirror 164 , and the target photon signal to be sent to the collection unit 140 can be obtained based on the long-pass dichroic mirror 165 .

As shown in FIG. 2 , corresponding to the collection units 140 and 141 are filter sets, which can perform back-end filtering on the target photon beam. 144 is a 1:1 non-polarizing beam splitter, which can split the target photon beam. 145, 146, 147, and 148 are achromatic lenses, which can focus and collimate the target photon beam. 149 is a small hole, which can perform spatial filtering on the target photon beam. 143 is a single photon counter, which can be used to realize scanning imaging. 142 is a CCD camera, which can be used to realize wide-field imaging.

As shown in FIG. 2 , 150 is a computer control unit, which 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 images and/or background-free widefield images. The computer control unit 150 can also be used to control the electronically 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 , to process the fluorescent signals sent back from the camera and the single photon counter.

Another aspect of the present invention provides a low-loss super-resolution imaging method.

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

As shown in Fig. 3, the method includes operations S310-S330.

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

In operation S320, the target fluorescence intensity corresponding to the pixel 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 tested is determined according to the multiple target fluorescence intensities corresponding to the multiple pixel points to be tested.

According to an embodiment of the present invention, as shown in FIG. 2 , in the low-loss super-resolution mode, adjusting the diameter and power of the first wavelength beam and the second wavelength beam can obtain a small focused beam at the sample to be tested. Using the single photon detector 143 to collect the fluorescent photons in the target photon signal, the light intensity change information of the fluorescent photon can be collected, and the light beam related to the target photon signal can be focused to the probe of the single photon detector 143 by the achromatic lens 148 . Whenever a fluorescent photon is measured, the single photon detector 143 can emit a TTL pulse signal. The TTL pulse signal can be recorded by the data acquisition card of the control unit 150, so as to obtain the fluorescence intensity count. When imaging, the pixel point of the sample to be measured can be moved to the detection position through the position scanning device, and the fluorescent photons radiated by each pixel point are collected, and then the pixel point position is used as the x-y coordinate, and the fluorescent light intensity is used as the point z-value for imaging. The position scanning device can be a piezoelectric ceramic displacement platform 133, and the piezoelectric ceramic displacement platform 133 can choose P-562.3CD (nanometer positioning system). For example, when imaging a target area with an area of 2μm*2μm, you can choose a step size of 10nm, divide the area into 200*200 pixels, and use the piezoelectric ceramic stage 133 to move the sample to 200*200 coordinates in sequence position, measure the fluorescence intensity of each coordinate point separately, and perform low-loss super-resolution imaging on the sample to be tested.

It should be noted that the control unit 150 needs to be used in conjunction with a data acquisition card and a pulse signal generator. The data acquisition card can use PCIe6363 series, and the pulse signal generator can use PCI pulser. The control program can be written based on LabVIEW software, which can be used to control the switch and position movement of the light source, obtain fluorescence intensity and position information, and perform post-processing imaging.

According to an embodiment of the present invention, when imaging each pixel to be detected, it is necessary to cycle through each area or each pixel to be detected multiple times to obtain sufficient fluorescent photon counts. That is, the beam switching control sequence needs to be looped many times. In this way, enough photon counts can be obtained, and a signal with a higher signal-to-noise ratio can be obtained, which is conducive to improving the imaging quality.

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

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

As shown in Fig. 4, the method includes operations S410-S420.

In operation S410, based on the background-free wide-field and low-loss super-resolution dual-purpose imaging device, a first image and a second image taken for the sample to be tested are acquired. The first image includes an image obtained when a sample to be tested including a second initialized fluorescent beacon is irradiated with a beam of the first wavelength, and the second image includes an image obtained when a sample including a second enhanced fluorescent beacon is irradiated with a beam of the first wavelength and a second wavelength. The image obtained in the case of the target image to be tested. The second initialization fluorescent beacon is obtained based on the first wavelength beam irradiating the fluorescent beacon in the sample to be tested, and the second enhanced fluorescent beacon is obtained based on the first wavelength beam and the second wavelength beam irradiating the second initialization fluorescent beacon.

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

According to an embodiment of the present invention, as shown in FIG. 2 , in the background-free wide-field mode, a CCD camera 142 can be used to collect fluorescent photons in the target photon signal, and the light beam related to the target photon signal can be focused by an achromatic lens 145 to the CCD camera 142 . The CCD camera 142 can transmit the light signal sensed by each pixel back to the control unit 150, and the control unit 150 can make difference between the images under the on and off states of the second wavelength light beam to obtain a background-free wide-field imaging image.

According to the embodiment of the present invention, in the wide-field mode, adjusting the adjustable beam expanders 117 and 127 to change the size and divergence angle of the first wavelength beam and the second wavelength beam can enlarge the focused irradiation range. In addition, the power of the second wavelength light beam can be changed by adjusting the angle of the electronically controlled rotating half-wave plate 125, so that the second position range becomes the entire first position range except for the central hole. A CCD camera is used to collect light intensity change information. Since the biological spontaneous background fluorescence is not affected by the second wavelength beam, the background signal can be filtered out to obtain a background-free wide-field image with a high signal-to-background ratio.

It should be noted that the specific implementation process of the background-free wide-field imaging method and the low-loss super-resolution imaging method in the embodiment of the present invention can refer to the functional modules of the background-free wide-field and low-loss super-resolution dual-purpose imaging device in the above-mentioned embodiments The specific implementation of , will not be repeated here.

Fig. 5 schematically shows a principle diagram of low-power continuous light-assisted imaging according to an embodiment of the present invention.

According to an embodiment of the present invention, NV has two charge states, a dark state NV ⁰ with weak fluorescence and a bright state NV ⁻ with strong fluorescence. When performing imaging, as shown in Figure 5, under the irradiation of 532 nm laser, 1064 nm laser can make NV switch between two charge states, and the layout of the two charge states is different when balanced under different power of 1064 nm light. Since the fluorescence intensity of the two charge states is different, the 1064 nm light power is different, and the fluorescence intensity of the NV equilibrium state is different. The fluorescence wavelength of NV ^- radiation is longer than that of NV ⁰ , and a 600 nm long-pass filter can be added to the filter set to filter out NV ⁰ fluorescence, so that only the fluorescence photons of NV ^- radiation can be detected.

In order to better understand the principles and ideas of the embodiments of the present invention, please refer to Figures 6 to 12. The following describes the dual-purpose imaging device for background-free wide-field and low-loss super-resolution using the embodiments of the present invention in conjunction with several specific application scenarios. The principle of imaging nano-diamond particles and nematodes that swallowed nano-diamond particles.

Fig. 6 schematically shows a schematic diagram of a scene of imaging nanodiamond particles according to an embodiment of the present invention.

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

As shown in FIG. 6 , the sample to be tested is nano-diamond particles 620 on a glass plate 610 , and the imaging mode is a low-loss super-resolution imaging mode. As shown in Figure 7, in the case of low-loss super-resolution imaging of nano-diamond particles, when measuring a pixel point to be measured, it can first be irradiated with a 532 nm Gaussian beam with a power of 20 μW for 200 μs, and the nanometer Initialization of NV charge state layout in diamond grains. Then, a 532 nm Gaussian beam with a power of 20 μW can be used to illuminate for 10 ms, and the fluorescence intensity can be recorded with a single photon counter. Next, a 532 nm Gaussian beam with a power of 20 μW and a 1064 nm hollow beam with a power of 10 mW can be irradiated together for 200 μs to increase and balance the NV ^- ratio in nanodiamonds outside the center of the beam, thereby enhancing the fluorescence count. Afterwards, a 532 nm Gaussian beam with a power of 20 μW and a 1064 nm hollow-core beam with a power of 10 mW can be co-irradiated for another 10 ms. The sequence of "initialization-counting-enhancement-counting" can be repeated multiple times, such as 10 times. Then, the average of the count enhancement values can be taken as the signal value of the pixel. In the case where the signal of the next pixel to be measured needs to be measured, the sample can be moved by controlling the piezoelectric ceramic stage. Since there are fewer NVs ⁱⁿ the center of the beam, the dark spot depression in the image can represent the location of the NVs. Optionally, a deconvolution algorithm can be used to extract the dark spots in the image and turn them into bright spots.

Fig. 8A schematically shows a low-loss super-resolution imaging image obtained by imaging nano-diamond particles using a traditional focusing imaging method according to an embodiment of the present invention.

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

FIG. 8C schematically shows the results of extracting dark spot depressions using a deconvolution algorithm for the low-loss super-resolution imaging image shown in FIG. 8B according to an embodiment of the present invention.

In traditional focusing imaging, referring to the imaging result shown in FIG. 8A , the resolution of nanodiamond particles is about 325 nm. Based on the sequence shown in FIG. 7 , the fluorescence of nanodiamond particles is specifically adjusted and imaged with structured light, and the result shown in FIG. 8B can be obtained. As shown in FIG. 8B , the nanodiamond forms a dark spot at the center of the light spot. Using the deconvolution algorithm, the result shown in Figure 8C can be obtained. As shown in FIG. 8C , the resolution of the obtained nanodiamond particle imaging result is about 100 nm.

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

According to an embodiment of the present invention, as shown in FIG. 9, by continuing to increase the power of the first wavelength beam and/or the second wavelength beam used to irradiate the sample to be tested, the resolution of the nanodiamond particles can be increased to about 30 nm , well below the diffraction limit of optical fluorescence microscopy.

Fig. 10 schematically shows a schematic diagram of a scene of imaging a nematode that has engulfed nanodiamond particles according to an embodiment of the present invention.

FIG. 11 schematically shows a sequence diagram of control signals and readout signals when imaging nematodes that have engulfed nanodiamond particles according to an embodiment of the present invention.

As shown in FIG. 10 , the sample to be tested is nano-diamond particles in nematodes, and the imaging mode is a background-free wide-field imaging mode. As shown in Figure 11, in the case of wide-field imaging of nematodes with a strong fluorescent background, you can first irradiate with a 532 nm Gaussian beam with a power of 100 μW for 100 ms, and the NV charge state distribution in the nanodiamond particles Home initialization, take the first image. Then, a 532nm Gaussian beam with a power of 100 μW and a 1064nm Gaussian beam with a power of 10 mW can be used to irradiate together for 200 μs to increase and balance the NV ^- ratio in the nanodiamonds in the area, and enhance the fluorescence count. A second image can then be taken by co-irradiating with a 532 nm Gaussian beam at 100 μW and a 1064 nm Gaussian beam at 10 mW for an additional 100 ms. Then, the difference between the second image and the first image can be taken as a frame of background-free wide-field image. Repeat this process to take multiple frames of images with a refresh time of 200.2 ms, and the bright spots in the images can represent nano-diamond particles.

Fig. 12A schematically shows a wide-field imaging image with background obtained by imaging nano-diamond particles endocytosed by nematodes using a traditional wide-field imaging method according to an embodiment of the present invention.

Fig. 12B schematically shows a background-free wide-field imaging image obtained by using the device of the present invention to image nanodiamond particles endocytosed by nematodes according to an embodiment of the present invention.

In traditional wide-field imaging, see the imaging results shown in Figure 12A, the nanodiamond particles are submerged in the autofluorescent background. Based on the sequence shown in FIG. 11 , the fluorescence of nanodiamond particles in nematodes is specifically adjusted and the structured light imaging is used to obtain a background-free wide-field imaging result as shown in FIG. 12B . Referring to Figure 12B, it can be confirmed that the background-free wide-field imaging based on the device and method provided by the present invention can remove the background well, improve the signal-to-background ratio, and can easily realize the extraction of nano-diamond particles from the fluorescent background of nematodes come out.

Through the above-mentioned embodiments of the present invention, a plenoptic dual-purpose imaging device and imaging method for background-free wide-field and low-loss super-resolution, suitable for in-situ imaging of biological samples, are provided. Related devices and methods use low-power continuous light to modulate the charge state of fluorescent beacons and then modulate the number of fluorescent signal photons, which can not only realize background-free wide-field imaging of the sample to be tested, but also achieve low-loss super-resolution imaging without affecting the sample to be tested. The nature of the book, the letter-to-back ratio is high. Compared with the existing technology, due to the long lifetime of the charge state, which can reach the order of seconds, the pump light with low power can control the charge state of the fluorescent beacon without affecting the properties of the sample to be tested, and has little damage to the sample. Good compatibility. In addition, the embodiment of the present invention adopts the all-optical method, the system structure and operation are simple, the use and assembly are convenient, the operation cost and expense cost are saved, and the user experience is improved.

It should be noted that the units and algorithm steps of the examples described in conjunction with the embodiments disclosed herein can be implemented by electronic hardware, computer software, or a combination of both. In order to clearly illustrate the interchangeability of hardware and software, the composition and steps of each example have been generally described in terms of functions in the above description. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art may use different methods to implement the described functions for each specific application, but such implementation should not be regarded as exceeding the scope of the present invention.

The steps of the methods or algorithms described in connection with the embodiments disclosed herein may be directly implemented by hardware, software modules executed by a processor, or a combination of both. Software modules can be placed in random access memory (RAM), internal memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, removable disk, CD-ROM, or any other Any other known storage medium.

The background-free wide-field and low-loss super-resolution dual-purpose imaging system and method provided by the present invention are described above in detail. In this paper, specific examples are used to illustrate the principle and implementation of the present invention, and the descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (8)

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.

Images