CN106501229B

CN106501229B - Optical super-resolution microscopic imaging system and method

Info

Publication number: CN106501229B
Application number: CN201611121968.5A
Authority: CN
Inventors: 陈向东; 孙方稳; 郭光灿
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2016-12-08
Filing date: 2016-12-08
Publication date: 2023-07-14
Anticipated expiration: 2036-12-08
Also published as: CN106501229A

Abstract

The embodiment of the invention discloses an optical super-resolution microscopic imaging system, which comprises an optical module and an imaging module; the optical module is used for irradiating the target to be imaged through the first wavelength light beam and the second wavelength light beam so as to initialize molecules of the target to be imaged into a first charge state and convert molecules in a first preset position range into a second charge state; irradiating molecules in a second preset position range of the target to be imaged by using a third wavelength light beam so as to radiate fluorescent signals; and imaging the target to be imaged according to the light intensity information of the fluorescent signal radiated by the target to be imaged. According to the technical scheme, the low-power continuous optical pump is used, super-resolution microscopic imaging of the target to be imaged is achieved, the property of the target to be imaged is not affected, the optical system and the electronic system are simple in structure, and the operation cost and the expense cost are saved. In addition, the embodiment of the invention also provides a corresponding using method aiming at the system, and the method has corresponding advantages.

Description

Optical super-resolution microscopic imaging system and method

Technical Field

The invention relates to the field of optical microscopic imaging, in particular to an optical super-resolution microscopic imaging system and method.

Background

Along with the development of science and technology, life science and technology are correspondingly developed, the requirements on precision and accuracy of biological cell imaging are higher and higher, and the research of micro-nano materials is well-developed. The spatial resolution of the traditional optical microscopic imaging is limited by the numerical aperture and the wavelength of light of the objective lens, the resolution can only reach about 300nm, and the requirement of the modern microscopic imaging on the resolution is far from being met.

Fluorescence is a phenomenon of luminescence that is common in nature. Fluorescence is the interaction of photons with molecules, i.e., after absorption of energy (light energy, electrical energy, chemical energy, etc.), a fluorescent molecule transitions from a ground state at the lowest energy to an excited state (first or second excited state) at a higher energy, electrons in the excited state are in a high energy state, are unstable, spontaneously transition back to the ground state in the nanosecond range of time, and release energy in the form of photon radiation, and the emitted light with this property is called fluorescence. The theoretical basis of fluorescence imaging is that the intensity of the fluorescent signal emitted after excitation of the fluorescent substance is in a linear relationship with the amount of fluorescein over a range. Conventional optical microscopy imaging is performed by detecting this spontaneously emitted fluorescent signal.

When a fluorescent molecule in an excited state is subjected to external radiation, it generates radiation of the same frequency, phase and polarization as the external radiation, i.e. an stimulated fluorescent radiation process, if this process competes for more than autofluorescent radiation, the spontaneous emitted fluorescence is not measured, a phenomenon called STED (stimulated emission depletion, stimulated radiation depletion). The optical microscopy based on the phenomenon belongs to super-resolution microscopic imaging technology, the imaging principle is changed from the molecular level, the bottleneck that the resolution cannot be improved due to the limitation of diffraction limit in the traditional optical microscopy imaging is broken through, and great development is achieved.

The STED super-resolution imaging principle and the optical path are shown in figures 1-3, the object to be imaged is excited by the first beam of laser to generate fluorescence, a circular spot (Rayleigh Li Ban) with diffraction limit is formed, the position is excited by the second beam of laser in a circular ring shape, the second beam of laser just can excite a non-central area of the spot formed at the same irradiation position, after the two beams of laser are overlapped, only fluorescence molecules in the central area can realize excited-state autofluorescence, surrounding molecules (namely the area irradiated by the second beam of laser) are in a loss state and do not emit light, so that the effective fluorescence excitation radius is greatly reduced, namely the diameter of the central fluorescence area of the spot can be infinitely small due to the invasion of the non-central depletion state area, the limit of Rayleigh Li Yanshe is broken through, and the imaging resolution is improved.

In the prior art, although STED super-resolution imaging method can meet the requirement of resolution, the energy required for converting molecules from a ground state to an excited state is relatively large, and strong pumping laser can influence the property of an object to be imaged and even cause damage; moreover, the method generally requires the use of pulsed lasers, and precise timing of the laser pulses requires more complex optical and electronic systems, increasing the operating costs and undoubtedly the cost of the charges.

Disclosure of Invention

The embodiment of the invention aims to provide an optical super-resolution microscopic imaging system, which uses low-power continuous optical pumping to realize super-resolution microscopic imaging of an object to be imaged, does not influence the property of the object to be imaged, has simple optical system and electronic system compared with STED, and saves the operation cost and the expense cost.

In order to solve the technical problems, the embodiment of the invention provides the following technical scheme:

the embodiment of the invention provides an optical super-resolution microscopic imaging system, which comprises the following components:

an optical module and an imaging module;

the optical module comprises a first pump light unit, a second pump light unit and a third pump light unit;

the first pump light unit is used for irradiating an object to be imaged by utilizing a first wavelength light beam so as to initialize molecules of the object to be imaged into a first charge state;

the second pump light unit is used for generating a second wavelength hollow light beam, and irradiating the target to be imaged by utilizing the second wavelength hollow light beam so as to convert molecules in a first preset position range of the target to be imaged into a second charge state;

the third pump light unit is used for irradiating molecules in a second preset position range of the target to be imaged by utilizing a third wavelength light beam so as to radiate fluorescent signals;

the imaging module is used for imaging the target to be imaged according to the light intensity information of the fluorescent signal radiated by the target to be imaged.

Preferably, the first pump light unit includes:

a light source for emitting the first wavelength light beam;

the lens group is used for focusing the first wavelength light beam to the modulator, collimating the light emitted by the modulator into parallel light beams and then making the parallel light beams incident on the target to be imaged;

the modulator is used for converting the first wavelength light beam into a first higher-order diffraction light beam in a preset time range.

Preferably, the second pump light unit includes:

a light source for emitting a second wavelength light beam;

the lens group is used for focusing the second wavelength light beam to the modulator, collimating the light emitted by the modulator into parallel light beams and then making the parallel light beams incident on the object to be imaged;

the modulator is used for converting the first wavelength light beam into a second high-order diffraction light beam in a preset time range;

and the spiral phase plate is used for converting the second wavelength light beam into the second wavelength hollow light beam.

Preferably, the third pump light unit includes:

a light source for emitting the third wavelength light beam;

the lens group is used for focusing the light beam with the third wavelength to the modulator, collimating the light emitted by the modulator into parallel light beams and then making the parallel light beams incident on the target to be imaged;

the modulator is used for converting the third wavelength light beam into a third high-order diffraction light beam in a preset time range.

Preferably, the optical module further includes:

and the beam combining unit is used for combining the three pump light beams into one beam through the bicolor sheet group and the reflecting mirror, and focusing the three pump light beams onto the target to be imaged through the objective lens.

Preferably, the imaging module includes:

the bicolor plate is used for separating the fluorescent signal radiated by the target to be imaged from the pump light signal;

the filter is used for filtering out pump light signals doped in the separated fluorescent signals;

the detector is used for collecting the filtered fluorescent signals and sending the fluorescent signals to the control unit;

a lens for focusing the filtered fluorescent signal onto the detector;

the position scanning unit is used for moving the pixel point to be imaged of the target to be imaged to a preset position so as to image the pixel point to be imaged;

the control unit is used for calculating the light intensity of the fluorescent signal and imaging the object to be imaged according to the light intensity of the fluorescent signal radiated by the object to be imaged and the corresponding position.

Preferably, the modulator is an acousto-optic modulator.

Preferably, the control unit comprises a data acquisition card and a pulse signal card.

Preferably, the detector is a single photon detector.

In another aspect, the embodiment of the invention provides an optical super-resolution microscopic imaging method, which includes:

illuminating an object to be imaged with a first wavelength beam to initialize molecules of the object to be imaged to a first charge state;

irradiating the target to be imaged by using a hollow light beam with a second wavelength so as to convert molecules in a first preset position range of the target to be imaged into a second charge state;

irradiating molecules in a second preset position range of the target to be imaged by using a third wavelength light beam so as to radiate fluorescent signals;

and imaging the target to be imaged according to the light intensity information of the fluorescent signal radiated by the target to be imaged.

The embodiment of the invention provides an optical super-resolution microscopic imaging system, which comprises an optical module and an imaging module; the optical module is used for irradiating the object to be imaged through the first wavelength light beam and the second wavelength light beam so as to initialize molecules of the object to be imaged into a first charge state and convert the molecules into a second charge state in a first preset position range; irradiating molecules in a second preset position range of the target to be imaged by using a third wavelength light beam so as to radiate fluorescent signals; and imaging the target to be imaged according to the light intensity information of the fluorescent signal radiated by the target to be imaged.

The invention has the advantage that super-resolution microscopic imaging of the target to be imaged is realized by using low-power continuous optical pumping. Compared with the prior art, because the service life of the charge state is longer than that of the excited state (a few nanoseconds), the second order can be achieved, so that the charge state of a target to be imaged can be controlled by pump light with lower power, and the property of a sample to be detected is not influenced by the pump light with lower power; in addition, the technical scheme does not need to perform accurate time sequence synchronization on laser pulses, so that the optical system and the electronic system are simpler in structure, more convenient to use and assemble, operation cost and cost are saved, and the use experience of a user is improved. In addition, the embodiment of the invention also provides a corresponding using method, and the method has corresponding advantages.

Drawings

For a clearer description of embodiments of the invention or of the prior art, the drawings that are used in the description of the embodiments or of the prior art will be briefly described, it being apparent that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained from them without inventive effort for a person skilled in the art.

Fig. 1-1 is a schematic diagram of STED super-resolution imaging provided by an embodiment of the present invention;

fig. 1-2 are optical path diagrams of STED super-resolution imaging and normal imaging provided by the embodiment of the invention;

FIGS. 1-3 are actual light path diagrams of STED super-resolution imaging provided by an embodiment of the invention;

FIG. 2 is a block diagram of an embodiment of an optical super-resolution microscopy imaging system according to an embodiment of the invention;

FIG. 3 is a block diagram of another embodiment of an optical super-resolution microscopy imaging system according to an embodiment of the invention;

FIG. 4 is a sequence diagram of control signals and read signals provided by an embodiment of the present invention;

FIG. 5 is a schematic diagram of an exemplary application scenario provided by an embodiment of the present invention;

FIG. 6 is a schematic diagram of an optical super-resolution microscopy imaging system providing the illustrative example of FIG. 5 in accordance with an embodiment of the invention;

FIG. 7 is an imaging schematic of the illustrative example of FIG. 5 provided in accordance with an embodiment of the present invention;

FIG. 8 is an imaging schematic of the illustrative example of FIG. 5 provided by an embodiment of the present invention;

fig. 9 is a schematic flow chart of an optical super-resolution microscopic imaging method according to an embodiment of the present invention.

Detailed Description

In order to better understand the aspects of the present invention, the present invention will be described in further detail with reference to the accompanying drawings and detailed description. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The terms "first," "second," "third," "fourth," and the like in the description and in the claims of this application and in the above-described figures, are used for distinguishing between different objects and not necessarily for describing a sequential or chronological order. Furthermore, the terms "comprise" and "have," as well as any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements but may include other steps or elements not expressly listed.

Referring first to fig. 2, fig. 2 is a block diagram of an optical super-resolution microscopic imaging system in a specific implementation manner provided in an embodiment of the present invention, where the embodiment of the present invention may include the following:

an optical super-resolution microscopic imaging system may include an optical module 21 and an imaging module 22.

The optical module 21 includes a first pump light unit 211, a second pump light unit 212, and a third pump light unit 213.

The first pump light unit 211 is configured to irradiate an object to be imaged with a first wavelength beam, so as to initialize molecules of the object to be imaged to a first charge state.

The second pump light unit 212 is configured to generate a second wavelength hollow beam, and irradiate the object to be imaged with the second wavelength hollow beam, so as to convert molecules in a first preset position range of the object to be imaged into a second charge state.

The third pump light unit 213 is configured to irradiate molecules within a second preset position range of the target to be imaged with a third wavelength beam, so as to radiate a fluorescent signal.

The object to be imaged is typically a fluorescent material or a material modified with a fluorescent material, such as fluorescent protein-labeled cells. Fluorescent materials typically exist in states with different amounts of charge, the fluorescence spectra and intensities of the different charge states being different, and the charge states typically have a longer lifetime, typically on the order of seconds, whereas the excited state of the molecule has a lifetime of only a few nanoseconds.

Most molecules of the fluorescent material are normally in a ground state energy level with the lowest energy, and when excited by energy (light energy, electrical energy, chemical energy, etc.), the fluorescent material absorbs the energy to make a transition. The energy absorbed by the different states of the molecule is different, and the energy required to be absorbed to transition to the desired molecular state can be determined according to the nature of the fluorescent material molecule. That is, light of different wavelengths can pump molecules of the same fluorescent material to different charge states, e.g., for nitrogen-vacancy (NV) color-centered fluorescent material in diamond, the charge state of NV is predominantly negatively charged NV ^- Neutral NV ⁰ Making the charge state of NV molecule become NV ^- Pumping with 637nm laser is required to change the charge state of NV molecules to NV ⁰ The pumping is performed using a 532nm laser.

The first wavelength light beam, the second wavelength hollow light beam and the third wavelength light beam can be laser light beams, and of course, any other light can be used, for example, infrared light, ultraviolet light, aurora, X-ray and the like; of course, other energy, such as electric energy and chemical energy, may be used, and the embodiment of the present invention is not limited in any way.

It should be noted that, from the above, it can be known that the first wavelength light beam (initial light) corresponds to the molecule of the object to be imaged in the first charge state; the second wavelength light beam (converted light) corresponds to molecules of the object to be imaged being in a second charge state.

The second wavelength hollow beam needs to be converted on the basis of the second wavelength beam, for example, the laser beam is a gaussian beam, and in order to obtain the hollow beam, the gaussian beam can be converted by a spiral phase plate. The phase plate modulates the phase of the light beam differently according to the angular distribution on the incident plane, thereby obtaining a hollow light beam.

The first preset position range of the object to be imaged is that the light beam irradiates other positions of the object to be imaged, which do not comprise the hollow corresponding irradiation positions of the hollow light beam, namely when the first wavelength light beam and the second wavelength light beam are light with the same shape, the first preset position range is that the first wavelength light beam irradiates the surface area of the object to be imaged minus the area of the hollow position of the hollow light beam of the second wavelength, which is mapped on the object to be imaged. For example, assuming that the first wavelength beam and the second wavelength beam are gaussian beams, when the first wavelength beam and the second wavelength beam are irradiated on the object to be imaged, the diameter of the beam is 1cm, and the hollow position of the second wavelength hollow beam is mapped on the area of the ring with the diameter of 0.1cm of the object to be imaged, the first preset position range is the area with the outer diameter of 1cm and the inner diameter of 0.1 cm.

The third wavelength beam is used as detection light to irradiate fluorescent material molecules in a second preset position range, so that the absorption light energy of the fluorescent material molecules is transited from a current charge state to an excited state, and then the fluorescent material molecules return to a ground state from the excited state, and energy is released in a photon radiation mode, namely fluorescence is emitted.

The second preset position range may be the first preset position range; the first wavelength light beam irradiates any other area of the surface area of the object to be imaged, which does not contain the first preset position range, or the second wavelength hollow light beam hollow position is mapped to the area of the object to be imaged; of course, any area of the surface area of the object to be imaged may be illuminated by any given beam. The area of the second predetermined location range may be determined according to the needs of the experimenter or the user.

The imaging module 22 is configured to image the target to be imaged according to light intensity information of fluorescent signals radiated by the target to be imaged.

The imaging module 22 sends the collected fluorescence to the control unit by acquiring and collecting the fluorescence, and the control unit performs imaging of the target to be imaged by software (e.g., MATLAB, labVIEW) based on the fluorescence intensity information by calculating the intensity of the collected fluorescence.

Optionally, in some implementations of this embodiment, the first pump unit 211 may include:

a light source 2111 for emitting the first wavelength light beam;

a lens group 2112 for focusing the first wavelength beam to a modulator, collimating the light coming out of the modulator into a parallel beam, and then making it incident on the object to be imaged;

the modulator 2113 is configured to convert the first wavelength light beam into a first higher-order diffracted light beam within a preset time range.

The second pump unit 212 includes:

a light source 2121 for emitting a second wavelength light beam;

a lens group 2122 for focusing the second wavelength beam to a modulator, collimating the light coming out of the modulator into a parallel beam, and then incident on the object to be imaged;

the modulator 2123, configured to convert the first wavelength light beam into a second higher-order diffracted light beam in a preset time range;

a spiral phase plate 2124 for converting the second wavelength light beam into the second wavelength hollow light beam.

The third pump unit 213 includes:

a light source 2131 for emitting the third wavelength light beam;

a lens group 2132 for focusing the light beam of the third wavelength to a modulator, collimating the light beam exiting the modulator into a parallel light beam, and then incident the parallel light beam on the object to be imaged;

the modulator 2133 is configured to convert the third wavelength light beam into a third higher-order diffracted light beam within a predetermined time range.

The light sources 2111, 2121 and 2131 may be laser light sources or other light sources. The laser light source may be a semiconductor laser, although other types of lasers may be used. Because of the advantages of high collimation, high brightness, good monochromaticity, high energy density and the like of the laser light source, the laser light source can be preferably adopted.

It should be noted that, the light sources 2111, 2121, 2131 may be different wavelengths emitted by the same machine, or may be three different machines.

The modulator is used to convert the current light beam into a higher-order diffracted light beam within a preset time frame, i.e. to control the switching and duration of each light source. The number of modulators corresponds to the number of light sources, for example, if three pump light beams are produced by one semiconductor laser. Only one modulator is required.

The modulator can be arranged at the focus of the light beam, and the three-dimensional position and angle of the modulator are adjusted, so that the light beam is diffracted after passing through the modulator, and a high-order diffraction light beam is generated. The modulator can be controlled by the control unit transmitting a preset signal, and when the modulator is signaled, higher-order diffracted light can be obtained. For example, when the modulator receives the high voltage TTL signal emitted by the control unit, the modulator converts the pump light into a high-order diffraction beam to be output; when a low voltage TTL signal is received, the modulator does not output a light beam.

Preferably, the modulator may employ an acousto-optic modulator, and the higher order diffracted light may be 1 st order diffracted light of the acousto-optic modulator, with a diffraction efficiency of about eighty percent and an extinction ratio of about 2000:1. the TTL control signal can be generated by a PCI card inserted into the control unit and controlled by a LabView program. The control of the irradiation time of the light source and the opening and closing of the light source are realized by adopting the modulator, so that the self operation of a user is avoided, the operation cost is saved, and the use experience of the user is improved; in addition, the adjusted light beam can obtain better imaging effect.

The lens group is mainly used for shaping the light emitted by the light source, so that the best imaging effect is achieved. For example, a convex lens with a focal length of 15cm may be used to focus three beams of light of different wavelengths from parallel light to the focal point of the convex lens, so that all or most of the light may pass through the next device. After passing through the modulator, the beam may be re-collimated into a parallel beam by another 15cm focal length convex lens.

Preferably, a VPP-1a spiral phase plate available from RPC Photonics, inc. of America, is used to obtain a hollow beam having a center optical power density to maximum optical density ratio of about 2:100.

it should be noted that, the three pump light beams may irradiate the imaging target at the same time, or may sequentially irradiate the imaging target, or may irradiate the imaging target according to a preset time, where the preset time is generally the irradiation time with the best effect summarized by multiple experiments. For example, the initial light irradiation time is 10 microseconds, the converted light irradiation time is 10 microseconds, and the probe light irradiation time is 1 millisecond.

As a specific implementation, referring to fig. 3, the optical module in the foregoing embodiment may further include:

The first wavelength light beam (initialization light) is projected to the first bicolor plate through the reflector, the second wavelength hollow light beam (conversion light) is incident to the first bicolor plate through the parallel light collimated by the convex lens, the third wavelength light beam (detection light) is incident to the second bicolor plate through the parallel light collimated by the convex lens, the initialization light and the conversion light are incident to the third bicolor plate after being incident to the second bicolor plate through the first bicolor plate, and three light beams emitted from the third bicolor plate are incident to the objective lens through the third bicolor plate, and the three light beams are focused to an object to be imaged through the objective lens. Preferably, the numerical aperture of the objective lens may be na=0.9. The position and the angle of the bicolor sheet are adjusted, so that the focusing points of the three laser beams after passing through the objective lens can be perfectly overlapped.

It should be noted that, when the three beams of light sequentially irradiate the object to be imaged in respective preset time, the beam combining unit is not required.

The three pump light beams are combined, so that all or most of the pump light beams are irradiated to the target to be imaged, more fluorescent signals are obtained, and the imaging of the target to be imaged is facilitated.

In other implementations of the present embodiment, referring to fig. 3, the imaging module 22 may include:

the fluorescence collecting unit 221 is configured to collect fluorescence photons of radiation of an object to be imaged, and specifically includes:

the bicolor chip 2211 is used for separating the fluorescence signal and the pump light signal of the target radiation to be imaged.

And a filter 2212 for filtering out the pump light signal incorporated in the separated fluorescent signal.

A detector 2213 for collecting the filtered fluorescence signal and transmitting the fluorescence signal to the control unit.

A lens 2214 for focusing the filtered fluorescent signal onto the detector.

The position scanning unit 222 is configured to move the pixel to be imaged to a preset position to image the pixel to be imaged.

The control unit 223 is configured to calculate the light intensity of the fluorescent signal, and image the target to be imaged according to the light intensity of the fluorescent signal radiated by the target to be imaged and the corresponding position.

Because the pumping light irradiates the target to be imaged, and then the target to be imaged spontaneously radiates fluorescence photons, the unavoidable pumping light and fluorescence coincide, the intensity of the fluorescence is weaker, and the fluorescence is difficult to identify after being mixed with the pumping light, so that the double-color plate is required to be separated; in addition, because the light intensity of the two is too large, less pump light is mixed in fluorescence, and the subsequent operations such as collection and the like are unfavorable, further filtration is needed through a filter plate, and the pump light doped in a fluorescence signal is filtered out, so that fluorescence photons with higher purity are obtained, and a better imaging effect is obtained.

It should be noted that, the two-color sheet 2211 and the third two-color sheet in the embodiment may be the same two-color sheet, that is, the two-color sheet 2211 is the third two-color sheet, so that the material and the cost can be saved, and the system structure can be simplified.

Preferably, the collection of fluorescent photons can be performed using a single photon detector, and the fluorescent light beam can be focused onto the single photon detector by a 7.5cm focal length convex lens. Each time a fluorescent photon is measured, the single photon detector will emit a TTL pulse signal. The TTL signal is recorded by a data acquisition card of the control unit, so that the fluorescent intensity value is obtained.

When imaging the target to be imaged, the whole target can be directly imaged, or the imaging can be performed according to each pixel point, and preferably, the second mode can be adopted. And the pixel point to be detected of the target to be imaged is moved to a detection position by a position scanning unit, fluorescence radiated by each pixel point is collected, and then the control unit is used for imaging according to the relation between the position of the pixel point and the light intensity. The position scanning system can be a piezoelectric ceramic displacement platform, the piezoelectric ceramic displacement platform can be purchased from German PI company P733.3 series, the position scanning system can be controlled by the control unit, namely, the control unit controls the piezoelectric displacement platform to move the sample to the next position, and the measurement of the fluorescence intensity of each pixel point is completed in sequence.

For example, in two-dimensional imaging, when imaging an object to be imaged having an area of 2 micrometers by 2 micrometers, the imaging area can be divided into 40 by 40 pixels, each having a size of 50 nanometers by 50 nanometers. And sequentially moving the sample to 40 x 40 coordinates by using a piezoelectric translation stage, respectively measuring the fluorescence intensity of each coordinate point, and carrying out optical super-resolution imaging on the target to be imaged.

The control unit can be composed of a computer, a data acquisition card and a pulse signal generation card, wherein the data acquisition card and the pulse signal generation card are integrated to a computer main board, the data acquisition card can be purchased from USB6343 series of America NI company, and the pulse signal generation card can be purchased from PCI pulse card produced by America Spincore company. The control system can be written based on LabVIEW software, can be used for controlling a light source switch and position movement, and the acquired fluorescence intensity and position information can also be used for realizing final imaging through the LabVIEW software.

It should be noted that, when imaging an object to be imaged or each pixel, it is generally necessary to cycle each block area or each pixel multiple times to obtain a sufficient fluorescent photon count. That is, the initial light, the converted light, and the probe light need to be circulated a plurality of times. Therefore, by obtaining enough fluorescence photons, a higher light intensity value is obtained, and super-resolution imaging of the target to be imaged is facilitated.

In one embodiment, for example, see fig. 5, fig. 5 is a sequence diagram of control signals and read signals. When collecting fluorescent light from a pixel, the initial light irradiates for 10 microseconds, the converted light irradiates for 20 microseconds, the detection light irradiates for 10 microseconds, and then the initial light, the converted light and the detection light circulate for a plurality of times within 50 milliseconds.

For a better understanding of the principles and concepts of the embodiments of the present invention, please refer to fig. 5 to 8, in which an optical super-resolution microscopic imaging system according to an embodiment of the present invention is used to perform optical super-resolution microscopic imaging on NV (nitrogen-vacancy) color-centered fluorescent materials in diamond.

NV has two charge states, negatively charged NV ^- And electrically neutral NV ⁰ . As shown in FIG. 5, a 637 nanometer laser can pump NV to NV with a 95% probability ⁰ A charge state; while a 532nm laser can pump NV to NV with 75% probability ^- A charge state. 589 nm lasers can be used to detect charge states. Due to NV ^- Fluorescence wavelength ratio NV of radiation ⁰ The fluorescence wavelength of the radiation is longer, and a 650 nanometer long-pass filter can be added in the light path to filter NV ⁰ Photons of radiation, thereby detecting only NV ^- Photons of radiation.

As shown in fig. 6, an optical path and a system diagram for performing optical super-resolution imaging on NV are shown. In the figure, 1 is a first wavelength light beam (initial light), 2 is a third wavelength light beam (detection light), 3 is a second wavelength light beam (converted light), and the three light beams are emitted simultaneously through a semiconductor laser; 4 is a convex lens for collimating the light beam; 5 is an acousto-optic modulator for controlling the opening and closing of the light beam and modulating the light beam into first-order diffraction light, the acousto-optic modulator is controlled by a pulse signal generating card 141 of the control unit; a spiral phase plate for modulating the second wavelength beam into a hollow beam; the reflecting mirror 10 and the double-color plates 7-1, 7-2 and 7-3 are used for combining three pump light beams and irradiating the three pump light beams on an object 9 to be imaged; the objective lens 8 is used for further focusing the combined light onto an object 9 to be imaged; the 7-3 bicolor sheets and the 11 filter sheets are used for separating the pump light in the fluorescence to obtain fluorescence with higher purity; the fluorescence is focused on a single photon detector 13 through convex lenses 12 and 4 to collect the fluorescence; the single photon detector collects a fluorescent photon and emits a TTL pulse signal, which is recorded by the data acquisition card 142 of the control unit 14, thereby obtaining a fluorescent intensity value.

The lifetime of the charge state is long, typically on the order of seconds, so a beam of lower power can be used to control the charge state. In the stimulated emission depletion technology, the excited state of the fluorescent material needs to be controlled, the service life of the fluorescent material is only a few nanoseconds, and the spontaneous emission rate is high. In order to achieve control of fluorescence intensity, the stimulated radiation depletion rate of its laser pump needs to be greater than its spontaneous radiation rate. The laser power of stimulated radiation depletion is relatively high, typically up to hundreds of milliwatts; in contrast, the laser power of the present invention requires only a few milliwatts to achieve higher resolution. Since both 637 and 532nm lasers can be used as the initializing light or converted light for imaging, there are two schemes to achieve super-resolution imaging.

Referring to fig. 7, super-resolution imaging is implemented by using 637nm laser as an initializing light and 532nm light as a converted light:

for fluorescence measurement at each pixel, NV is first initialized to NV with a 637nm Gaussian beam with a power of 2 mW ⁰ The duration of the beam may be 10 microseconds; then a 532nm hollow beam of 30 mW was used to convert the charge state of NV except the beam center to NV ^- The duration of the beam may also be 10 microseconds; finally, a 589 nanometer Gaussian beam pump with a power of 40 microwatts is used for measuring NV by a single photon counter ^- Photon number corresponding to charge state. The "initialize-convert-probe" sequence loops 50 milliseconds and then turns onAnd controlling the NV target to be imaged to move by the overvoltage translation stage, and measuring the fluorescence intensity of the next pixel point. Since the NV of the beam center is always at NV ⁰ State, and detected is NV ^- Fluorescence in the state, each dark spot in the resulting image represents an NV. Under such pumping conditions, the resolution of the resulting single NV is about 25 nm, well below the diffraction limit of around 350 nm.

Referring to fig. 8, a 532nm laser is used as an initializing light, and a 637nm laser is used as a converted light to realize super-resolution imaging:

first, initializing NV with a 532nm Gaussian beam with 2 mW power ^- The duration of the beam may be 10 microseconds; then the charge state of NV except the beam center is converted to NV by using a 30 mW 637 nanometer hollow beam ⁰ The duration of the beam is 10 microseconds; finally, a 589 nm Gaussian beam pump with 40 microwatts was used to measure NV ^- Photon number radiated by the charge state. The fluorescence measurement time for each pixel is also 50 milliseconds. NV due to beam centre being NV ^- The charge state, each bright spot in the resulting image represents an NV. Under such pumping conditions, the resolution of the resulting single NV is up to about 45 nm, again well below the diffraction limit.

As can be seen from the above, the embodiment of the invention uses the low-power continuous optical pump, which not only realizes super-resolution microscopic imaging of the target to be imaged, but also does not affect the property of the target to be imaged.

The embodiment of the invention also provides a corresponding use method for the optical super-resolution microscopic imaging system. The optical super-resolution microscopic imaging method described below and the optical super-resolution microscopic imaging system described above can be referred to correspondingly to each other.

Referring to fig. 9, fig. 9 is a flow chart of an optical super-resolution microscopic imaging method according to an embodiment of the present invention, where the embodiment of the present invention may include the following:

s901: illuminating an object to be imaged with a first wavelength beam to initialize molecules of the object to be imaged to a first charge state;

s902: irradiating the target to be imaged by using a hollow light beam with a second wavelength so as to convert molecules in a first preset position range of the target to be imaged into a second charge state;

s903: irradiating molecules in a second preset position range of the target to be imaged by using a third wavelength light beam so as to radiate fluorescent signals;

s904: and imaging the target to be imaged according to the light intensity information of the fluorescent signal radiated by the target to be imaged.

The specific implementation process of the optical super-resolution microscopic imaging method according to the embodiment of the present invention may refer to the specific implementation of each functional module of the optical super-resolution microscopic imaging system in the above embodiment, and will not be described herein.

In this specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, so that the same or similar parts between the embodiments are referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.

Those of skill would further appreciate that the various illustrative elements and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both, and that the various illustrative elements and steps are described above generally in terms of functionality in order to clearly illustrate the interchangeability of hardware and software. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the solution. 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. The software modules may be disposed 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 optical super-resolution microscopic imaging system and the method provided by the invention are described in detail. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

Claims

1. An optical super-resolution microscopy imaging system, comprising:

an optical module and an imaging module;

2. The system of claim 1, wherein the first pump unit comprises:

a light source for emitting a first wavelength light beam;

3. The system of claim 1, wherein the second pump unit comprises:

a light source for emitting a second wavelength light beam;

4. The system of claim 1, wherein the third pump unit comprises:

a light source for emitting a light beam of a third wavelength;

5. The system of any one of claims 2-4, wherein the optical module further comprises:

6. The system of claim 5, wherein the imaging module comprises:

a lens for focusing the filtered fluorescent signal onto the detector;

7. The system of claim 6, wherein the modulator is an acousto-optic modulator.

8. The system of claim 7, wherein the control unit comprises a data acquisition card and a pulse signal card.

9. The system of claim 8, wherein the detector is a single photon detector.

10. An optical super-resolution microscopic imaging method, comprising: