CN117074382A - Single beam imaging apparatus and method - Google Patents
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- 238000003384 imaging method Methods 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title abstract description 17
- 230000005284 excitation Effects 0.000 claims abstract description 42
- 230000005764 inhibitory process Effects 0.000 claims abstract description 16
- 230000001629 suppression Effects 0.000 claims description 29
- 230000010287 polarization Effects 0.000 claims description 16
- 235000012489 doughnuts Nutrition 0.000 claims description 6
- 230000005540 biological transmission Effects 0.000 claims description 3
- 230000008569 process Effects 0.000 abstract description 11
- 230000003287 optical effect Effects 0.000 description 18
- 230000005281 excited state Effects 0.000 description 14
- 238000013500 data storage Methods 0.000 description 13
- 230000005283 ground state Effects 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 3
- 238000001459 lithography Methods 0.000 description 3
- 230000008832 photodamage Effects 0.000 description 3
- 239000011232 storage material Substances 0.000 description 3
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- 239000000975 dye Substances 0.000 description 2
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- 238000005329 nanolithography Methods 0.000 description 2
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- 238000004590 computer program Methods 0.000 description 1
- 238000001839 endoscopy Methods 0.000 description 1
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- 239000002105 nanoparticle Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
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Abstract
The application discloses a single-beam imaging device and a method, and belongs to the technical field of microscopic imaging. The application adopts a single light beam, utilizes the principle of time multiplexing the wavefront of the continuous wave light beam and the time inhibition process of the fluorophor, carries out time modulation between short time and long time, and simultaneously carries out wavefront modulation between excitation and inhibition to realize imaging, simplifies the structure of an imaging device and improves the imaging super-resolution.
Description
Technical Field
The application relates to the technical field of microscopic imaging, in particular to a single-beam imaging device and a single-beam imaging method.
Background
The basic principle of the stimulated emission loss (stimulated emission depletion, STED) microscope technology is that two beams of laser are adopted to irradiate a sample at the same time, wherein one beam of laser is used for exciting fluorescent molecules, so that the fluorescent molecules in the range of the focal Airy spot of an objective lens are in an excited state; meanwhile, another ring-shaped loss laser with zero central light intensity is overlapped with the laser, so that fluorescent molecules in an excited state at the edge region of the focal point Airy spot of the objective lens return to a ground state through an excited radiation loss process and do not spontaneously radiate fluorescence, and only fluorescent molecules in the central region can spontaneously radiate fluorescence, so that a fluorescent luminous point with a super diffraction limit is obtained.
The application of STED and super-resolution technologies including super-resolution photoinduced inhibition nano-lithography (super resolution photoinduction-inhibition nanolithography, SPIN) technology enables revolutionary changes to the fields of optical imaging and optical data storage (Operational Data Store, ODS) and breaks through Abbe diffraction limit. In super-resolution technology, STED and SPIN increase resolution by reducing the effective Point Spread Function (PSF) through the on-off control of two lasers, which revolutionized the applications related to point scanning, such as three-dimensional optical imaging, endoscopy, lithography, and optical data storage. However, both beam imaging methods require high power levels of pulsed lasers or high power continuous lasers to perform the shutdown (suppression) process, resulting in photodamage and photobleaching of the optical data storage material. Furthermore, the use of two different lasers in STED-like microscopes requires special filters, phase plates, dichroic mirrors, lasers and chromatic correction, complicating the system.
Disclosure of Invention
Aiming at the problem of complex structure of the existing imaging device using two different laser beams, a single-beam imaging device and a single-beam imaging method which aim at simultaneous excitation and suppression are provided.
The application provides a single beam imaging device, which is applied to imaging a sample with triplet state performance and comprises the following components:
the continuous wave laser is used for emitting a continuous wave laser beam and is incident to the time wavefront multiplexer;
the time wavefront multiplexer is used for modulating an incident continuous wave laser beam into an excitation light pulse and a suppression light pulse, and the excitation light pulse and the suppression light pulse are sequentially switched to be incident to the imaging device;
and the imaging device is used for sequentially switching the excitation light pulse and the inhibition light pulse to be incident to the sample, modulating the time of the excitation light pulse and the inhibition light pulse to be incident to the sample, and detecting the light emitted by the sample for imaging.
Preferably, the excitation light pulse is a gaussian distributed pulse.
Preferably, the suppression light pulses are doughnut distributed pulses.
Preferably, the time wavefront multiplexer comprises an acousto-optic modulator, a polarizer, an electro-optic modulator, a first polarizing beam splitter, a second polarizing beam splitter, a first mirror, a spiral phase plate and a second mirror;
an acousto-optic modulator, a polarizer, an electro-optic modulator and a first polarization beam splitter are sequentially arranged in the transmission direction of laser beams emitted by the continuous wave laser, one beam of light split by the first polarization beam splitter is incident on a first reflecting mirror, light reflected by the first reflecting mirror is incident on a spiral phase plate, light emitted by the spiral phase plate is incident on a second reflecting mirror, light reflected by the second reflecting mirror is incident on the second polarization beam splitter, and one beam of light split by the second polarization beam splitter is excitation light pulses or suppression light pulses.
Preferably, the time wavefront multiplexer further comprises a function generator for driving the acousto-optic modulator and the electro-optic modulator.
Preferably, the imaging device comprises a third reflecting mirror, a photoelectric detector, a 1/4 wave plate and an imaging objective lens;
the excitation light pulse and the inhibition light pulse separated by the second polarization beam splitter are transmitted through the third reflector and are incident to the imaging objective lens through the 1/4 wave plate in sequence;
the light emitted by the storage module is incident to the third reflector through the imaging objective lens and the 1/4 wave plate, and then is reflected by the third reflector and then is incident to the photoelectric detector.
Preferably, the photodetector is a single photon avalanche diode.
The application also provides an imaging method of the single-beam imaging device, which comprises the following steps:
opening a sample with triplet state performance, modulating continuous wave laser into excitation light pulses to be incident on the sample, modulating the time of the excitation light pulses to be incident on the sample, and closing the sample to realize short-time incidence of the excitation light pulses;
opening the sample, modulating the continuous wave laser into a suppression light pulse to be incident on the sample, modulating the time for the suppression light pulse to be incident on the sample, and closing the sample to realize the long-time incidence of the suppression light pulse;
light emitted from the sample is detected for imaging.
Preferably, the excitation light pulse is a gaussian distributed pulse.
Preferably, the suppression light pulses are doughnut distributed pulses.
The beneficial effects of the technical scheme are that:
in the technical scheme, the application adopts a single beam, utilizes the principle of time multiplexing Continuous Wave (CW) beam wave front and fluorophore time inhibition process, carries out time modulation between short time (excitation) and long time (inhibition), and simultaneously carries out wave front modulation between excitation and inhibition to realize imaging, simplifies the structure of an imaging device and improves the imaging super-resolution.
Drawings
FIG. 1 is a schematic view of an optical path of an embodiment of a single beam imaging apparatus according to the present application;
FIG. 2a is a schematic diagram of the energy transfer path of a short pulse incident sample according to the present application;
FIG. 2b is a schematic diagram of the energy transfer path of a long pulse incident sample according to the present application;
FIG. 3a is a graph of peak pulse energy versus fluorescence intensity for a long pulse in accordance with the present application;
FIG. 3b is a graph of pulse peak energy versus full width at half maximum of fluorescence for a long pulse in accordance with the present application;
fig. 4 is a flowchart of an imaging method of a single beam imaging device according to an embodiment of the present application.
Detailed Description
Advantages of the application are further illustrated in the following description, taken in conjunction with the accompanying drawings and detailed description.
Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples are not representative of all implementations consistent with the present disclosure. Rather, they are merely examples of apparatus and methods consistent with some aspects of the present disclosure as detailed in the accompanying claims.
The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
It should be understood that although the terms first, second, third, etc. may be used in this disclosure to describe various information, these information should not be limited to these terms. These terms are only used to distinguish one type of information from another. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the present disclosure. The word "if" as used herein may be interpreted as "at … …" or "at … …" or "responsive to a determination", depending on the context.
In the description of the present application, it should be understood that the numerical references before the steps do not identify the order in which the steps are performed, but are merely used to facilitate description of the present application and to distinguish between each step, and thus should not be construed as limiting the present application.
The application provides a single-beam imaging device for imaging a sample with triplet state performance, which comprises: continuous wave lasers (CW), time wavefront multiplexers, and imaging devices.
The continuous wave laser is used for emitting a continuous wave laser beam and is incident to the time wavefront multiplexer;
the time wavefront multiplexer is used for modulating an incident continuous wave laser beam into an excitation light pulse and a suppression light pulse, and the excitation light pulse and the suppression light pulse are sequentially switched to be incident to the imaging device;
the imaging device is used for sequentially switching the excitation light pulse and the inhibition light pulse to be incident on the sample, modulating the time of the excitation light pulse and the inhibition light pulse to be incident on the sample, realizing short-time incidence of the excitation light pulse and long-time incidence of the inhibition light pulse, and detecting light emitted by the sample for imaging.
Taking the optical data storage unit ODS having triplet performance as an example, the sample of the present embodiment uses a single beam, uses a time-multiplexed Continuous Wave (CW) beam wavefront, performs time modulation between a short time (excitation) and a long time (suppression), performs wavefront modulation between an excitation light pulse and a suppression light pulse, forms a high-resolution write spot, performs lithography on the optical data storage unit ODS, and performs scanning on the optical data storage unit ODS to perform reading.
The principle of the time-suppression process is based on the interaction mechanism between the different energy states of fluorophores or nanomaterials and metastable dark states or dark states (e.g. triplet states of organic dyes). Consider the general case of a three-level system where the sample has a ground state S0, an excited state S1, and a dark state S2. The excited state S1 has two paths for energy transfer. Referring to fig. 2a, when a short pulse is used to enter a sample, electrons in a ground state S0 are excited to an excited state S1 by laser light, and light is emitted when the electrons are transferred from the excited state S1 to the ground state S0; referring to fig. 2b, when a long pulse is used to enter a sample, electrons in the ground state S0 are excited to the excited state S1 by the laser, electrons in the excited state S1 can be transferred to the dark state S2 through a crossover process, and electrons in the dark state cannot emit light or photons with different energies. Referring to fig. 3a-3b, when the short pulse is fixed as 500nm, the long pulse is restrained along with the increase of pulse peak energy, the turn-off capability is reflected, the effect of the short pulse is weakened, the full width at half maximum of the fluorescence is reduced, the effective light spot is reduced, and the resolution is improved.
This principle can be demonstrated with color-converting nanoparticles that exhibit a time-suppressed process caused by intermediate states crossing time modulation. The simulation result shows that the resolution of the optical data storage unit ODS reaches 25nm at a loss power of 40.0 mw.
In the embodiment, the time wavefront multiplexer is used for switching the long pulse and the short pulse of a beam of light source, and the optical resolution is improved by matching the long pulse with the adjustment of the wavefront and matching the short pulse with the adjustment of the wavefront. The long pulse in this embodiment is a light pulse with gaussian distribution, excitation is achieved, and the short pulse is a light pulse with doughnut distribution, and suppression is achieved. The pulse length in this embodiment can be achieved by turning on and off the sample, avoiding the use of high power levels of pulsed lasers or high power continuous lasers to turn off, resulting in photodamage and photobleaching of the optical data storage material.
Further, as shown in fig. 1, the time wavefront multiplexer of the present embodiment includes an acousto-optic modulator 2, a polarizer 3, an electro-optic modulator 4, a first polarizing beam splitter 6, a second polarizing beam splitter 10, a first reflecting mirror 7, a spiral phase plate 8, and a second reflecting mirror 9;
the continuous wave laser 1 emits laser beam, and has acousto-optic modulator 2, polarizer 3, electro-optic modulator 4, first polarization beam splitter 6 in order in transmission direction, and first polarization beam splitter 6 divides a beam of light to be incident on first speculum 7, and the light that first speculum 7 reflection was incident on spiral phase plate 8, and the light that spiral phase plate 8 was emergent is incident on second speculum 9, and the light that second speculum 9 reflection was incident on second polarization beam splitter 10, and the first beam of light that second polarization beam splitter 10 divides is excitation light pulse or suppresses the light pulse.
The continuous wave laser according to the present embodiment generates polarized modulated light by electro-optical modulation, and the polarized modulated light generates time-wavefront multiplexed light by polarization beam splitting and a spiral phase plate.
Further, the time wavefront multiplexer of the present embodiment further includes a function generator for driving the acousto-optic modulator 2 and the electro-optic modulator 4.
The imaging device of the present embodiment includes a third reflecting mirror 11, a photodetector 13, a 1/4 wave plate 12, and an imaging objective lens 14; the excitation light pulse and the inhibition light pulse separated by the second polarization beam splitter 10 are sequentially transmitted by the third reflecting mirror 11 and are incident to the imaging objective lens 14 through the 1/4 wave plate 12; the light emitted from the storage module is incident to the third mirror 11 through the imaging objective lens 14 and the 1/4 wave plate 12, and then is reflected by the third mirror 11 and then is incident to the photodetector 13. The photodetector 13 of the present embodiment may be implemented using a single photon avalanche diode.
The switching period of the wavefront multiplexer in this embodiment may be 100Hz to 1MHz, the pulse length may be 1ms to 1ps, and the pulse interval may be 100ms to 10ps.
Referring to fig. 4, the present embodiment further provides an imaging method of a single beam imaging device, including the following steps:
a1, opening a sample with triplet state performance, modulating continuous wave laser into excitation light pulses to be incident on the sample, modulating the time of the excitation light pulses to be incident on the sample, and closing the sample to realize short-time incidence of the excitation light pulses;
a2, opening the sample, modulating the continuous wave laser into a suppression light pulse to be incident on the sample, modulating the time for the suppression light pulse to be incident on the sample, and closing the sample to realize the long-time incidence of the suppression light pulse;
a3, detecting light emitted by the sample for imaging.
Taking the optical data storage unit ODS having triplet performance as an example, the sample of the present embodiment uses a single beam, uses a time-multiplexed Continuous Wave (CW) beam wavefront, performs time modulation between a short time (excitation) and a long time (suppression), performs wavefront modulation between an excitation light pulse and a suppression light pulse, forms a high-resolution write spot, performs lithography on the optical data storage unit ODS, and performs scanning on the optical data storage unit ODS to perform reading.
The principle of the time-suppression process is based on the interaction mechanism between the different energy states of fluorophores or nanomaterials and metastable dark states or dark states (e.g. triplet states of organic dyes). Consider the general case of a three-level system where the sample has a ground state S0, an excited state S1, and a dark state S2. The excited state S1 has two paths for energy transfer. Referring to fig. 2a, when a short pulse is used to enter a sample, electrons in a ground state S0 are excited to an excited state S1 by laser light, and light is emitted when the electrons are transferred from the excited state S1 to the ground state S0; referring to fig. 2b, when a long pulse is used to enter a sample, electrons in the ground state S0 are excited to the excited state S1 by the laser, electrons in the excited state S1 can be transferred to the dark state S2 through a crossover process, and electrons in the dark state cannot emit light or photons with different energies. Referring to fig. 3a-3b, when the short pulse is fixed as 500nm, the long pulse is restrained along with the increase of pulse peak energy, the turn-off capability is reflected, the effect of the short pulse is weakened, the full width at half maximum of the fluorescence is reduced, the effective light spot is reduced, and the resolution is improved.
In the embodiment, the time wavefront multiplexer is used for switching the long pulse and the short pulse of a beam of light source, and the optical resolution is improved by matching the long pulse with the adjustment of the wavefront and matching the short pulse with the adjustment of the wavefront. The long pulse in this embodiment is a light pulse with gaussian distribution, excitation is achieved, and the short pulse is a light pulse with doughnut distribution, and suppression is achieved. The pulse length in this embodiment can be achieved by turning on and off the sample, avoiding the use of high power levels of pulsed lasers or high power continuous lasers to turn off, resulting in photodamage and photobleaching of the optical data storage material.
The read-write resolution of the optical data storage unit ODS of this embodiment may range from 25-250nm, with a minimum spot size of 200nm in the existing optical storage unit.
The apparatus embodiments described above are merely illustrative, wherein elements illustrated as separate elements may or may not be physically separate, and elements shown as elements may or may not be physical elements, may be located in one place, or may be distributed over at least two network elements. Some or all of the modules can be selected according to actual needs to achieve the purpose of the embodiment of the application. Those of ordinary skill in the art will understand and implement the present application without undue burden.
From the above description of embodiments, it will be apparent to those skilled in the art that the embodiments may be implemented by means of software plus a general purpose hardware platform, or may be implemented by hardware. Those skilled in the art will appreciate that all or part of the processes implementing the methods of the above embodiments may be implemented by a computer program for instructing relevant hardware, where the program may be stored in a computer readable storage medium, and where the program may include processes implementing the embodiments of the methods described above. The storage medium may be a magnetic disk, an optical disk, a Read-only memory (ROM), a random access memory (RandomAccessMemory, RAM), or the like.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (10)
1. A single beam imaging apparatus for imaging a sample having triplet properties, comprising:
the continuous wave laser is used for emitting a continuous wave laser beam and is incident to the time wavefront multiplexer;
the time wavefront multiplexer is used for modulating an incident continuous wave laser beam into an excitation light pulse and a suppression light pulse, and the excitation light pulse and the suppression light pulse are sequentially switched to be incident to the imaging device;
and the imaging device is used for sequentially switching the excitation light pulse and the inhibition light pulse to be incident to the sample, modulating the time of the excitation light pulse and the inhibition light pulse to be incident to the sample, and detecting the light emitted by the sample for imaging.
2. The single beam imaging apparatus of claim 1 wherein the excitation light pulses are gaussian distributed pulses.
3. The single beam imaging apparatus of claim 1 wherein the suppression light pulses are doughnut distributed pulses.
4. The single beam imaging apparatus of claim 1 wherein the time wavefront multiplexer comprises an acousto-optic modulator, a polarizer, an electro-optic modulator, a first polarizing beam splitter, a second polarizing beam splitter, a first mirror, a spiral phase plate, and a second mirror;
an acousto-optic modulator, a polarizer, an electro-optic modulator and a first polarization beam splitter are sequentially arranged in the transmission direction of laser beams emitted by the continuous wave laser, one beam of light split by the first polarization beam splitter is incident on a first reflecting mirror, light reflected by the first reflecting mirror is incident on a spiral phase plate, light emitted by the spiral phase plate is incident on a second reflecting mirror, light reflected by the second reflecting mirror is incident on the second polarization beam splitter, and one beam of light split by the second polarization beam splitter is excitation light pulses or suppression light pulses.
5. The single beam imaging apparatus of claim 4 wherein the time wavefront multiplexer further comprises a function generator for driving the acousto-optic modulator and the electro-optic modulator.
6. The single beam imaging apparatus of claim 4, wherein the imaging apparatus comprises a third mirror, a photodetector, a 1/4 wave plate, and an imaging objective lens;
the excitation light pulse and the inhibition light pulse separated by the second polarization beam splitter are transmitted by the third reflector and are incident to the imaging objective lens through the 1/4 wave plate in sequence;
the light emitted by the storage module is incident to the third reflector through the imaging objective lens and the 1/4 wave plate, and then is reflected by the third reflector and then is incident to the photoelectric detector.
7. The single beam imaging apparatus of claim 6 wherein the photodetector is a single photon avalanche diode.
8. An imaging method of a single beam imaging apparatus, comprising:
opening a sample with triplet state performance, modulating continuous wave laser into excitation light pulses to be incident on the sample, modulating the time of the excitation light pulses to be incident on the sample, and closing the sample to realize short-time incidence of the excitation light pulses;
opening the sample, modulating the continuous wave laser into a suppression light pulse to be incident on the sample, modulating the time for the suppression light pulse to be incident on the sample, and closing the sample to realize the long-time incidence of the suppression light pulse;
light emitted from the sample is detected for imaging.
9. The imaging method of a single beam imaging apparatus of claim 8, wherein the excitation light pulses are gaussian distributed pulses.
10. The imaging method of a single beam imaging apparatus of claim 8, wherein the suppression light pulses are doughnut distributed pulses.
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