Single-beam imaging apparatus and method

Abstract

The application discloses a single-beam imaging device and method, and belongs to the technical field of microscopic imaging. The application adopts a single beam, utilizes the principle of time multiplexing continuous wave beam wavefront and fluorophore time suppression process, carries out time modulation between short time and long time, carries out wavefront modulation between excitation and suppression to realize imaging, simplifies the structure of the imaging device, and improves imaging super-resolution.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and more particularly to a single-beam imaging device and method. Background Technology

[0002] The basic principle of stimulated emission depletion (STED) microscopy is to simultaneously irradiate the sample with two laser beams. One laser beam is used to excite fluorescent molecules, putting them in an excited state within the Airy disk area at the objective lens focal point. At the same time, another ring-shaped depletion laser beam with zero intensity at the center is superimposed on the STED beam. This causes the fluorescent molecules in the excited state at the edge of the Airy disk at the objective lens focal point to return to the ground state through stimulated emission depletion, without spontaneously emitting fluorescence. Therefore, only the fluorescent molecules in the central region can spontaneously emitting fluorescence, thus obtaining super-diffraction-limited fluorescent emission points.

[0003] The invention of super-resolution technologies, including STED and super-resolution photoinduction-inhibition nanolithography (SPIN), has revolutionized the fields of optical imaging and operational data storage (ODS), breaking the Abbe diffraction limit. In super-resolution technologies, STED and SPIN improve resolution by reducing the effective point spread function (PSF) through the switching control of two laser beams, revolutionizing point-scanning applications such as 3D optical imaging, endoscopy, lithography, and optical data storage. However, these two beam imaging methods require high-power levels of pulsed or high-power continuous lasers for the switching (suppression) process, leading to optical damage and photobleaching of the optical data storage materials. Furthermore, using two different laser beams in STED-like microscopes requires specialized filters, phase plates, dichroscopes, lasers, and chromatic aberration correction, complicating the system. Summary of the Invention

[0004] To address the structural complexity of existing imaging devices that use two different laser beams, a single-beam imaging device and method designed to simultaneously excite and suppress laser beams are provided.

[0005] This invention provides a single-beam imaging device for imaging samples with triplet properties, comprising:

[0006] A continuous-wave laser is used to emit a continuous-wave laser beam and direct it onto a time-wavefront multiplexer.

[0007] A time wavefront multiplexer is used to modulate an incident continuous wave laser beam into an excitation pulse and a suppression pulse, which are then sequentially switched and incident on the imaging device.

[0008] An imaging device is used to sequentially switch excitation light pulses and suppression light pulses to be incident on the sample, while modulating the timing of the excitation light pulses and suppression light pulses incident on the sample. The imaging device is also used to detect the light emitted from the sample for imaging.

[0009] Preferably, the excitation light pulse is a Gaussian distributed pulse.

[0010] Preferably, the suppression light pulse is a donut-shaped pulse.

[0011] Preferably, the time wavefront multiplexer includes an acousto-optic modulator, a polarizer, an electro-optic modulator, a first polarization beamsplitter, a second polarization beamsplitter, a first reflector, a spiral phase plate, and a second reflector.

[0012] In the transmission direction of the laser beam emitted by the continuous wave laser, an acousto-optic modulator, a polarizer, an electro-optic modulator, and a first polarization beam splitter are arranged sequentially. A beam of light split from the first polarization beam splitter is incident on a first reflecting mirror. The light reflected by the first reflecting mirror is incident on a spiral phase plate. The light emitted from the spiral phase plate is incident on a second reflecting mirror. The light reflected by the second reflecting mirror is incident on a second polarization beam splitter. A beam of light split from the second polarization beam splitter is either an excitation light pulse or a suppression light pulse.

[0013] Preferably, the time wavefront multiplexer further includes a function generator for driving the acousto-optic modulator and the electro-optic modulator.

[0014] Preferably, the imaging device includes a third reflecting mirror, a photodetector, a quarter-wave plate, and an imaging objective lens;

[0015] The excitation and suppression light pulses split by the second polarization beam splitter are transmitted sequentially through the third mirror and incident on the imaging objective lens through the quarter-wave plate;

[0016] The light emitted from the storage module passes through the imaging objective and the quarter-wave plate and is incident on the third reflecting mirror. After being reflected by the third reflecting mirror, it is then incident on the photodetector.

[0017] Preferably, the photodetector is a single-photon avalanche diode.

[0018] The present invention also provides an imaging method for a single-beam imaging device, comprising:

[0019] The sample with triplet properties is opened, and a continuous wave laser is modulated into an excitation light pulse and incident on the sample. The time of the excitation light pulse incident on the sample is modulated, and the sample is closed to achieve short-time incident of the excitation light pulse.

[0020] Open the sample, modulate the continuous wave laser into a suppression light pulse and incident it on the sample, modulate the duration of the suppression light pulse incident on the sample, close the sample, and thus achieve long-term incident suppression light pulse;

[0021] Imaging is performed by detecting the light emitted from the sample.

[0022] Preferably, the excitation light pulse is a Gaussian distributed pulse.

[0023] Preferably, the suppression light pulse is a donut-shaped pulse.

[0024] The beneficial effects of the above technical solution are as follows:

[0025] In this technical solution, the present invention uses a single beam and utilizes the principle of time-multiplexed continuous wave (CW) beam wavefront and fluorophore time suppression process to perform time modulation between short time (excitation) and long time (suppression), and simultaneously perform wavefront modulation between excitation and suppression to achieve imaging, which simplifies the structure of the imaging device and improves the super-resolution of imaging. Attached Figure Description

[0026] Figure 1 This is an optical path structure diagram of one embodiment of the single-beam imaging device described in this invention;

[0027] Figure 2a This is a schematic diagram of the energy transfer path of the short-pulse incident sample in this invention;

[0028] Figure 2b This is a schematic diagram of the energy transfer path of the long-pulse incident sample in this invention;

[0029] Figure 3a This is a graph showing the pulse peak energy versus fluorescence intensity of the long pulse in this invention.

[0030] Figure 3b This is a graph showing the pulse peak energy versus fluorescence full width at half maximum (FWHM) of the long pulse in this invention.

[0031] Figure 4 This is a flowchart of an embodiment of the imaging method of the single-beam imaging device described in this invention. Detailed Implementation

[0032] The advantages of the present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments.

[0033] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this disclosure as detailed in the appended claims.

[0034] The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. The singular forms “a,” “the,” and “the” as used in this disclosure and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0035] It should be understood that although the terms first, second, third, etc., may be used in this disclosure to describe various information, such information should not be limited to these terms. These terms are used only to distinguish information of the same type from one another. For example, without departing from the scope of this disclosure, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0036] In the description of this invention, it should be understood that the numerical labels before the steps do not indicate the order in which the steps are performed, but are only used to facilitate the description of this invention and to distinguish each step, and therefore should not be construed as a limitation of this invention.

[0037] To address the shortcomings of using two different laser beams in a STED-like microscope, which requires special filters, phase plates, dichroic mirrors, lasers, and chromatic aberration correction, making the system complex, this invention proposes a single-beam imaging device for imaging samples with triplet properties, comprising: a continuous wave laser (CW), a time wavefront multiplexer, and an imaging device.

[0038] A continuous-wave laser is used to emit a continuous-wave laser beam and direct it onto a time-wavefront multiplexer.

[0039] A time wavefront multiplexer is used to modulate an incident continuous wave laser beam into an excitation pulse and a suppression pulse, which are then sequentially switched and incident on the imaging device.

[0040] The imaging device is used to sequentially switch excitation light pulses and suppression light pulses to be incident on the sample, while modulating the incident time of the excitation light pulses and suppression light pulses on the sample, so as to achieve short-time incident excitation light pulses and long-time incident suppression light pulses. The imaging device is also used to detect the light emitted from the sample for imaging.

[0041] The sample of this embodiment takes an optical data storage unit (ODS) with triplet performance as an example. It uses a single beam and utilizes the wavefront of a time-multiplexed continuous wave (CW) beam to perform time modulation between a short time (excitation) and a long time (suppression). Wavefront modulation is performed between the excitation light pulse and the suppression light pulse to form a high-resolution write spot for photolithography to write to the optical data storage unit (ODS). A high-resolution read spot is formed to scan the optical data storage unit (ODS) to read.

[0042] The principle of time-suppression is based on the interaction mechanism between different energy states of fluorophores or nanomaterials and metastable dark or dark states (such as the triplet state of organic dyes). Consider the general case of a three-level system with a ground state S0, an excited state S1, and a dark state S2. The excited state S1 has two energy transfer pathways. See [reference needed]. Figure 2a As shown, when a short pulse is incident on the sample, electrons in the ground state S0 are excited to the excited state S1 by the laser, and then transfer from the excited state S1 back to the ground state S0 to emit light; see reference Figure 2b As shown, when a long pulse is incident on the sample, electrons in the ground state S0 are excited to the excited state S1 by the laser. Electrons in the excited state S1 can transfer to the dark state S2 through a crossover process. Electrons in the dark state cannot emit light or photons of different energies. (See also...) Figures 3a-3b As shown, when the short pulse is fixed at 500 nm, the fluorescence intensity of the long pulse is suppressed as the pulse peak energy increases, which reflects the turn-off capability. The effect of the short pulse is weakened, the full width at half maximum of the fluorescence peak decreases, the effective spot size is reduced, and the resolution is improved.

[0043] This principle can be demonstrated using color-converting nanoparticles that exhibit a time-suppression process caused by the crossover of intermediate states and time modulation. Simulation results show that the resolution of the optical data storage unit (ODS) reaches 25 nm with a power loss of 40.0 mW.

[0044] This embodiment uses a time-wavefront multiplexer to switch between long and short pulses from a light source. Optical resolution is improved by adjusting the long pulse in conjunction with wavefront modulation, and by adjusting the short pulse in conjunction with wavefront modulation. In this embodiment, the long pulse is a Gaussian-distributed light pulse for excitation, and the short pulse is a donut-distributed light pulse for suppression. The pulse length in this embodiment can be controlled by turning the sample on and off, avoiding the use of high-power pulsed lasers or high-power continuous lasers for shutdown, which could lead to optical damage and photobleaching of the optical data storage material.

[0045] Furthermore, such as Figure 1 The time wavefront multiplexer of this embodiment includes an acousto-optic modulator 2, a polarizer 3, an electro-optic modulator 4, a first polarization beamsplitter 6, a second polarization beamsplitter 10, a first reflector 7, a spiral phase plate 8, and a second reflector 9.

[0046] A continuous wave laser 1 emits a laser beam and is sequentially arranged with an acousto-optic modulator 2, a polarizer 3, an electro-optic modulator 4, and a first polarization beam splitter 6. A beam of light split from the first polarization beam splitter 6 is incident on a first reflecting mirror 7. The light reflected from the first reflecting mirror 7 is incident on a spiral phase plate 8. The light emitted from the spiral phase plate 8 is incident on a second reflecting mirror 9. The light reflected from the second reflecting mirror 9 is incident on a second polarization beam splitter 10. A beam of light split from the second polarization beam splitter 10 is either an excitation pulse or a suppression pulse.

[0047] In this embodiment, the continuous wave laser generates polarization-modulated light through electro-optic modulation, and the polarization-modulated light is used to generate time-wavefront multiplexed light through polarization beam splitting and spiral phase plate.

[0048] Furthermore, the time wavefront multiplexer in this embodiment also includes a function generator for driving the acousto-optic modulator 2 and the electro-optic modulator 4.

[0049] The imaging device in this embodiment includes a third reflecting mirror 11, a photodetector 13, a quarter-wave plate 12, and an imaging objective lens 14. The excitation and suppression light pulses split by the second polarization beam splitter 10 are sequentially transmitted through the third reflecting mirror 11 and incident on the imaging objective lens 14 via the quarter-wave plate 12. Light emitted from the storage module is incident on the third reflecting mirror 11 via the imaging objective lens 14 and the quarter-wave plate 12, and then reflected by the third reflecting mirror 11 before being incident on the photodetector 13. The photodetector 13 in this embodiment can be implemented using a single-photon avalanche diode.

[0050] In this embodiment, the switching period of the wavefront multiplexer can be from 100 Hz to 1 MHz, the pulse length can be from 1 ms to 1 ps, and the pulse interval can be from 100 ms to 10 ps.

[0051] See Figure 4 As shown, this embodiment also provides an imaging method for a single-beam imaging device, including the following steps:

[0052] A1. Open the sample with triplet properties, modulate the continuous wave laser into an excitation light pulse and incident it on the sample, modulate the time when the excitation light pulse is incident on the sample, close the sample, and realize the short-time incident of the excitation light pulse.

[0053] A2. Open the sample, modulate the continuous wave laser into a suppression light pulse and incident it on the sample, modulate the duration of the suppression light pulse incident on the sample, close the sample, and achieve long-term incident suppression light pulse;

[0054] A3. Imaging is performed by detecting the light emitted from the sample.

[0055] The sample of this embodiment takes an optical data storage unit (ODS) with triplet performance as an example. It uses a single beam and utilizes the wavefront of a time-multiplexed continuous wave (CW) beam to perform time modulation between a short time (excitation) and a long time (suppression). Wavefront modulation is performed between the excitation light pulse and the suppression light pulse to form a high-resolution write spot for photolithography to write to the optical data storage unit (ODS). A high-resolution read spot is formed to scan the optical data storage unit (ODS) to read.

[0056] The principle of time-suppression is based on the interaction mechanism between different energy states of fluorophores or nanomaterials and metastable dark or dark states (such as the triplet state of organic dyes). Consider the general case of a three-level system with a ground state S0, an excited state S1, and a dark state S2. The excited state S1 has two energy transfer pathways. See [reference needed]. Figure 2a As shown, when a short pulse is incident on the sample, electrons in the ground state S0 are excited to the excited state S1 by the laser, and then transfer from the excited state S1 back to the ground state S0 to emit light; see reference Figure 2b As shown, when a long pulse is incident on the sample, electrons in the ground state S0 are excited to the excited state S1 by the laser. Electrons in the excited state S1 can transfer to the dark state S2 through a crossover process. Electrons in the dark state cannot emit light or photons of different energies. (See also...) Figures 3a-3b As shown, when the short pulse is fixed at 500 nm, the fluorescence intensity of the long pulse is suppressed as the pulse peak energy increases, which reflects the turn-off capability. The effect of the short pulse is weakened, the full width at half maximum of the fluorescence peak decreases, the effective spot size is reduced, and the resolution is improved.

[0057] This embodiment uses a time-wavefront multiplexer to switch between long and short pulses from a light source. Optical resolution is improved by adjusting the long pulse in conjunction with wavefront modulation, and by adjusting the short pulse in conjunction with wavefront modulation. In this embodiment, the long pulse is a Gaussian-distributed light pulse for excitation, and the short pulse is a donut-distributed light pulse for suppression. The pulse length in this embodiment can be controlled by turning the sample on and off, avoiding the use of high-power pulsed lasers or high-power continuous lasers for shutdown, which could lead to optical damage and photobleaching of the optical data storage material.

[0058] In this embodiment, the read / write resolution range of the optical data storage unit (ODS) can be 25-250nm, while the smallest spot size in existing optical storage units is 200nm.

[0059] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units; that is, they may be located in one place or distributed across at least two network units. Some or all of the modules can be selected to achieve the purpose of the embodiments of this application, depending on actual needs. Those skilled in the art can understand and implement this without any inventive effort.

[0060] Through the above description of the embodiments, those skilled in the art can clearly understand that each embodiment can be implemented using software and a general-purpose hardware platform, or of course, using hardware. Those skilled in the art will understand that all or part of the processes in the above embodiments can be implemented by a computer program instructing related hardware. The program can be stored in a computer-readable storage medium, and when executed, it can include the processes of the embodiments of the above methods. The storage medium can be a magnetic disk, optical disk, read-only memory (ROM), or random access memory (RAM), etc.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. An imaging method using a single-beam imaging device, characterized in that, The single-beam imaging device is used for imaging samples with triplet properties, and the device includes: A continuous-wave laser is used to emit a single continuous-wave laser beam and direct it onto a time-wavefront multiplexer. A time-wavefront multiplexer is used to modulate an incident continuous-wave laser beam into an excitation pulse and a suppression pulse, which are then sequentially switched and incident on the imaging device. An imaging device is used to sequentially switch excitation light pulses and suppression light pulses to be incident on the sample, while modulating the timing of the excitation light pulses and suppression light pulses incident on the sample. The imaging device is also used to detect the light emitted from the sample for imaging. The time wavefront multiplexer includes an acousto-optic modulator, a polarizer, an electro-optic modulator, a first polarization beamsplitter, a second polarization beamsplitter, a first reflector, a spiral phase plate, and a second reflector. A continuous wave laser emits a laser beam and, in sequence, an acousto-optic modulator, a polarizer, an electro-optic modulator, and a first polarization beam splitter are arranged along the transmission direction. A beam of light split from the first polarization beam splitter is incident on a first reflecting mirror. The light reflected from the first reflecting mirror is incident on a spiral phase plate. The light emitted from the spiral phase plate is incident on a second reflecting mirror. The light reflected from the second reflecting mirror is incident on a second polarization beam splitter. A beam of light split from the second polarization beam splitter is either an excitation light pulse or a suppression light pulse. The switching period of the wavefront multiplexer is 100Hz to 1MHz, the pulse length is 1ms to 1ps, and the pulse interval is 100ms to 10ps; The imaging device includes a third semi-transparent mirror, a photodetector, a quarter-wave plate, and an imaging objective lens; The excitation and suppression light pulses split by the second polarization beam splitter are transmitted sequentially through the third semi-transparent and semi-reflective mirror and then incident on the imaging objective lens through the quarter-wave plate. The light emitted from the sample passes through the imaging objective and the quarter-wave plate and is incident on the third semi-transparent mirror. After being reflected by the third semi-transparent mirror, it is incident on the photodetector. The imaging method includes: The sample with triplet properties is opened, and a continuous wave laser is modulated into an excitation light pulse and incident on the sample. The time of the excitation light pulse incident on the sample is modulated, and the sample is closed to achieve short-time incident of the excitation light pulse. Open the sample, modulate the continuous wave laser into a suppression light pulse and incident it on the sample, modulate the duration of the suppression light pulse incident on the sample, close the sample, and thus achieve long-term incident suppression light pulse; Imaging is performed by detecting the light emitted from the sample; The excitation light pulse is a Gaussian distributed pulse, and the suppression light pulse is a donut-shaped distributed pulse.

2. The imaging method of the single-beam imaging device according to claim 1, characterized in that, The time wavefront multiplexer also includes a function generator for driving the acousto-optic modulator and the electro-optic modulator.

3. The imaging method of the single-beam imaging device according to claim 1, characterized in that, The photodetector is a single-photon avalanche diode.

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