Stimulated emission depletion and super-resolution fluorescence lifetime imaging method

Abstract

Disclosed in the present invention is a stimulated emission depletion and super-resolution fluorescence lifetime imaging method, which is applied to the technical field of optical microscope imaging. The method provided in the present invention combines the stimulated emission depletion and fluorescence lifetime imaging principles, a depletion laser pulse frequency used therein is half of an excitation light pulse frequency, and a time interval between two pulse sequences is adjusted, such that a depletion laser pulse follows an adjacent excitation light pulse; and analysis processing is performed on collected fluorescence lifetime data, such that a super-resolution fluorescence lifetime image is obtained, thereby solving the problem of the fluorescence lifetime of a stimulated emission depletion and fluorescence lifetime imaging microscope being significantly shortened under the action of a high-energy depletion laser, and thus accurately reflecting real information of a fluorochrome and a micro-environment. Therefore, the innovative technique provides important technical support for life science research, significantly improves the accuracy and effectiveness of research, and promotes further development in the field of biomedicine.

Description

A method for super-resolution fluorescence lifetime imaging using stimulated emission depletion Technical Field

[0001] The present invention belongs to the technical field of optical microscope imaging, and particularly relates to a new method of fluorescence lifetime microscopy imaging that utilizes the principle of stimulated emission loss to break through the optical diffraction limit and combines fluorescence lifetime detection to achieve spatial super-resolution. Background Art

[0002] The development of super-resolution fluorescence microscopy has greatly promoted the progress of biomedical research. Traditional optical microscopes are limited by the Abbe diffraction limit, and their resolution cannot exceed half the excitation wavelength. Therefore, to a certain extent, it hinders their in-depth study of the fine structure and dynamic processes inside cells. In recent years, several super-resolution microscopy technologies such as stimulated emission depletion microscopy, structured light illumination microscopy, single-molecule localization microscopy and minimum flux microscopy have been introduced. These technologies have broken through the optical diffraction limit, significantly improved imaging resolution, and expanded the application areas of microscopic imaging.

[0003] Stimulated emission depletion microscopy was proposed by German scientist Stefan Herr. It is the first far-field super-resolution microscopy technology. It uses the stimulated emission depletion effect to achieve precise control of the excitation area of ​​fluorescent molecules, thereby breaking the optical diffraction limit and improving spatial resolution. In stimulated emission depletion microscopy, an excitation laser is first used to irradiate the fluorescent molecules in the ground state into an excited state, and then a loss laser with a ring-shaped wavefront is introduced to make the fluorescent molecules in the laser irradiation area return to the ground state by stimulated emission, retaining only the spontaneous fluorescence signal in the center area of ​​the excitation spot. By adjusting the intensity and spatial distribution of the stimulated emission depletion laser, the imaging resolution can be improved to tens of nanometers or even higher.

[0004] Fluorescence lifetime imaging microscopy is an imaging technology based on the fluorescence lifetime characteristics of luminescent materials. By measuring the time required for fluorescent molecules to return from the excited state to the ground state, it provides rich information about the molecular environment, interactions, and microscopic dynamics. Unlike traditional intensity imaging, fluorescence lifetime imaging microscopy is not affected by changes in fluorescence intensity. Therefore, it has important application value in biomedical research, especially quantitative analysis and biological system research. However, the spatial resolution of fluorescence lifetime imaging microscopy is limited by the diffraction limit of traditional microscopes and cannot meet the needs of high-resolution research on the complex internal structures of cells and their dynamic processes.

[0005] In order to overcome the limitations of traditional fluorescence lifetime imaging microscopy technology in spatial resolution, researchers combined stimulated emission depletion microscopy with fluorescence lifetime imaging microscopy technology and proposed a stimulated emission depletion fluorescence lifetime imaging method. This method realizes fluorescence lifetime measurement at sub-diffraction resolution by introducing fluorescence lifetime imaging technology on the basis of stimulated emission depletion microscopy. However, due to the stimulated radiation effect of high-energy loss laser, the fluorescence lifetime measured by fluorescence lifetime imaging microscopy is significantly shortened, and its value is significantly lower than the normal fluorescence lifetime of the fluorescent material, and cannot accurately reflect the true information of the fluorescent dye and the microenvironment. This has caused great obstacles to research in the biomedical field. Therefore, the development of a stimulated emission depletion super-resolution fluorescence lifetime imaging technology that can maintain normal fluorescence lifetime information can provide important technical support for the precision research of life sciences. Summary of the Invention

[0006] The purpose of the present invention is to propose a stimulated emission depletion super-resolution fluorescence lifetime imaging method, which has the advantage of overcoming the limitations of traditional fluorescence lifetime imaging microscopes in terms of spatial resolution.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions: a stimulated emission depletion super-resolution fluorescence lifetime imaging method, including a laser scanning confocal imaging system combining stimulated emission depletion and fluorescence lifetime imaging, the laser scanning confocal imaging system combining stimulated emission depletion and fluorescence lifetime imaging comprising:

[0008] Excitation laser, pulsed laser generated by picosecond laser;

[0009] Loss laser, a pulsed laser generated by a picosecond laser, has a wavelength longer than the excitation laser and a pulse frequency half that of the excitation laser. It emits synchronized pulsed lasers after being triggered by the control system of the excitation laser.

[0010] Half-wave plate, used to adjust the polarization direction of the laser;

[0011] Polarization beam splitter, used for laser beam splitting, can be used in conjunction with a half-wave plate to control the energy ratio of reflected and transmitted laser light;

[0012] a baffle for blocking the laser beam;

[0013] Reflector, used to change the transmission direction of laser;

[0014] Vortex phase plate, used to modulate the wavefront of lossy laser light and convert Gaussian light into ring light;

[0015] The corner reflector is used to adjust the pulse interval between the excitation laser and the loss laser. That is, the position of the corner reflector is moved to extend or shorten the optical path of the annular loss laser spot, thereby controlling the time when the loss laser pulse reaches the sample.

[0016] Color mirror 1, used to reflect the excitation laser and transmit the fluorescence signal;

[0017] Dichroic mirror 2, used to reflect the loss laser and transmit the excitation laser and fluorescence signal;

[0018] Galvanometer x, used for performing horizontal synchronous scanning of the two laser beams;

[0019] Galvanometer y is used to perform longitudinal synchronous scanning of the two laser beams and work with galvanometer x to achieve area array imaging of the sample;

[0020] The scanning lens is placed after the galvanometer and is used to collect the laser beam for area array scanning;

[0021] Tube lens, which works with the objective lens to form a microscope system;

[0022] The objective lens is used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample;

[0023] The stage is used to place and fix the sample to be tested and to control the three-dimensional movement of the sample;

[0024] A lens, used to focus the light beam;

[0025] Filters are used to transmit fluorescence, remove stray light other than fluorescence, and improve the image signal-to-noise ratio;

[0026] Detector 1, using a photomultiplier tube or avalanche photodiode, is used to collect signals and amplify the fluorescence signal;

[0027] Detector 2 is used to detect the laser reflected by the polarization beam splitter in the excitation light path as a reference signal in fluorescence lifetime imaging;

[0028] Time-correlated single-photon counter, used to record the spatiotemporal information of fluorescence signals;

[0029] Computer, used to control the software to acquire images, store data and process image data.

[0030] The present invention is further configured as follows: the excitation laser (black solid line) is emitted from the laser and split into two beams by a polarization beam splitter. The reflected light is collected by detector 2 and used as a reference signal for fluorescence lifetime imaging. The transmitted light is reflected by dichroic mirror 1. The loss laser (black dashed line) is wavefront modulated by a vortex phase plate to generate a ring-shaped laser spot.

[0031] The two excitation laser beams and the loss laser beam meet at dichroic mirror 2 and precisely overlap in space. After being excited, the sample emits a fluorescence signal (black dotted line). The signals collected by detectors 1 and 2 are transmitted to a time-correlated single-photon counter, and the data is saved to a computer.

[0032] The present invention is further configured as follows: by turning on the excitation laser and the loss laser, the control system of the excitation laser is used to synchronously trigger the loss laser, so that the pulse sequences of the two laser beams are kept synchronized, and the loss can be changed by moving the corner reflector.

[0033] The optical path of the laser is used to adjust the pulse interval between the two laser beams.

[0034] The present invention is further configured as follows: the pulse frequency of the excitation laser is twice the pulse frequency of the loss laser, the wavelength of the loss laser is longer than that of the excitation laser, the excitation laser outputs a Gaussian pulse laser, and after the laser is emitted, it is split into two by a polarization beam splitter, and the reflected light is used as a reference signal for fluorescence lifetime imaging. The transmitted light is reflected by the dichroic mirror 1, transmitted by the dichroic mirror 2, scanned by the galvanometer x and y, and then focused on the sample after passing through the scanning lens, the tube lens and the objective lens in sequence.

[0035] The present invention is further configured as follows: the loss laser is triggered by the control system of the excitation laser to emit a Gaussian pulse laser, the loss laser energy in the optical path is controlled by a combination of a half-wave plate and a polarization beam splitter, the time when the loss laser pulse reaches the sample is adjusted by a corner reflector, and the loss laser is converted from a Gaussian type to a ring laser through a vortex phase plate. After being reflected by the dichroic mirror 2, it is scanned by the galvanometer x and y, and is focused on the sample through a scanning lens, a tube lens, and an objective lens in sequence.

[0036] The present invention is further configured such that the light spots of the excitation laser and the loss laser are precisely overlapped in space after being focused by the objective lens, and the time-correlated single-photon counter simultaneously collects the fluorescence signal collected by detector 1 and the reference signal collected by detector 2, and transmits the data to a computer for storage and processing.

[0037] The present invention is further configured as follows: when the excitation laser is turned on, only the excitation light pulse sequence constitutes confocal fluorescence lifetime imaging, and the fluorescence emission shows a single exponential decay. When both the excitation laser and the loss laser are turned on, since the pulse frequency of the excitation laser is twice the pulse frequency of the loss laser, the loss laser pulse is positioned after the adjacent excitation laser pulse by adjusting the corner reflector, ensuring that the pulse interval between the adjacent excitation laser pulses matches the excited state vibrational relaxation time of the fluorescent dye, which is on the order of hundreds of picoseconds. At this time, the fluorescence emission simultaneously includes a confocal fluorescence decay curve and a stimulated emission depletion super-resolution fluorescence decay curve. Under the action of the stimulated emission effect, the number of fluorescence photons is significantly reduced, and the fluorescence emission shows a multi-exponential decay trend.

[0038] The present invention is further configured such that the pulse frequency of the depletion laser is used as the detection period (T) of the fluorescence signal. Thus, one detection period includes two excitation light pulses and one depletion laser pulse, and the detection period can be divided into two parts, namely, region 1 and region 2, according to the fluorescence decay curve.

[0039] In region 1, the molecules are only exposed to the excitation laser, and the resulting fluorescence photons constitute the standard confocal fluorescence imaging signal;

[0040] In region 2, the molecules are affected by the combined effects of the excitation laser and the depletion laser, and their fluorescence photons appear as stimulated emission depletion microscopy fluorescence imaging signals. Therefore, through fluorescence lifetime imaging detection, the fluorescence intensity information IC(x,y) and fluorescence lifetime information τC(x,y) of confocal fluorescence imaging, as well as the fluorescence intensity information IS(x,y) and fluorescence lifetime information τS(x,y) of stimulated emission depletion fluorescence imaging can be obtained simultaneously in each pixel at one time.

[0041] The present invention is further configured as follows: after obtaining the above information, the fluorescence lifetime information τC(x,y) in confocal fluorescence imaging and the fluorescence intensity information IS(x,y) in stimulated emission depletion fluorescence imaging are respectively obtained using an image processing algorithm, τC(x,y) represents normal fluorescence lifetime information, and IS(x,y) represents super-resolution fluorescence intensity information, and an all-one matrix ones(x,y) with the same dimension and pixels as the acquired image is generated, and then the fluorescence intensity IS(x,y) and the fluorescence lifetime τC(x,y) are normalized, and the normalized fluorescence intensity and fluorescence lifetime information are respectively used as brightness and color and merged with the all-one matrix into a three-channel HSV color image.

[0042] The present invention is further configured such that: the HSV image is converted into an RGB image to obtain an intensity-weighted fluorescence lifetime image, that is, a stimulated emission depletion super-resolution fluorescence lifetime image that contains both normal fluorescence lifetime information and super-resolution structural information.

[0043] In summary, the present invention has the following beneficial effects:

[0044] This method combines the principles of stimulated emission depletion and fluorescence lifetime imaging, uses a loss laser pulse frequency that is half the frequency of the excitation light pulse, and adjusts the time interval between the two pulse sequences so that the loss laser pulses are located adjacent to each other.

[0045] After the excitation light pulse, the collected fluorescence lifetime data is analyzed and processed to obtain a super-resolution fluorescence lifetime image, which solves the problem of significantly shortened fluorescence lifetime in stimulated emission depletion fluorescence lifetime imaging microscopy under the action of high-energy loss laser, and accurately reflects the real information of fluorescent dyes and microenvironment. This innovative technology will provide important technical support for life science research, significantly improve the accuracy and effectiveness of research, and promote further development in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG1 is a schematic diagram of a stimulated emission depletion super-resolution fluorescence lifetime imaging system implemented in the present invention;

[0047] FIG2 is a schematic diagram of a laser pulse timing diagram and a fluorescence decay curve implemented in the present invention;

[0048] FIG3 is a schematic diagram (a) of the principle of stimulated emission depletion super-resolution fluorescence lifetime imaging implemented in the present invention;

[0049] FIG4 is a schematic diagram (b) showing the principle of stimulated emission depletion super-resolution fluorescence lifetime imaging implemented in the present invention. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below with reference to the accompanying drawings.

[0051] 1-4 , a stimulated emission depletion (SED) super-resolution fluorescence lifetime imaging method includes a laser scanning confocal imaging system that combines stimulated emission depletion (SED) and fluorescence lifetime imaging. The laser scanning confocal imaging system that combines stimulated emission depletion (SED) and fluorescence lifetime imaging includes:

[0052] Excitation laser, pulsed laser generated by picosecond laser;

[0053] Loss laser, a pulsed laser generated by a picosecond laser, has a wavelength longer than the excitation laser and a pulse frequency half that of the excitation laser. It emits synchronized pulsed lasers after being triggered by the control system of the excitation laser.

[0054] Half-wave plate, used to adjust the polarization direction of the laser;

[0055] Polarization beam splitter, used for laser beam splitting, can be used in conjunction with a half-wave plate to control the energy ratio of reflected and transmitted laser light;

[0056] a baffle for blocking the laser beam;

[0057] Reflector, used to change the transmission direction of laser;

[0058] Vortex phase plate, used to modulate the wavefront of lossy laser light and convert Gaussian light into ring light;

[0059] The corner reflector is used to adjust the pulse interval between the excitation laser and the loss laser. That is, the position of the corner reflector is moved to extend or shorten the optical path of the annular loss laser spot, thereby controlling the time when the loss laser pulse reaches the sample.

[0060] Dichroic mirror 1, used to reflect the excitation laser and transmit the fluorescence signal;

[0061] Dichroic mirror 2, used to reflect the loss laser and transmit the excitation laser and fluorescence signal;

[0062] Galvanometer x, used for performing horizontal synchronous scanning of the two laser beams;

[0063] Galvanometer y is used to perform longitudinal synchronous scanning of the two laser beams and work with galvanometer x to achieve area array imaging of the sample;

[0064] The scanning lens is placed after the galvanometer and is used to collect the laser beam for area array scanning;

[0065] Tube lens, which works with the objective lens to form a microscope system;

[0066] The objective lens is used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample;

[0067] The stage is used to place and fix the sample to be tested and to control the three-dimensional movement of the sample;

[0068] A lens, used to focus the light beam;

[0069] Filters are used to transmit fluorescence, remove stray light other than fluorescence, and improve the image signal-to-noise ratio;

[0070] Detector 1, using a photomultiplier tube or avalanche photodiode, is used to collect signals and amplify the fluorescence signal;

[0071] Detector 2 is used to detect the laser reflected by the polarization beam splitter in the excitation light path as a reference signal in fluorescence lifetime imaging;

[0072] Time-correlated single-photon counter, used to record the spatiotemporal information of fluorescence signals;

[0073] Computer, used to control the software to acquire images, store data and process image data.

[0074] As shown in Figure 1, the black solid line in the figure represents the excitation laser. After the excitation laser is emitted from the laser, it is divided into two beams by a polarization beam splitter. The reflected light is collected by detector 2 and used as a reference signal for fluorescence lifetime imaging. The transmitted light is reflected by the dichroic mirror 1. The black dotted line represents the loss laser. The wavefront modulation of the vortex phase plate generates a ring laser spot. The above two laser beams meet at the dichroic mirror 2 and overlap precisely in space. The black dotted line represents the fluorescence signal reflected back after the sample is excited. The signals collected by detectors 1 and 2 are transmitted to a time-correlated single photon counter, and the data is saved to a computer. The pulse interval between the Gaussian excitation light and the ring loss laser can be adjusted by moving the position of the corner reflector;

[0075] During operation, turn on the excitation laser and the loss laser, and use the control system of the excitation laser to synchronously trigger the loss laser so that the pulse sequences of the two laser beams remain synchronized (the control system of the loss laser can also be used to synchronously trigger the excitation laser). By moving the corner reflector, the optical path of the loss laser can be changed and the pulse interval between the two laser beams can be adjusted.

[0076] As shown in Figure 1, there are two laser light sources with different wavelengths, namely the excitation laser and the loss laser. The pulse frequency of the excitation laser is twice that of the loss laser, and the wavelength of the loss laser is longer than that of the excitation laser (usually located at the tail end of the fluorescent dye emission spectrum). The excitation laser outputs a Gaussian pulse laser. After the laser is emitted, it is split into two by a polarization beam splitter. The reflected light serves as the reference signal for fluorescence lifetime imaging. The transmitted light is reflected by dichroic mirror 1, transmitted by dichroic mirror 2, scanned by the galvanometer x and y, and then focuses on the sample after passing through the scanning lens, tube lens, and objective lens in sequence.

[0077] The loss laser is triggered by the control system of the excitation laser and emits a Gaussian pulse laser. The loss laser energy in the optical path is controlled by a combination of a half-wave plate and a polarization beam splitter. The time when the loss laser pulse reaches the sample is adjusted by a corner reflector. The loss laser is converted from a Gaussian pulse to a ring laser through a vortex phase plate. After being reflected by the dichroic mirror 2, it is scanned by the galvanometer x and y, and focused on the sample through the scanning lens, tube lens, and objective lens in sequence.

[0078] The two laser beams are focused by the objective lens and precisely overlap in space. The time-correlated single-photon counter simultaneously collects the fluorescence signal collected by detector 1 and the reference signal collected by detector 2, and transmits the data to the computer for storage and processing.

[0079] [Corrected 30.04.2025 in accordance with Rule 91] As shown in Figure 2, when only the excitation laser is on, only the excitation light pulse sequence (the black solid line on the upper axis in (a)) constitutes confocal fluorescence lifetime imaging, and the fluorescence emission exhibits a single exponential decay, as shown by the black solid line on the lower axis in (a). When both lasers are on, because the excitation laser pulse frequency is twice that of the depletion laser pulse frequency, the depletion laser pulse (the black dashed line on the upper axis in (b)) is positioned after the adjacent excitation laser pulse by adjusting the corner reflector to ensure that the pulse interval between adjacent excitation laser pulses matches the excited-state vibrational relaxation time of the fluorescent dye, which is on the order of hundreds of picoseconds. At this time, the fluorescence emission includes both the confocal fluorescence decay curve (the black solid line on the lower axis in (b)) and the super-resolution fluorescence decay curve of stimulated emission depletion microscopy (the black dashed line on the lower axis in (b)). Due to the stimulated emission effect, the number of fluorescence photons in stimulated emission depletion microscopy is significantly reduced, and the fluorescence emission exhibits a multi-exponential decay trend.

[0080] As shown in Figures 3 and 4, this method includes two steps:

[0081] S1. Extraction of normal fluorescence lifetime information and super-resolution intensity information;

[0082] S2. Synthesis of super-resolution fluorescence lifetime images;

[0083] The pulse frequency of the depletion laser is used as the detection period (T) of the fluorescence signal. Therefore, one detection period includes two excitation light pulses and one depletion laser pulse. As can be seen from Figure 3 (a) and Figure 4 (b), the detection period can be divided into two parts according to the fluorescence decay curve, namely, region 1 and region 2.

[0084] [Corrected 30.04.2025 according to rule 91] In region 1, the molecules are only exposed to the excitation laser light, and the resulting fluorescence photons constitute the standard confocal fluorescence imaging signal (solid black line);

[0085] [Corrected 30.04.2025 according to Rule 91] In region 2, the molecules are acted upon by both the excitation laser and the depletion laser, and their fluorescence photons appear as stimulated emission depletion microscopy fluorescence imaging signals (double-dashed line). Therefore, fluorescence lifetime imaging detection allows for simultaneous acquisition of fluorescence intensity information IC(x,y) and fluorescence lifetime information τC(x,y) for confocal fluorescence imaging, as well as fluorescence intensity information IS(x,y) and fluorescence lifetime information τS(x,y) for stimulated emission depletion fluorescence imaging, at each pixel.

[0086] After obtaining the above information, the fluorescence lifetime information τC(x,y) in confocal fluorescence imaging and the fluorescence intensity information IS(x,y) in stimulated emission depletion fluorescence imaging are obtained using image processing algorithms. τC(x,y) represents the normal fluorescence lifetime information, while IS(x,y) represents the super-resolution fluorescence intensity information. An all-one matrix ones(x,y) with the same dimensions and pixels as the acquired image is generated. The fluorescence intensity IS(x,y) and fluorescence lifetime τC(x,y) are then normalized, and the normalized fluorescence intensity and fluorescence lifetime information are merged with the all-one matrix as brightness and color, respectively, to form a three-channel HSV color image.

[0087] Finally, the HSV image is converted into an RGB image to obtain an intensity-weighted fluorescence lifetime image, that is, a stimulated emission depletion super-resolution fluorescence lifetime image that contains both normal fluorescence lifetime information and super-resolution structural information.

[0088] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A stimulated emission depletion super-resolution fluorescence lifetime imaging method, which combines stimulated emission depletion and fluorescence lifetime imaging The laser scanning confocal imaging system is characterized by: The laser scanning confocal imaging system combining stimulated emission depletion and fluorescence lifetime imaging includes: Excitation laser, pulsed laser generated by picosecond laser; Loss laser, a pulsed laser generated by a picosecond laser, has a wavelength longer than the excitation laser and a pulse frequency half that of the excitation laser. It emits synchronized pulsed lasers after being triggered by the control system of the excitation laser. Half-wave plate, used to adjust the polarization direction of the laser; Polarization beam splitter, used for laser beam splitting, can be used with half-wave plate to control the energy ratio of reflected and transmitted laser; baffle, used to block laser beam; Reflector, used to change the transmission direction of laser; Vortex phase plate, used to modulate the wavefront of lossy laser light and convert Gaussian light into ring light; The corner reflector is used to adjust the pulse interval between the excitation laser and the loss laser. That is, the position of the corner reflector is moved to extend or shorten the optical path of the annular loss laser spot, thereby controlling the time when the loss laser pulse reaches the sample. Dichroic mirror 1, used to reflect the excitation laser and transmit the fluorescence signal; Dichroic mirror 2, used to reflect the loss laser and transmit the excitation laser and fluorescence signal; Galvanometer x, used for performing horizontal synchronous scanning of the two laser beams; Galvanometer y is used to perform longitudinal synchronous scanning of the two laser beams and work with galvanometer x to achieve area array imaging of the sample; The scanning lens is placed after the galvanometer and is used to collect the laser beam for area array scanning; Tube lens, which works with the objective lens to form a microscope system; The objective lens is used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample; The stage is used to place and fix the sample to be tested and to control the three-dimensional movement of the sample; A lens, used to focus the light beam; Filters are used to transmit fluorescence, remove stray light other than fluorescence, and improve the image signal-to-noise ratio; Detector 1, using a photomultiplier tube or avalanche photodiode, is used to collect signals and amplify the fluorescence signal; Detector 2 is used to detect the laser reflected by the polarization beam splitter in the excitation light path as a reference signal in fluorescence lifetime imaging; Time-correlated single-photon counter, used to record the spatiotemporal information of fluorescence signals; Computer, used to control the software to acquire images, store data and process image data; After the excitation laser is emitted from the laser, it is split into two beams by a polarization beam splitter. The reflected light is collected by detector 2 and used as a reference signal for fluorescence lifetime imaging. The transmitted light is reflected by dichroic mirror 1, and the loss laser is modulated by the wavefront of the vortex phase plate to generate a ring-shaped laser spot. The excitation laser and the loss laser meet at the dichroic mirror 2 and overlap precisely in space. The sample is excited and emits a fluorescence signal. The signals collected by detectors 1 and 2 are transmitted to a time-correlated single-photon counter, and the data is saved to a computer. By turning on the excitation laser and the loss laser, the control system of the excitation laser is used to synchronously trigger the loss laser so that the pulse sequences of the two laser beams remain synchronized. By moving the corner reflector, the optical path of the loss laser can be changed and the pulse interval between the two laser beams can be adjusted. The pulse frequency of the excitation laser is twice that of the loss laser. The wavelength of the loss laser is longer than that of the excitation laser. The excitation laser outputs a Gaussian pulse laser. After the laser is emitted, it is split into two by a polarization beam splitter. The reflected light is used as a reference signal for fluorescence lifetime imaging. The transmitted light is reflected by dichroic mirror 1, transmitted by dichroic mirror 2, scanned by galvanometer x and galvanometer y, and then passes through the scanning The lens, tube lens, and objective lens are then focused on the sample; The loss laser is triggered by the control system of the excitation laser and emits a Gaussian pulse laser. The loss laser energy in the optical path is controlled by a combination of a half-wave plate and a polarization beam splitter. The time when the loss laser pulse reaches the sample is adjusted by a corner reflector. The loss laser is converted from a Gaussian pulse to a ring laser through a vortex phase plate. After being reflected by dichroic mirror 2, it is scanned by galvanometer x and galvanometer y, and focused on the sample through a scanning lens, a tube lens, and an objective lens in sequence. The excitation laser and depletion laser spots are focused by the objective lens so that they coincide precisely in space. A time-correlated single-photon counter simultaneously collects the fluorescence signal from detector 1 and the reference signal from detector 2, and transmits the data to a computer for storage and processing. When the excitation laser is turned on, only the excitation light pulse sequence constitutes confocal fluorescence lifetime imaging, and the fluorescence emission exhibits a single exponential decay. When both the excitation laser and the depletion laser are turned on, since the excitation laser pulse frequency is twice that of the depletion laser pulse frequency, the depletion laser pulse is positioned after the adjacent excitation laser pulse by adjusting the corner reflector to ensure that the pulse interval between adjacent excitation laser pulses matches the excited state vibrational relaxation time of the fluorescent dye, which is on the order of hundreds of picoseconds. At this time, the fluorescence emission includes both the confocal fluorescence decay curve and the stimulated emission depletion super-resolution fluorescence decay curve. Under the action of the stimulated emission effect, the number of fluorescence photons is significantly reduced, and the fluorescence emission exhibits a multi-exponential decay trend. The pulse frequency of the depletion laser is used as the detection period (T) of the fluorescence signal. Therefore, one detection period contains two excitation light pulses and one depletion laser pulse. According to the fluorescence decay curve, the detection period can be divided into two parts, namely region 1 and region 2. In region 1, the molecules are only exposed to the excitation laser, and the resulting fluorescence photons constitute the standard confocal fluorescence imaging signal; In region 2, the molecules are acted upon by both the excitation laser and the depletion laser, and their fluorescence photons appear as stimulated emission depletion microscopy (SLE) fluorescence imaging signals. Therefore, through fluorescence lifetime imaging detection, the fluorescence intensity information IC(x,y) and fluorescence lifetime information τC(x,y) of confocal fluorescence imaging, as well as the fluorescence intensity information IS(x,y) and fluorescence lifetime information τS(x,y) of SLE fluorescence imaging, can be obtained simultaneously in each pixel. After obtaining the above information, the fluorescence lifetime information τC(x,y) in confocal fluorescence imaging and the fluorescence intensity information IS(x,y) in stimulated emission depletion fluorescence imaging are obtained using image processing algorithms. τC(x,y) represents the normal fluorescence lifetime information, while IS(x,y) represents the super-resolution fluorescence intensity information. An all-one matrix ones(x,y) with the same dimensions and pixels as the acquired image is generated. The fluorescence intensity IS(x,y) and fluorescence lifetime τC(x,y) are then normalized, and the normalized fluorescence intensity and fluorescence lifetime information are merged with the all-one matrix as brightness and color, respectively, to form a three-channel HSV color image. By converting the HSV image into an RGB image, an intensity-weighted fluorescence lifetime image can be obtained, that is, a stimulated emission depletion super-resolution fluorescence lifetime image that contains both normal fluorescence lifetime information and super-resolution structural information.

Images