WO2021243754A1

WO2021243754A1 - Super-resolution imaging method and imaging system based on low-power stimulated emission depletion

Info

Publication number: WO2021243754A1
Application number: PCT/CN2020/096779
Authority: WO
Inventors: 严伟; 王璐玮; 屈军乐; 王佳林; 张佳
Original assignee: 深圳大学
Priority date: 2020-06-04
Filing date: 2020-06-18
Publication date: 2021-12-09
Also published as: CN111579486A; CN111579486B

Abstract

Provided are a super-resolution imaging method and imaging system based on low-power stimulated emission depletion. The method comprises: by adjusting the position of a corner reflector (106) arranged in a first optical path, extending the optical distance of depletion laser light propagated in the first optical path, converting low-power Gaussian-type depletion laser light into annular depletion laser light, and the annular depletion laser light coinciding with excitation laser light and then focusing on and irradiating a sample, such that acquired fluorescence service life data includes both a confocal signal and a super-resolution signal; and separating a confocal image and an initial super-resolution image from a fluorescence signal by means of data processing, analyzing and processing the confocal image and the initial super-resolution image according to an image processing rule, and thus obtaining a target super-resolution image, the resolution of which has been further improved. By means of the method, damage to a biological sample is reduced by using low-power depletion laser light, a photobleaching effect on a fluorescent dye is reduced, the effective time of super-resolution imaging is prolonged, and a high-resolution image including fine structural features can be obtained.

Description

Super-resolution imaging method and imaging system based on low-power stimulated emission loss

This application is based on a Chinese patent application with an application number of 202010500061.X and an application date of June 4, 2020, and claims its priority. The entire content of this application is hereby incorporated into this application as a whole.

Technical field

This application relates to the technical field of super-resolution optical microscopy imaging, in particular to a super-resolution imaging method and imaging system based on low-power stimulated emission loss.

Background technique

Live cell and tissue imaging is very important to the research in the field of biomedicine. In addition to correct cell culture conditions and sample preparation methods, advanced imaging methods and imaging systems can guarantee the greatest degree of protection while maintaining the biological characteristics of the observed samples. Obtain the authenticity and validity of the information. Optical microscope has the advantages of non-contact, non-damage and specificity. It is an important symbol of the beginning of the development of modern natural science. It can be well applied to imaging of living cells and tissues. However, the diffraction of light limits the resolution capability of optical microscopes, making it impossible to clearly distinguish microscopic biological structures with a size below 200 nm. Super-resolution optical microscopy (SRM) technology inherits the non-contact and specific advantages of optical microscopy. Through physical and chemical principles, the resolution of optical microscopy is increased by 1 to 2 orders of magnitude, and life can be understood at the molecular level. The law of development and change, revealing the cellular and molecular mechanisms of biological resistance/drug resistance and intervention therapy is one of the most significant breakthroughs in the field of optical microscopy in this century. In recent years, the rapid development of super-resolution optical imaging technology has made optical microscopes more closely related to the fields of biomedicine, but the existing technology has extremely strict requirements on samples (preparation) and fluorescent dyes, which limits the use of in vivo biological imaging. Applications.

In 1994, German scientist Stefan W. Hell proposed stimulated emission depletion (STED) microscopy technology based on Einstein's radiation theory. Based on the non-linear relationship between fluorescence saturation and excited fluorescence stimulated emission, STED technology uses a second wavelength red-shifted laser to selectively dissipate excited molecules in advance, and improve imaging by compressing the effective point spread function of the excitation spot Resolution, theoretically, nanometer-level resolution can be achieved in three-dimensional space. As the first far-field super-resolution imaging method proposed theoretically and realized in experiments, STED technology has the advantages of fast imaging and no need for post-image reconstruction. However, in order to avoid the impact of re-excitation effect on the quality of super-resolution images, the loss laser wavelength is usually at the tail end of the fluorescent dye emission spectrum. Due to the extremely small stimulated radiation cross section at the tail end of the emission spectrum, STED technology requires extremely high loss energy (usually more than three orders of magnitude higher than the energy of the excitation light) to improve resolution. Excessive laser energy can cause photobleaching and phototoxicity, and cause damage to fluorescent probes and biological tissues, thus limiting the application of this technology in live cell and tissue imaging.

Fluorescence lifetime imaging microscopy (FLIM) is a microscope imaging technique that describes the spatial distribution of fluorescence lifetimes. It can be applied to the imaging of fixed cells and living cells, revealing more biological information (such as the microenvironment within the cell). Variety). FLIM technology based on time-correlated single photon counting (TCSPC) divides the pulse period into multiple equally spaced time channels, and converts it into memory based on the time difference of the photon relative to the reference signal reaching the detector. Address, the fluorescence decay curve of the dye is obtained after the accumulation of the photon signal. Combining the two imaging technologies of STED and FLIM, the intensity and lifetime information of the fluorescence signal can be obtained at the same time, and the dual-mode STED-FLIM super-resolution fluorescence lifetime imaging can be realized. In confocal fluorescence lifetime imaging (Confocal-FLIM), the fluorescence lifetime curve decays single exponentially with time; but in STED super-resolution fluorescence lifetime imaging (STED-FLIM), due to the stimulated radiation effect of the depleted laser on excited molecules , The fluorescence lifetime curve decays exponentially with time. Although STED-FLIM can achieve super-resolution fluorescence lifetime imaging, too high laser energy changes the fluorescence lifetime characteristics of the fluorescent dye itself, so it cannot truly reflect the changes in the microenvironment, resulting in the inability to present fine structural features when imaging the sample , Resulting in poor imaging effects and difficult to obtain high-resolution images.

Application content

The embodiments of the present application provide a super-resolution imaging method and imaging system based on low-power stimulated emission loss, aiming to solve the high demand for loss laser power in the stimulated radiation loss super-resolution imaging method and super-resolution images under high laser power. The problem of poor quality.

In the first aspect, an embodiment of the present application provides a super-resolution imaging method based on low-power stimulated emission loss, which includes:

Place the sample dyed with fluorescent dye on the stage and adjust the position of the corner reflector in the first light path;

Emitting an excitation laser and a loss laser, the excitation laser and the loss laser are both Gaussian pulse lasers, wherein the wavelength of the loss laser is greater than the wavelength of the excitation laser;

After converting the loss laser propagating along the first optical path into a ring loss laser and overlapping the excitation laser propagating along the second optical path, focus and irradiate the sample;

Simultaneously collecting the excitation light pulse signal of the excitation laser and the fluorescence signal generated after the sample is irradiated, and the fluorescence signal contains the time information and spatial information of the fluorescence photon;

Separating a confocal image and an initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;

The confocal image and the initial super-resolution image are analyzed and processed according to preset image processing rules to obtain a higher-resolution target super-resolution image.

In the second aspect, the embodiments of the present application provide a super-resolution imaging system based on low-power stimulated emission loss, which includes:

Signal acquisition device and imaging processing terminal;

The signal acquisition device is configured to acquire the excitation light pulse signal as a reference signal, and acquire the fluorescent signal generated after the sample is irradiated;

The imaging processing terminal is configured to process the excitation light pulse signal and the fluorescence signal collected by the signal collection device to obtain the target super-resolution image.

The embodiments of the present application provide a super-resolution imaging method and imaging system based on low-power stimulated emission loss. By adjusting the position of the corner reflector in the first optical path to extend the optical path of the loss laser propagating in the first optical path, the low-power Gaussian loss laser is converted into a ring loss laser and overlaps with the excitation laser to focus and irradiate the sample. Make the collected fluorescence lifetime data include confocal signal and super-resolution signal at the same time. The confocal image and the initial super-resolution image are separated from the fluorescence signal through data processing, and the confocal image and the initial super-resolution image are analyzed and processed according to the image processing rules to obtain the target super-resolution image with further improved resolution. Through the above methods, the use of low-power loss lasers reduces the damage to biological samples, reduces the photobleaching effect of fluorescent dyes, and prolongs the effective time of super-resolution imaging. Combined with image enhancement processing, high resolutions containing fine structural features are obtained. Images have achieved good technical results in the actual application process.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present application more clearly, the following will briefly introduce the drawings used in the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present application. Ordinary technicians can obtain other drawings based on these drawings without creative work.

FIG. 1 is a schematic flowchart of a super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;

2 is a schematic diagram of a sub-flow of a super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application;

3 is a schematic diagram of a sub-process of a super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;

4 is a schematic diagram of a sub-process of a super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application;

5 is a schematic diagram of a super-resolution imaging system based on low-power stimulated emission loss according to an embodiment of the application;

FIG. 6 is a schematic block diagram of an imaging processing terminal provided by an embodiment of the application;

FIG. 7 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;

FIG. 8 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss according to an embodiment of the application;

9 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application;

FIG. 10 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application.

detailed description

The technical solutions in the embodiments of the present application will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of them. Based on the embodiments in this application, all other embodiments obtained by a person of ordinary skill in the art without creative work shall fall within the protection scope of this application.

It should be understood that when used in this specification and appended claims, the terms "including" and "including" indicate the existence of the described features, wholes, steps, operations, elements and/or components, but do not exclude one or The existence or addition of multiple other features, wholes, steps, operations, elements, components, and/or collections thereof.

It should also be understood that the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit the application. As used in the specification of this application and the appended claims, unless the context clearly indicates other circumstances, the singular forms "a", "an" and "the" are intended to include plural forms.

It should be further understood that the term "and/or" used in the specification and appended claims of this application refers to any combination of one or more of the items listed in the associated and all possible combinations, and includes these combinations .

Please refer to FIG. 1 and FIG. 5. FIG. 1 is a schematic flowchart of a super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of this application, and FIG. 5 is a low-power stimulated emission loss-based method provided by an embodiment of this application. Schematic diagram of the super-resolution imaging system. The super-resolution imaging method based on low-power stimulated emission loss is applied to an imaging system. The imaging system includes a signal acquisition device 10 and an imaging processing terminal 20. The method combines the signal acquisition device 10 with application software installed in the imaging processing terminal 20 For execution, the imaging system is a system device used to implement a super-resolution imaging method based on low-power stimulated emission loss to achieve high-resolution imaging of the sample, and the signal acquisition device 10 is used to emit excitation laser and loss The laser detects the sample and collects the excitation light pulse signal and the fluorescence signal. The imaging processing terminal 20 is used to obtain the excitation light pulse signal and the fluorescence signal collected by the signal collection device and perform imaging processing to obtain the target super-resolution image. Of terminal devices, such as workstations, desktop computers, laptops, tablets, or mobile phones.

As shown in Figure 1, the method includes steps S110 to S160.

S110: Place the sample dyed by the fluorescent dye on the stage and adjust the position of the corner reflector in the first light path.

Place the sample dyed with fluorescent dye on the stage and adjust the position of the corner reflector in the first light path. First, use fluorescent dyes to dye the sample. Specifically, the sample can be biological materials such as living cells, viruses or tissues. Fluorescent dyes are dyes that are excited by light and generate fluorescent photons. Adjusting the position of the corner reflector can prolong the loss. The optical path of the laser in the first optical path.

FIG. 10 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application. Specifically, as shown in FIG. 10, when the corner reflector is not adjusted, the corner reflector is located in the position in FIG. 10 ① Where the optical path length of the lossy laser propagating along the first optical path (the time required for the light to travel a certain distance along a certain path) is τ ₁ , at this time the optical path interval between the excitation laser pulse and the loss laser pulse is Δτ ₁ (excitation The time difference between the laser pulse and the loss laser pulse acting on the sample); adjust the position of the corner reflector to position ② in Figure 10, and the distance between position ① and position ② is S, then the optical path of the loss laser propagating along the first optical path is τ ₁ +2S/c, where c is the speed of light, then the optical path interval between the excitation laser pulse and the loss laser pulse at this time is Δτ ₂ =Δτ ₁ +2S/c.

S120. Emit an excitation laser and a loss laser, both of the excitation laser and the loss laser are Gaussian pulse lasers, wherein the wavelength of the loss laser is greater than the wavelength of the excitation laser.

Both excitation laser and loss laser can be emitted at the same time. Both lasers are pulsed lasers, and the pulse frequency is the same (such as 80MHz). The frequency of the laser is inversely proportional to the pulse period of the laser. The pulse period should include at least a complete autofluorescence process ( Usually on the order of nanoseconds and above). The longer the fluorescence lifetime, the larger the pulse period of the laser, and the smaller the pulse frequency of the laser. Wherein, the power of the excitation laser is less than the power of the loss laser. Specifically, the power of the loss laser here is at least an order of magnitude (such as less than 10mW) lower than the traditional stimulated radiation loss super-resolution imaging (~100mW). The power of is related to the spectral characteristics of the luminescent material, usually 0.1-100μW. The greater the power of the loss laser, the smaller the central zero-intensity area of the ring-shaped spot in the ring-shaped image obtained, and the greater the peak intensity. The pulse width of the excitation laser and the pulse width of the loss laser are both on the order of one hundred picoseconds. For example, the pulse width of the excitation laser and the pulse width of the loss laser can both be in the range of 0.1-1 nanoseconds. The wavelength of the laser is greater than the wavelength of the excitation laser, and the pulse width is the width of the excitation laser pulse or the loss laser pulse. In order to achieve super-resolution imaging of the sample, the pulse width of the excitation laser and the pulse width of the loss laser must be controlled to be 100 picoseconds Magnitude (100 picoseconds = 0.1 nanosecond).

S130. After converting the lossy laser light propagating along the first optical path into a ring-shaped lossy laser light and overlapping the excitation laser light propagating along the second optical path, focus and irradiate the sample.

Specifically, a spiral phase plate can be installed after the corner reflector of the first optical path, and the loss laser of the Gaussian pulse laser is converted into ring loss laser through the spiral phase plate, and the ring loss laser coincides with the excitation laser propagating along the second optical path. The focal planes of the two laser beams precisely coincide in space and then focus and irradiate the sample. After the dyed sample is irradiated, the fluorescent dye will produce a fluorescent signal.

S140. Collect the excitation light pulse signal of the excitation laser and the fluorescence signal generated after the sample is irradiated at the same time, and the fluorescence signal contains time information and spatial information of the fluorescence photon.

At the same time, the excitation light pulse signal of the excitation laser and the fluorescence signal generated after the sample is irradiated are collected. The collected excitation light pulse signal of the excitation laser is used as the starting point of fluorescence lifetime detection; the fluorescent dye is irradiated and spontaneously emits fluorescence photons Signal, the obtained fluorescent photon signal constitutes the above-mentioned fluorescent signal. The fluorescent signal contains the time information and spatial information of the fluorescent photon. The spatial information of the fluorescent photon is the specific position of the radiated fluorescent photon on the two-dimensional plane. Information, the intensity of the fluorescent photon emitted by the fluorescent molecule gradually decreases with time within a single pulse period, and the time information of the fluorescent photon is the time information when the relative reference signal of the collected fluorescent photon reaches the detector.

S150. Separate a confocal image and an initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule.

The excitation light pulse signal and the fluorescence signal are respectively transmitted to the imaging processing terminal, and the excitation light pulse signal and the fluorescence signal are analyzed and processed through the imaging processing terminal to obtain a super-resolution image for high-resolution imaging of the sample. Specifically, firstly, the fluorescence signal is segmented according to the segmentation rule and the excitation light pulse signal to obtain the confocal image and the initial super-resolution image. The confocal image is the fluorescence lifetime imaging generated by the excitation laser irradiating the sample, and the initial super-resolution image is For STED (Stimulated Emission Depletion) super-resolution fluorescence lifetime imaging produced by irradiating the sample with the excitation laser and ring loss laser at the same time.

In one embodiment, as shown in FIG. 2, step S150 includes sub-steps S151, S152, S153, and S154.

S151. Use the collected time point of the excitation light pulse signal as the start time of fluorescence lifetime detection, and obtain the intensity change of the fluorescence photon on the time channel to obtain the fluorescence decay curve of the fluorescence signal.

The time at which the excitation light pulse signal is detected is regarded as the start time of fluorescence lifetime detection, that is, as the zero point of the time channel, and the intensity change of the fluorescent photon on the time channel is obtained according to the start time, that is, the time is taken as the horizontal The coordinate is to obtain the intensity change of the fluorescent photon through the accumulation of the number of photons. A time channel is a unit time (for example, a time channel can be set to 0.05 nanoseconds). The ordinate is the intensity value of the fluorescent photon, and the intensity of the fluorescent photon can be It is reflected by the number of fluorescent photons collected by time accumulation in each time channel. The greater the number of fluorescent photons in a certain time channel, the higher the intensity of fluorescent photons, and finally the fluorescence decay curve of the fluorescent signal is obtained.

FIG. 7 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application. A sample of fluorescent beads with a diameter of 23nm was used for the experiment. The wavelength of the excitation laser was 635nm, the power was 35μW, the wavelength of the loss laser was 730nm, and the power was 10mW. The frequency of the excitation laser and the frequency of the loss laser were both 80MHz. The pulse width of the wide and depleted laser is about 0.3 nanoseconds (ns), and the optical path interval Δτ ₂ between the excitation laser pulse and the depleted laser pulse is 2 nanoseconds. The fluorescence decay curve of the obtained fluorescence signal is shown in Figure 7 .

S152: Obtain the time channel position of the intensity mutation in the fluorescence attenuation curve as a segmentation point according to the segmentation rule.

Obtain the time channel position of the intensity mutation in the fluorescence decay curve according to the segmentation rule as the segmentation point. When the excitation laser and ring loss laser irradiate the sample at the same time, the signal intensity of the fluorescence signal will have a sudden change, and the sudden change of signal intensity can be reflected in the obtained fluorescence decay curve. Obtain the time channel position of the intensity change in the fluorescence decay curve as By dividing the point, the fluorescence signal can be divided.

In an embodiment, as shown in FIG. 3, step S152 includes sub-steps S1521, S1522, and S1523.

S1521, calculate the slope value of each point in the fluorescence decay curve according to the slope value calculation formula to obtain the corresponding slope curve; S1522, calculate the slope change value of each point in the slope curve according to the slope change value calculation formula S1523. Obtain a time channel position in the fluorescence decay curve corresponding to the slope change value with the largest value as the segmentation point.

Specifically, the fluorescence decay curve is composed of multiple points, and the slope calculation formula can calculate the slope value of each point in the fluorescence decay curve to obtain the slope curve. For example, the slope calculation formula can be A _r =(y _r+2 -y _r-2 )/(x _r+2 -x _r-2 ), where A _r is the rth point in the calculated fluorescence attenuation curve The slope value of y _r+2 is the ordinate of the point on the right side of the rth point 2 pixels apart from it, and y _r-2 is the ordinate of the point on the left side of the rth point 2 pixels apart from it, x _r+2 is the abscissa of the point 2 pixels away from the right of the rth point, x _r+2 is the abscissa of the point 2 pixels away from the left of the rth point, where r is arbitrary In the case of points, x _r+2 -x _{r-2 is} always equal to 5. If there is no point on the left of the rth point, then y _r-2 = y _r , x _r-2 = x _r ; if there is no point on the right of the rth point, then y _r+2 = y _r , x _r+2 = X _r . The slope calculation formula can also be a calculation formula for deriving points in the fluorescence attenuation curve. The slope change value calculation formula is used to calculate the slope change value of each point in the slope curve. The calculation method of the slope change value is the same as the calculation method of the above slope value. A non-negative value corresponding to a slope change value is obtained, and the time channel position corresponding to the time channel position in the fluorescence decay curve of the slope change value with the largest value among the non-negative values is obtained as the division point, that is, the slope change value with the largest value is obtained The corresponding abscissa value in the fluorescence decay curve is used as the dividing point.

For example, for the fluorescence attenuation curve shown in FIG. 7, the abscissa value of the dividing point obtained by the above method is τ _x =3ns, then 3ns can be used as the dividing point to divide the fluorescence signal.

S153. Compose the confocal image according to the spatial information of the fluorescent photons located before the dividing point in the fluorescent signal.

S154. Compose the initial super-resolution image according to the spatial information of the fluorescent photons located after the dividing point in the fluorescent signal.

Taking the segmentation point as the reference, the spatial information of the fluorescent photons from the start time to the segmentation point in the fluorescence signal is obtained to form a confocal image, and the spatial information of the fluorescence photons after the segmentation point in the fluorescence signal is obtained to form an initial Super-resolution image.

FIG. 8 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application. For example, after segmenting the obtained fluorescent signal, a confocal image of the sample is obtained as shown in Fig. 8(a), and a corresponding initial super-resolution image is obtained as shown in Fig. 8(b).

S160. Analyze and process the confocal image and the initial super-resolution image according to a preset image processing rule to obtain a high-resolution target super-resolution image.

The confocal image and the initial super-resolution image are analyzed and processed through the image processing rules, which can greatly improve the resolution of imaging the sample and obtain the target super-resolution image of the sample.

In an embodiment, as shown in FIG. 4, step S160 includes sub-steps S161, S162, and S163.

S161. Subtract the intensity value of the initial super-resolution image from the intensity value of the confocal image to obtain a ring-shaped image.

The field of view of the confocal image, the initial super-resolution image, and the resulting ring image is the same (the image size is the same). Specifically, the pixel value of a pixel in the confocal image is subtracted from the pixel value corresponding to the pixel in the initial super-resolution image to obtain the pixel difference value of the pixel, and the pixel difference value of each pixel in the confocal image is obtained and combined You can get a corresponding ring image.

For example, the focused image in Figure 8 is named image A, the initial super-resolution image is named image B, and the resulting ring image is named image C, then C=AB, and the resulting ring image is shown in Figure 8 ( c) as shown.

S162. Multiply the ring image and the enhancement coefficient in the image processing rule to obtain an enhanced ring image.

Specifically, the pixel value of each pixel in the ring image is multiplied by the enhancement coefficient to obtain the corresponding enhanced ring image. Wherein, the enhancement coefficient is a coefficient value preset by the user, the value of the enhancement coefficient is greater than 1, and the enhancement coefficient may be an integer or a decimal number.

FIG. 9 is a schematic diagram of the use effect of the super-resolution imaging method based on low-power stimulated emission loss provided by an embodiment of the application. For example, if the enhancement factor is 1, the ring image is shown in Figure 9(1), and the enhanced ring image obtained at this time is the same as the ring image C; if the enhancement factor is 2, the enhanced ring image obtained at this time is as As shown in Fig. 9(2); taking the enhancement coefficient as 4, the enhanced ring image obtained at this time is as shown in Fig. 9(3).

S163. Subtract the intensity value of the enhanced annular image from the intensity value of the initial super-resolution image to obtain the target super-resolution image.

The obtained super-resolution image of the target also has the same field of view as the confocal image. Specifically, the pixel value of a pixel in the initial super-resolution image is subtracted from the pixel value corresponding to the pixel in the ring image to obtain the pixel difference value of the pixel, and the initial super-resolution image and the pixel value of each pixel in the ring image are obtained. The pixel difference can be combined to obtain the corresponding target super-resolution image.

For example, if the enhancement factor is 1, the obtained ring image can be expressed as 1×C, and the target super-resolution image obtained at this time is shown in Figure 9 (4); if the enhancement factor is 2, the obtained ring image can be expressed as 2×C, the target super-resolution image obtained at this time is shown in Figure 9(5); taking the enhancement coefficient as 4, the obtained ring image can be expressed as 4×C, and the target super-resolution image obtained at this time is shown in Figure 9(5). Shown in 9(6).

The super-resolution imaging method based on low-power stimulated emission loss provided by the embodiment of the present application extends the optical path of the lossy laser propagating in the first optical path by adjusting the position of the corner reflector in the first optical path, and reduces the low-power The Gaussian loss laser is converted into a ring loss laser and coincides with the excitation laser to focus and irradiate the sample, so that the collected fluorescence lifetime data contains both confocal and super-resolution signals. The confocal image and the initial super-resolution image are separated from the fluorescence signal through data processing, and the confocal image and the initial super-resolution image are analyzed and processed according to the image processing rules to obtain the target super-resolution image with further improved resolution. Through the above methods, the use of low-power loss lasers reduces the damage to biological samples, reduces the photobleaching effect of fluorescent dyes, and prolongs the effective time of super-resolution imaging. Combined with image enhancement processing, high resolutions containing fine structural features are obtained. Images have achieved good technical results in the actual application process.

The embodiment of the present application also provides a super-resolution imaging system based on low-power stimulated emission loss. The super-resolution imaging system based on low-power stimulated emission loss can be used to realize the aforementioned super-resolution imaging method based on low-power stimulated emission loss. Any embodiment of. Specifically, please refer to FIGS. 5-6. FIG. 5 is a schematic diagram of a super-resolution imaging system based on low-power stimulated emission loss according to an embodiment of the application, and FIG. 6 is a schematic diagram of an imaging processing terminal provided by an embodiment of the application. In a block diagram, the imaging system includes a signal acquisition device 10 and an imaging processing terminal 20.

The signal acquisition device 10 is used for acquiring the excitation light pulse signal as a reference signal, and acquiring the fluorescent signal generated after the sample is irradiated.

Specifically, the signal acquisition device includes an excitation laser 101, a loss laser 102, a beam splitter 103, a first dichroic mirror 104, a second dichroic mirror 105, the corner reflector 106, a spiral phase plate 107, a scanning galvanometer 108, The objective lens 109, the stage 110, the preamplifier 111, the first detector 112, the second detector 113, and a time dependent single photon counter (TCSPC) 114.

Wherein, the excitation laser 101 is used to emit Gaussian pulse excitation laser; the loss laser 102 is used to emit Gaussian pulse loss laser; the spectroscope is used to split the excitation laser light to A part of the excitation laser light is propagated along the second optical path, and the other part of the excitation laser light is injected into the second detector; The laser light is reflected to propagate to the spiral phase plate; the spiral phase plate 107 is used to convert the incident loss laser light of the Gaussian pulse into ring loss laser light so that it propagates to the second dichroic mirror The first dichroic mirror 104 is used to reflect the excitation laser propagating along the second optical path to make it propagate to the second dichroic mirror, and to transmit the fluorescent signal generated after the sample is irradiated The second dichroic mirror 105 is used to reflect the ring loss laser and transmit the excitation laser propagating along the second optical path so that the two lasers overlap and propagate to the scanning galvanometer, and The fluorescence signal is transmitted; the scanning galvanometer 108 is used to synchronously scan the incident excitation laser and ring loss laser to realize the area array imaging of the sample; the objective lens 109 is used to perform the incident laser After focusing, illuminate the sample; the stage 110 is used to place and fix the sample, and to move the sample three-dimensionally; the first detector 112 is used to detect and collect the fluorescent dye emitted after being irradiated The fluorescent photon signal; the second detector 113 is used to detect the incident excitation laser light to obtain the excitation light pulse signal; the preamplifier 111 is used to detect the fluorescent photon from the first detector The signal is amplified and filtered; the time-correlated single photon counter (TCSPC) 114 is used for signal storage and fluorescence lifetime imaging to obtain the fluorescence signal.

More specifically, an electrical connection is made between the excitation laser 101 and the loss laser 102, and the excitation laser 101 is triggered synchronously by the loss laser 102, so that the two laser pulses maintain a stable pulse interval.

The imaging processing terminal 20 is configured to process the excitation light pulse signal and the fluorescence signal collected by the signal collection device to obtain the target super-resolution image.

The imaging processing terminal 20 is a terminal device used to obtain the excitation light pulse signal and the fluorescence signal collected by the signal acquisition device and then perform imaging processing to obtain the target super-resolution image, such as a workstation, a desktop computer, a notebook computer, a tablet computer, or a mobile phone. Wait.

The imaging processing terminal 20 may perform the following steps: separate a confocal image and an initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule; The focused image and the initial super-resolution image are analyzed and processed to obtain a high-resolution target super-resolution image.

In an embodiment, as shown in FIG. 6, the imaging processing terminal 20 includes a fluorescent signal dividing unit 210 and an image processing unit 220.

The fluorescence signal segmentation unit 210 is used to separate the confocal image and the initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and preset segmentation rules; the image processing unit 220 is used to separate the confocal image and the initial super-resolution image according to the preset image The processing rules analyze and process the confocal image and the initial super-resolution image to obtain a higher-resolution target super-resolution image.

The above are only specific implementations of this application, but the scope of protection of this application is not limited to this. Anyone familiar with the technical field can easily think of various equivalents within the technical scope disclosed in this application. Modifications or replacements, these modifications or replacements shall be covered within the scope of protection of this application. Therefore, the protection scope of this application shall be subject to the protection scope of the claims.

Claims

A super-resolution imaging method based on low-power stimulated emission loss, applied to an imaging system, characterized in that the method includes:

Place the sample dyed with fluorescent dye on the stage and adjust the position of the corner reflector in the first light path;

Emitting an excitation laser and a loss laser, the excitation laser and the loss laser are both Gaussian pulse lasers, wherein the wavelength of the loss laser is greater than the wavelength of the excitation laser;

After converting the loss laser propagating along the first optical path into a ring loss laser and overlapping the excitation laser propagating along the second optical path, focus and irradiate the sample;

Simultaneously collecting the excitation light pulse signal of the excitation laser and the fluorescence signal generated after the sample is irradiated, and the fluorescence signal contains the time information and spatial information of the fluorescence photon;

Separating a confocal image and an initial super-resolution image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;

The confocal image and the initial super-resolution image are analyzed and processed according to preset image processing rules to obtain a higher-resolution target super-resolution image.
The super-resolution imaging method based on low-power stimulated emission loss according to claim 1, wherein the confocal image is separated from the fluorescent signal according to the excitation light pulse signal and a preset segmentation rule And the initial super-resolution image, including:

Taking the collected time point of the excitation light pulse signal as the start time of fluorescence lifetime detection, and obtaining the intensity change of the fluorescence photon on the time channel to obtain the fluorescence decay curve of the fluorescence signal;

Acquiring, according to the segmentation rule, the time channel position of the intensity mutation in the fluorescence decay curve as a segmentation point;

Composing the confocal image according to the spatial information of the fluorescent photons located before the dividing point in the fluorescent signal;

The initial super-resolution image is composed according to the spatial information of the fluorescent photons located after the dividing point in the fluorescent signal.
The super-resolution imaging method based on low-power stimulated emission loss according to claim 1, wherein said analyzing and processing said confocal image and said initial super-resolution image according to preset image processing rules, In order to obtain high-resolution super-resolution images of the target, including:

Subtracting the intensity value of the initial super-resolution image from the intensity value of the confocal image to obtain a ring image;

Multiplying the ring image and the enhancement coefficient in the image processing rule to obtain an enhanced ring image;

The intensity value of the enhanced annular image is subtracted from the intensity value of the initial super-resolution image to obtain the target super-resolution image.
The super-resolution imaging method based on low-power stimulated emission loss according to claim 3, wherein the value of the enhancement coefficient is greater than 1.
The super-resolution imaging method based on low-power stimulated emission loss according to claim 2, wherein the segmentation rule includes a slope value calculation formula and a slope change value calculation formula, and the segmentation rule obtains the The time channel position of the intensity mutation in the fluorescence decay curve is used as the dividing point, including:

Calculate the slope value of each point in the fluorescence decay curve according to the slope value calculation formula to obtain the corresponding slope curve;

Calculate the slope change value of each point in the slope curve according to the slope change value calculation formula;

Obtain the time channel position corresponding to the one of the slope change values with the largest value in the fluorescence decay curve as the segmentation point.
The super-resolution imaging method based on low-power stimulated emission loss according to any one of claims 1-5, wherein the power of the loss laser is greater than the power of the excitation laser.
The super-resolution imaging method based on low-power stimulated emission loss according to claim 6, wherein the pulse width of the excitation laser and the pulse width of the loss laser are both on the order of one hundred picoseconds.
A super-resolution imaging system based on low-power stimulated emission loss, which is used to implement the super-resolution imaging method based on low-power stimulated emission loss according to any one of claims 1-7, characterized in that , The imaging system includes a signal acquisition device and an imaging processing terminal;

The signal acquisition device is configured to acquire the excitation light pulse signal as a reference signal, and acquire the fluorescent signal generated after the sample is irradiated;

The imaging processing terminal is configured to process the excitation light pulse signal and the fluorescence signal collected by the signal collection device to obtain the target super-resolution image.
The super-resolution imaging system based on low-power stimulated emission loss according to claim 8, wherein the imaging processing terminal comprises:

A fluorescent signal segmentation unit for separating a confocal image and an initial super-resolution image from the fluorescent signal according to the excitation light pulse signal and a preset segmentation rule;

The image processing unit is configured to analyze and process the confocal image and the initial super-resolution image according to preset image processing rules to obtain a higher-resolution target super-resolution image.
The super-resolution imaging system based on low-power stimulated emission loss according to claim 8, wherein the signal acquisition device includes an excitation laser, a lossy laser, a beam splitter, a first dichroic mirror, a second dichroic mirror, and a The corner reflector, the spiral phase plate, the scanning galvanometer, the objective lens, the stage, the preamplifier, the first detector, the second detector, and the time-dependent single photon counter;

The excitation laser is used to emit a Gaussian pulse excitation laser;

The lossy laser is used to emit a lossy laser with Gaussian pulses;

The beam splitter is configured to split the excitation laser light so that a part of the excitation laser light propagates along the second optical path, and the other part of the excitation laser light enters the second detector;

The corner reflector is used to reflect the lossy laser light propagating along the first optical path so as to propagate to the spiral phase plate;

The spiral phase plate is used to convert the incident loss laser of the Gaussian pulse into a ring loss laser so that it can propagate to the second dichroic mirror;

The first dichroic mirror is used to reflect the excitation laser propagating along the second optical path so that it can propagate to the second dichroic mirror, and transmit the fluorescent signal generated after the sample is irradiated;

The second dichroic mirror is used to reflect the ring-loss laser light and transmit the excitation laser light propagating along the second optical path so that the two laser beams overlap and then propagate to the scanning galvanometer, and to Fluorescence signal is transmitted;

The scanning galvanometer is used to synchronously scan the incident excitation laser and ring loss laser to realize the area array imaging of the sample;

The objective lens is used to focus the incident laser light and irradiate the sample;

The stage is used to place and fix a sample, and to move the sample in three dimensions;

The first detector is used to detect and collect the fluorescent photon signal emitted by the fluorescent dye after being irradiated;

The second detector is used to detect incident excitation laser light to obtain the excitation light pulse signal;

The preamplifier is used to amplify and filter the fluorescence photon signal from the first detector;

The time-dependent single-photon counter is used for signal storage and fluorescence lifetime imaging to obtain the fluorescence signal, wherein the fluorescence signal contains time information and spatial information of the fluorescence photons.