WO2021243755A1 - Fluorescence difference super-resolution imaging method and imaging system - Google Patents

Abstract

A fluorescence difference super-resolution imaging method and imaging system. By means of adjusting the position of a corner reflector in a system, the optical path of propagation of one Gaussian pulse laser beam in a first light path is prolonged or shortened, and the Gaussian pulse laser beam is converted into an annular pulse laser, and is then focused and irradiates a sample, and the other Gaussian pulse laser beam propagates along a second light path, and is focused and irradiates the sample, wherein the pulse interval between the annular pulse laser and the Gaussian pulse laser, which irradiate the sample, is greater than the fluorescence lifetime of fluorescent dye; an exciting light pulse signal and a fluorescence signal are respectively collected by means of two detectors; a first image and a second image are separated from the fluorescence signal; and the first image and the second image are subjected to analysis processing according to an image processing rule, so as to obtain a target super-resolution image, the resolution of which is further improved. By means of the method, damage to a biological sample is reduced by using a low-power pulse laser, and the problem of a reduction in the quality of a super-resolution image caused by pixel mismatching during traditional fluorescence difference super-resolution imaging is solved.

Description

Fluorescence differential super-resolution imaging method and imaging system

This application is based on a Chinese patent application with an application number of 202010500069.6 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 fluorescence differential super-resolution imaging method and imaging system.

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. In 2013, Professor Kuang Cuifang of Zhejiang University proposed a fluorescence emission difference (FED) super-resolution imaging method based on the idea of compressing the excitation light spot expansion function, which subtracted one from the confocal image excited by the Gaussian spot The negative confocal image excited by the ring-shaped spot realizes super-resolution imaging at very low laser energy. Because it does not involve the stimulated emission process, the method is simple and effective, and the imaging system is simple and low in cost. However, fluorescence difference requires two images to be collected at the same position of the sample. Switching between imaging modes and the movement of the sample itself during imaging will cause pixel mismatch (that is, the incomplete coincidence of the Gaussian spot and the ring spot), which can cause serious problems. Therefore, it is not suitable for long-term imaging studies of biological samples such as living cells, and it is impossible to observe biological samples for a long time and obtain high-resolution images.

Application content

The embodiments of the present application provide a fluorescence differential super-resolution imaging method and imaging system, which are designed to solve the problem of being unable to observe biological samples for a long time and obtain high-quality super-resolution images due to pixel mismatch in the existing fluorescence differential super-resolution imaging method. The problem.

In the first aspect, an embodiment of the present application provides a fluorescence differential super-resolution imaging method, 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;

The Gaussian pulsed laser is emitted and split to obtain two Gaussian pulsed lasers. One of the Gaussian pulsed lasers propagates along the second optical path and then focuses on the sample, and the other Gaussian pulsed laser propagates along the first optical path. After being converted into a ring pulse laser, the sample is focused and irradiated, wherein the pulse interval between the ring pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is greater than the fluorescence lifetime of the fluorescent dye;

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

Separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;

The first image and the second image are analyzed and processed according to a preset image processing rule to obtain a high-resolution target super-resolution image.

In the second aspect, an embodiment of the present application provides a fluorescence differential super-resolution imaging system, which includes:

Signal acquisition device and imaging processing terminal;

The signal acquisition device is configured to acquire the excitation light pulse signal of the Gaussian pulsed laser 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 fluorescence differential super-resolution imaging method and imaging system. By adjusting the position of the corner reflector in the first optical path, the optical path of a beam of Gaussian pulse laser propagating in the first optical path is extended or shortened, and the Gaussian pulse laser propagating along the first optical path is converted into a ring pulse laser and then focused The sample is irradiated, and another beam of Gaussian pulsed laser propagates along the second optical path to focus and irradiate the sample. The pulse interval between the ring pulsed laser irradiating the sample and the Gaussian pulsed laser irradiating the sample is greater than the fluorescence lifetime of the fluorescent dye, and the collected fluorescence signal contains Time information and spatial information of fluorescent photons. The first image and the second image are separated from the fluorescence signal through data processing, and the first image and the second 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 lasers reduces the damage to biological samples, reduces the photobleaching effect of fluorescent dyes, and extends the effective time of super-resolution imaging. Combined with image enhancement processing, high-resolution images containing fine structural features are obtained. It is especially suitable for long-term observation of biological samples and obtaining high-resolution images, and has achieved good technical effects 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 fluorescence differential super-resolution imaging method provided by an embodiment of the application;

2 is a schematic diagram of a sub-flow of a fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 3 is a schematic diagram of a sub-flow of a fluorescence differential super-resolution imaging method provided by an embodiment of the application;

4 is a schematic diagram of a sub-flow of a fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 5 is a schematic diagram of a sub-flow of a fluorescence differential super-resolution imaging method provided by an embodiment of the application;

6 is a schematic diagram of a sub-flow of a fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 7 is a schematic diagram of a sub-flow of a fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 8 is a schematic diagram of a fluorescence differential super-resolution imaging system provided by an embodiment of the application;

FIG. 9 is a schematic block diagram of an imaging processing terminal provided by an embodiment of the application;

10 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by an embodiment of the application;

11 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 12 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 13 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by an embodiment of the application;

FIG. 14 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method 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 FIGS. 1 and 8. FIG. 1 is a schematic flowchart of a fluorescence differential super-resolution imaging method provided by an embodiment of this application, and FIG. 8 is a schematic diagram of a fluorescence differential super-resolution imaging system provided by an embodiment of this application. The fluorescence differential super-resolution imaging method is applied to an imaging system. The imaging system includes a signal acquisition device 10 and an imaging processing terminal 20. The method is executed by the signal acquisition device 10 in combination with application software installed in the imaging processing terminal 20, and the imaging system is It is a system device used to implement a fluorescence differential super-resolution imaging method to achieve high-resolution imaging of the sample. The signal acquisition device 10 is used to emit a Gaussian pulsed laser to detect the sample and collect excitation light pulse signals and Fluorescence signal device, the imaging processing terminal 20 is a terminal device used to obtain the excitation light pulse signal and fluorescent signal collected by the signal acquisition device and then perform imaging processing to obtain the target super-resolution image, such as workstations, desktop computers, notebook computers, and tablets. Computer or mobile phone, etc.

As shown in Figure 1, the method includes steps S110 to S150.

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, the sample is dyed with fluorescent dye. Specifically, the sample can be biological materials such as living cells, viruses or tissues. The fluorescent dye is the material that generates autofluorescence signal after being irradiated by the laser. The corner reflector is set in the first light path. The Gaussian pulsed laser can propagate along the first optical path and the second optical path respectively. Adjusting the position of the corner reflector can extend or shorten the optical path of the Gaussian pulsed laser in the first optical path, so as to change the Gaussian pulsed laser in the first optical path and The optical path interval for propagation of the second optical path.

14 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by the embodiments of the application. Specifically, as shown in FIG. 14, when the corner reflector is not adjusted, the corner reflector is located at position ① in FIG. The optical path of the first optical path (the time required for light to travel a certain distance along a certain path) is τ ₁ , and the optical path interval between the first optical path and the second optical path is Δτ ₁ (the optical path interval is also equal to The pulse interval time between the ring pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample); adjust the position of the corner reflector to the position ② in Figure 14, the distance between the position ① and the position ② Is S, the optical path length of the Gaussian pulse 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 first optical path and the second optical path at this time is Δτ ₂ =Δτ ₁ +2S/c.

S120. Emit and split the Gaussian pulsed laser to obtain two Gaussian pulsed lasers. One of the Gaussian pulsed lasers propagates along the second optical path and then focuses and irradiates the sample, and the other Gaussian pulsed laser is along the first optical path. After being propagated and converted into a ring pulse laser, the sample is focused and irradiated.

The Gaussian pulsed laser is emitted and split to obtain two Gaussian pulsed lasers. One of the Gaussian pulsed lasers propagates along the second optical path and then focuses on the sample, and the other Gaussian pulsed laser propagates along the first optical path. After being converted into a ring pulse laser, the sample is focused and irradiated, wherein the pulse interval between the ring pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is greater than the fluorescence lifetime of the fluorescent dye.

Specifically, a spiral phase plate can be arranged before the corner reflector of the first optical path, and the Gaussian pulse laser propagating along the first optical path can be converted into a ring pulse laser through the spiral phase plate. A beam of Gaussian pulsed laser propagated by the two optical paths is focused at different times and irradiates the sample. After the dyed sample is irradiated, the fluorescent dye will generate a fluorescent signal. The ring pulse laser can irradiate the sample before the Gaussian pulsed laser or after The sample is irradiated with a Gaussian pulsed laser. The emitted laser is a Gaussian pulsed laser (for example, the pulse frequency is 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 longer the pulse period of the laser, and the smaller the frequency of the laser. Among them, the power of the Gaussian pulsed laser is related to the spectral characteristics of the luminescent material, usually 0.1-100 μW. The greater the power of the Gaussian pulsed laser, the smaller the center zero-intensity area of the ring spot in the ring image obtained, and the greater the peak intensity. The pulse interval between the ring pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample needs to be Longer than the fluorescence lifetime of fluorescent dyes. The pulse width of the Gaussian pulsed laser is in the order of hundreds of picoseconds. For example, the value range of the Gaussian pulsed laser can be 0.1-1 nanosecond. In order to realize super-resolution imaging of the sample, the Gaussian pulsed laser needs to be controlled to be 100 picoseconds. Magnitude (100 picoseconds = 0.1 nanosecond).

S130: Collect the excitation light pulse signal of the Gaussian pulsed laser and the fluorescent signal generated after the sample is irradiated at the same time, and the fluorescent signal contains time information and spatial information of fluorescent photons.

At the same time, the excitation light pulse signal of the Gaussian pulsed laser and the fluorescence signal generated after the sample is irradiated are collected. The collected excitation light pulse signal of the Gaussian pulsed laser is used as the starting point of fluorescence lifetime detection; the spontaneous radiation is generated after the fluorescent dye is irradiated Fluorescent photon 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 radiated fluorescent photon on a two-dimensional plane. Specific location 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 collected fluorescent photon reaches the detector relative to the reference signal.

S140. Separate a first image and a second 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, first, the fluorescence signal is segmented according to the segmentation rule and the excitation light pulse signal to obtain the first image and the second image. If the ring pulse laser is followed by Gaussian pulsed laser to irradiate the sample, the first image obtained is a confocal image, and the second image is a ring image. The confocal image is the fluorescence lifetime imaging produced by irradiating the sample with the Gaussian pulsed laser , The ring image is the fluorescence signal image generated by irradiating the sample with the Gaussian pulsed laser and then irradiating the sample with the ring pulsed laser; if the ring pulsed laser irradiates the sample before the Gaussian pulsed laser, the first image obtained is The ring image, the ring image is the ring fluorescence lifetime imaging produced by the ring pulse laser irradiating the sample, and the second image is the confocal image.

In an embodiment, as shown in FIG. 2, step S140 includes sub-steps S141, S142, S143, and S144.

In this embodiment, the ring pulse laser is followed by Gaussian pulse laser to irradiate the sample. The first image obtained is a confocal image, and the second image is a ring image. The segmentation rules include fluorescence intensity interval, fluorescence intensity threshold, and time threshold. .

S141. 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 more the number of fluorescent photons in a certain time channel, the higher the intensity of the fluorescent photons, and the fluorescence decay curve of the fluorescent signal is obtained.

FIG. 10 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method 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 Gaussian pulsed laser was 635nm, the power was 35μW, the pulse frequency of the laser was 40MHz, and the pulse width was 0.3 nanoseconds (ns). The optical path interval Δτ ₂ is 12.5 nanoseconds, and the fluorescence decay curve of the obtained fluorescence signal is shown in Fig. 10(a).

S142. Determine a division point of the fluorescence attenuation curve according to the division rule.

The division point of the fluorescence attenuation curve is determined according to the division rule. Specifically, the cutoff time of fluorescence lifetime detection can be determined according to the fluorescence decay curve and the segmentation rule, the time channel position corresponding to the intermediate point of the start time and the end time is taken as the segmentation point, and the fluorescence signal is segmented according to the segmentation point.

In an embodiment, as shown in FIG. 3, step S142 includes sub-steps S1421, S1422, S1423, and S1424.

S1421, judge whether the fluorescence intensity of each time channel of the fluorescence decay curve is within the fluorescence intensity interval, and obtain the time channel whose fluorescence intensity is within the fluorescence intensity interval as the first time channel; S1422, Determine whether the fluorescence intensity of the time channel separated from the first time channel by the time threshold in the attenuation curve is less than the fluorescence intensity threshold; S1423, if the fluorescence intensity of the time channel separated from the first time channel by the time threshold is If the fluorescence intensity is less than the fluorescence intensity threshold, use the first time channel as the cut-off time of the fluorescence lifetime detection; S1424. Use the time channel position corresponding to the midpoint of the start time and the cut-off time as the Split point.

Specifically, the fluorescence decay curve is composed of multiple points, each point is located in a time channel, and each point corresponds to a fluorescence intensity value. It can be judged whether the fluorescence intensity value of each time channel in the fluorescence decay curve is within the fluorescence intensity interval, and the time channel whose fluorescence intensity is within the fluorescence intensity interval is used as the first time channel, and the time interval from the first time channel is obtained. Threshold the fluorescence intensity of the time channel, and judge whether the fluorescence intensity is less than the fluorescence intensity threshold, if it is smaller, the first time channel is used as the cut-off time for fluorescence lifetime detection, and the cut-off time obtained according to the above judgment method has one and only one , And use the time channel position corresponding to the midpoint of the start time and the end time as the split point.

For example, for the fluorescence decay curve shown in Figure 10, the cut-off time is 25ns and the start time is 0 using the above method. Then the intermediate point between the start time and the end time is τ _x =12.5ns, which will be equal to 12.5ns A corresponding time channel position is used as the split point.

S143. Compose the confocal image according to the spatial information of the fluorescent photons located before the dividing point in the fluorescent signal.

S144. Compose the ring image according to the spatial information of the fluorescent photons located after the dividing point in the fluorescent signal.

Take the segmentation point as the reference, obtain the spatial information of the fluorescence photons from the start time to the segmentation point in the fluorescence signal to form a confocal image, and obtain the spatial information composition of the fluorescence photons from the segmentation point to the cut-off time in the fluorescence signal An annular image.

For example, as shown in Figure 10, after segmenting the obtained fluorescence signal, a confocal image of the sample is obtained as shown in Figure 10(b), and a corresponding ring image is obtained as shown in Figure 10(c). As shown, the confocal image can be named image A, and the ring image can be named image B.

S150. Analyze and process the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image.

The analysis and processing of the first image and the second image through the image processing rules can greatly improve the resolution of imaging the sample, and obtain the target super-resolution image of the sample. The field of view of the confocal image and the ring image is the same (the image size is the same).

In one embodiment, as shown in FIG. 4, step S150 includes sub-steps S151 and S152.

S151. 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. 11 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by an embodiment of the application. For example, if the enhancement factor is 1, the ring image is shown in Figure 11(a), and the enhanced ring image (which can be expressed as 1×B) is the same as the ring image B; the enhancement factor is 2, this The enhanced ring image (which can be expressed as 2×B) is shown in Fig. 11(b); taking the enhancement coefficient as 4, the enhanced ring image (which can be expressed as 4×B) obtained at this time is shown in Fig. 11(b). c) as shown.

S152: Subtract the intensity value of the enhanced annular image from the intensity value of the confocal image to obtain the target super-resolution image.

The obtained target super-resolution image and the confocal image have the same field of view (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 ring 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, namely A corresponding super-resolution image of a target can be obtained.

For example, if the enhancement factor is 1, the resulting enhanced ring image can be expressed as 1×B, and the target super-resolution image can be expressed as A-1×B. At this time, the target super-resolution image obtained is shown in Figure 11(d) ; Taking the enhancement factor as 2, the resulting enhanced ring image can be expressed as 2×B, and the target super-resolution image can be expressed as A-2×B, and the target super-resolution image obtained at this time is shown in Figure 11(e); Taking the enhancement factor as 4, the resulting enhanced ring image can be expressed as 4×B, and the target super-resolution image can be expressed as A-4×B, and the target super-resolution image obtained at this time is shown in Figure 11(f).

In another embodiment, as shown in FIG. 5, step S140 includes sub-steps S1401, S1402, S1403, and S1404.

In this embodiment, the ring pulse laser irradiates the sample before the Gaussian pulse laser. The first image obtained is a ring image, and the second image is a confocal image. The segmentation rule includes a fluorescence intensity threshold and an intensity difference threshold.

S1401: 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.

This process is the same as S141, and will not be repeated here.

FIG. 12 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method 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 Gaussian pulsed laser was 635nm, the power was 35μW, the pulse frequency of the laser was 80MHz, and the pulse width was 0.3 nanoseconds (ns). The optical path interval Δτ ₂ is 3 nanoseconds, and the fluorescence decay curve of the obtained fluorescence signal is shown in Fig. 12(a).

S1402. Determine a division point of the fluorescence attenuation curve according to the division rule.

The division point of the fluorescence attenuation curve is determined according to the division rule. Specifically, a corresponding point in the fluorescence attenuation curve can be determined as the segmentation point according to the segmentation rule.

In an embodiment, as shown in FIG. 6, step S1402 includes sub-steps S14021, S14022, and S14023.

S14021. Determine whether the fluorescence intensity of each time channel of the fluorescence decay curve is not less than the fluorescence intensity threshold, and obtain a curve segment in the fluorescence decay curve that is not less than the fluorescence intensity threshold as a target curve segment; S14022, Determine whether the absolute value of the fluorescence intensity difference between each target time channel and two adjacent time channels in the target curve segment is greater than the intensity difference threshold; S14023, if the target time channel is The absolute value of the difference in fluorescence intensity between two adjacent time channels is greater than the intensity difference threshold, and the position of the target time channel is used as the segmentation point.

Specifically, the fluorescence decay curve is composed of multiple points, each point is located in a time channel, and each point corresponds to a fluorescence intensity value. It can be judged whether the fluorescence intensity value of each time channel in the fluorescence decay curve is not less than the fluorescence intensity threshold value, and the curve segment with the fluorescence intensity not less than the fluorescence intensity threshold value is used as the target curve segment, and each target time channel in the target curve segment is judged Whether the absolute value of the fluorescence intensity difference between the two adjacent time channels is greater than the intensity difference threshold is judged, if both are greater, the target time channel is used as the segmentation point, and the segmentation point obtained according to the above judgment method has and There is only one.

For example, for the fluorescence decay curve shown in FIG. 12, the time corresponding to a target time channel determined by the above method is τ _x '=4 ns, then the target time channel position is taken as the dividing point.

S1403. Compose the ring image according to the spatial information of the fluorescent photons located before the dividing point in the fluorescent signal.

S1404, composing the confocal image according to the spatial information of the fluorescent photons located after the dividing point in the fluorescent signal.

Take the segmentation point as the reference, acquire the spatial information of the fluorescent photons from the start time to the segmentation point in the fluorescence signal to form a ring image, and acquire the spatial information composition of the fluorescence photons from the segmentation point to the cut-off time in the fluorescence signal A confocal image.

For example, as shown in Figure 12, after the obtained fluorescence signal is divided, a ring image of the sample is obtained as shown in Figure 12(b), and a corresponding confocal image is obtained as shown in Figure 10(c). Show.

In this embodiment, as shown in FIG. 7, step S150 includes sub-steps S1501 and S1502.

S1501. 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. 13 is a schematic diagram of the use effect of the fluorescence differential super-resolution imaging method provided by an embodiment of the application. For example, if the enhancement factor is 1, the ring image is shown in Figure 13(a), and the enhanced ring image (which can be expressed as 1×B) is the same as the ring image B; the enhancement factor is 1.25, this The enhanced ring image (which can be expressed as 1.25×B) is shown in Fig. 13(b); taking the enhancement coefficient as 1.5, the enhanced ring image obtained at this time (which can be expressed as 1.5×B) is shown in Fig. 13( c) as shown.

S1502: Subtract the intensity value of the enhanced annular image from the intensity value of the confocal image to obtain the target super-resolution image.

The obtained super-resolution image of the target has the same field of view as the confocal image. Specifically, the pixel value of a pixel in the confocal 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 pixel difference value of each pixel in the confocal image is obtained and combined, namely A corresponding super-resolution image of a target can be obtained.

For example, if the enhancement factor is 1, the target super-resolution image can be expressed as A-1×B, and the target super-resolution image obtained at this time is shown in Figure 13(d); if the enhancement factor is 1.25, the target super-resolution image can be Denoted as A-1.25×B, the target super-resolution image obtained at this time is shown in Figure 13(e); taking the enhancement factor as 1.5, the target super-resolution image can be expressed as A-1.5×B, and the target super-resolution image obtained at this time The super-resolution image is shown in Figure 13(f).

The fluorescence differential super-resolution imaging method provided by the embodiment of the present application extends or shortens the optical path of a beam of Gaussian pulsed laser in the first optical path by adjusting the position of the corner reflector in the first optical path, and moves along the first optical path. The Gaussian pulsed laser propagating on one optical path is converted into a ring pulsed laser to focus and irradiate the sample, and the other Gaussian pulsed laser propagates along the second optical path to focus and irradiate the sample. The pulse interval between the ring pulsed laser irradiating the sample and the Gaussian pulsed laser irradiating the sample Greater than the fluorescence lifetime of the fluorescent dye, the collected fluorescence signal contains the time information and spatial information of the fluorescence photon. The first image and the second image are separated from the fluorescence signal through data processing, and the first image and the second 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 method, the problem of pixel mismatch during intensity difference is solved. The use of low-power Gaussian pulsed laser reduces the damage to biological samples, reduces the photobleaching effect of fluorescent dyes, and prolongs the effective time of super-resolution imaging. Conducive to the realization of long-term live cell dynamic super-resolution imaging research, combined with image enhancement processing to obtain high-resolution images containing fine structural features, greatly improving the imaging quality of target super-resolution images, especially suitable for long-term observation and observation of biological samples Obtain high-resolution images, and achieved good technical results in the actual application process.

The embodiments of the present application also provide a fluorescence differential super-resolution imaging system, which can be used to implement any embodiment of the aforementioned fluorescence differential super-resolution imaging method. Specifically, please refer to FIGS. 8-9. FIG. 8 is a schematic diagram of a fluorescence differential super-resolution imaging system provided by an embodiment of the application, and FIG. 9 is a schematic block diagram of an imaging processing terminal provided by an embodiment of the application. The imaging system It 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 of the Gaussian pulse laser as a reference signal, and acquiring the fluorescent signal generated after the sample is irradiated.

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

Wherein, the laser 101 is used to emit Gaussian pulsed laser; the first beam splitter 102 is used to split the Gaussian pulsed laser to obtain two Gaussian pulsed lasers. The second optical path propagates, and another beam of the Gaussian pulsed laser is along the first optical path; the second beam splitter 103 is used to split the Gaussian pulsed laser that propagates along the second optical path, so that a part of the The Gaussian pulsed laser is incident on the second detector, and another part of the Gaussian pulsed laser is reflected and propagated to the third beam splitter; the spiral phase plate 107 is used to transfer all the pulses propagating along the first optical path. The Gaussian pulse laser is converted into a ring pulse laser and propagated to the corner reflector; the corner reflector 106 is used to reflect the incident ring pulse laser so that it propagates to the third beam splitter; The third beam splitter 104 is configured to reflect the ring pulse laser and transmit the Gaussian pulse laser propagating along the second optical path so that two laser beams can propagate along the same path; the dichroic mirror 105. Used to reflect the ring pulse laser and the Gaussian pulse laser propagating along the second optical path so that the two laser beams can propagate to the scanning galvanometer along the same path, and perform processing on the fluorescence signal Transmission; the scanning galvanometer 108 is used to synchronously scan the incident Gaussian pulsed laser and ring pulsed laser to realize the area array imaging of the sample; the objective lens 109 is used to focus the incident laser light and then irradiate the The sample; the stage 110 is used to place and fix the sample, and the sample is three-dimensionally moved; the first detector 112 is used to detect and collect the fluorescent photon signal emitted by the fluorescent dye after being irradiated by the laser The second detector 113 is used to detect the incident Gaussian pulsed laser to obtain the excitation light pulse signal; the preamplifier 111 is used to perform the fluorescence photon signal from the first detector Amplification and filtering; the time-correlated single photon counter (TCSPC) 114 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.

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 first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule; The image and the second 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 first image and the second image from the fluorescence signal according to the excitation light pulse signal and the preset segmentation rule; the image processing unit 220 is used to process the image according to the preset The first image and the second image are analyzed and processed by rules to obtain a high-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 (10)

1. A fluorescence differential super-resolution imaging method, applied to an imaging system, is 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;

   The Gaussian pulsed laser is emitted and split to obtain two Gaussian pulsed lasers. One of the Gaussian pulsed lasers propagates along the second optical path and then focuses on the sample, and the other Gaussian pulsed laser propagates along the first optical path. After being converted into a ring pulse laser, the sample is focused and irradiated, wherein the pulse interval between the ring pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is greater than the fluorescence lifetime of the fluorescent dye;

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

   Separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;

   The first image and the second image are analyzed and processed according to a preset image processing rule to obtain a high-resolution target super-resolution image.
2. The fluorescence differential super-resolution imaging method according to claim 1, wherein the Gaussian pulse laser propagating along the second optical path irradiates the sample before the ring pulse laser, and the first image is a confocal image The second image is a ring image, and the separation of the first image and the second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule includes:

   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;

   Determining the dividing point of the fluorescence attenuation curve according to the dividing rule;

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

   The annular image is formed according to the spatial information of the fluorescent photons located after the dividing point in the fluorescent signal.
3. The fluorescence differential super-resolution imaging method of claim 2, wherein the first image and the second image are analyzed and processed according to a preset image processing rule to obtain a high-resolution target Super-resolution images, including:

   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 confocal image to obtain the target super-resolution image.
4. The fluorescence differential super-resolution imaging method according to claim 2, wherein the segmentation rule includes a fluorescence intensity interval, a fluorescence intensity threshold, and a time threshold, and the segmentation point of the fluorescence attenuation curve is determined according to the segmentation rule ,include:

   Judging whether the fluorescence intensity of each time channel of the fluorescence decay curve is within the fluorescence intensity interval, and acquiring the time channel whose fluorescence intensity is within the fluorescence intensity interval as the first time channel;

   Judging whether the fluorescence intensity of the time channel separated from the first time channel by the time threshold in the fluorescence decay curve is less than the fluorescence intensity threshold;

   If the fluorescence intensity of the time channel separated from the first time channel by the time threshold is less than the fluorescence intensity threshold, use the first time channel as the cut-off time of the fluorescence lifetime detection;

   The time channel position corresponding to the midpoint of the start time and the end time is used as the division point.
5. The fluorescence differential super-resolution imaging method according to claim 1, wherein the Gaussian pulse laser propagating along the second optical path irradiates the sample before the ring pulse laser, and the first image is a ring image The second image is a confocal image, and the separation of the first image and the second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule includes:

   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;

   Determining the dividing point of the fluorescence attenuation curve according to the dividing rule;

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

   The confocal image is composed according to the spatial information of the fluorescent photons located after the dividing point in the fluorescent signal.
6. The fluorescence differential super-resolution imaging method of claim 5, wherein the first image and the second image are analyzed and processed according to a preset image processing rule to obtain a high-resolution target Super-resolution images, including:

   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 confocal image to obtain the target super-resolution image.
7. The fluorescence differential super-resolution imaging method according to claim 5, wherein the segmentation rule includes a fluorescence intensity threshold and an intensity difference threshold, and determining the segmentation point of the fluorescence attenuation curve according to the segmentation rule includes:

   Determine whether the fluorescence intensity of each time channel of the fluorescence decay curve is not less than the fluorescence intensity threshold, and obtain a curve segment in the fluorescence decay curve that is not less than the fluorescence intensity threshold as a target curve segment;

   Judging whether the absolute value of the fluorescence intensity difference between each target time channel and two adjacent time channels in the target curve segment is greater than the intensity difference threshold;

   If the absolute value of the fluorescence intensity difference between the target time channel and two adjacent time channels is greater than the intensity difference threshold, the position of the target time channel is used as the segmentation point.
8. A fluorescence differential super-resolution imaging system for realizing the fluorescence differential super-resolution imaging method according to any one of claims 1-7, wherein the imaging system includes a signal acquisition device and imaging processing terminal;

   The signal collection device is used to collect and obtain the excitation light pulse signal of the Gaussian pulsed laser as a reference signal, and collect the fluorescence 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.
9. The fluorescence differential super-resolution imaging system according to claim 8, wherein the imaging processing terminal comprises:

   A fluorescent signal segmentation unit for separating a first image and a second 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 first image and the second image according to preset image processing rules to obtain a high-resolution target super-resolution image.
10. The fluorescence differential super-resolution imaging system according to claim 8, wherein the signal acquisition device comprises a laser, a first beam splitter, a second beam splitter, a third beam splitter, a dichroic mirror, the corner reflector, Spiral phase plate, scanning galvanometer, objective lens, said stage, preamplifier, first detector, second detector, time-dependent single photon counter;

    The laser is used to emit Gaussian pulsed laser;

    The first beam splitter is used to split the Gaussian pulsed laser to obtain two Gaussian pulsed lasers. One of the Gaussian pulsed lasers propagates along the second optical path, and the other Gaussian pulsed laser travels along the path. Narrate the first optical path;

    The second beam splitter is used to split the Gaussian pulsed laser light propagating along the second optical path, so that a part of the Gaussian pulsed laser light enters the second detector, and the other part of the Gaussian pulsed laser light is reflected And spread to the third beam splitter;

    The spiral phase plate is used to convert the Gaussian pulse laser propagating along the first optical path into a ring pulse laser and propagate to the corner reflector;

    The corner reflector is used to reflect the incident ring pulse laser so that it can propagate to the third beam splitter;

    The third beam splitter is configured to reflect the ring pulse laser and transmit the Gaussian pulse laser propagating along the second optical path so that two laser beams can propagate along the same path;

    The dichroic mirror is used to reflect the ring pulse laser and the Gaussian pulse laser propagating along the second optical path so that the two laser beams can propagate to the scanning galvanometer along the same path, and to Fluorescence signal is transmitted;

    The scanning galvanometer is used to synchronously scan the incident Gaussian pulsed laser and ring pulsed 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 by the laser;

    The second detector is used to detect the incident Gaussian pulsed laser 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.

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