CN111521596B - Fluorescence differential super-resolution imaging method and imaging system - Google Patents
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Abstract
The invention discloses a fluorescence differential super-resolution imaging method and an imaging system. The position of a corner reflector in the system is adjusted to prolong or shorten the optical path of one beam of Gaussian pulse laser transmitted in a first optical path and convert the optical path into annular pulse laser to focus and irradiate a sample, the other beam of Gaussian pulse laser transmits and focuses and irradiates the sample along a second optical path, the pulse interval between the annular pulse laser and the Gaussian pulse laser irradiating the sample is longer than the fluorescence life of the fluorescent dye, and an excitation light pulse signal and a fluorescence signal are acquired by two detectors respectively. And separating the first image and the second image from the fluorescence signal, and analyzing and processing the first image and the second image according to an image processing rule to obtain a target super-resolution image with further improved resolution. By the method, the damage to the biological sample is reduced by adopting the low-power pulse laser, and the problem of the quality reduction of the super-resolution image caused by pixel mismatch in the traditional fluorescence differential super-resolution imaging is solved.
Description
Technical Field
The invention relates to the technical field of super-resolution optical microscopic imaging, in particular to a fluorescence differential super-resolution imaging method and an imaging system.
Background
The living cells and the tissue images are important for the research in the biomedical field, and besides the correct cell culture conditions and sample preparation methods, the advanced imaging method and the advanced imaging system can furthest ensure the authenticity and the effectiveness of the acquired information on the basis of keeping the biological characteristics of the observed sample. The optical microscope has the advantages of non-contact, no damage and specificity, is an important mark at the beginning of the development of recent natural science, and can be well applied to the imaging of living cells and tissues. However, diffraction of light limits the resolution capabilities of optical microscopes, making it impossible to clearly distinguish microscopic biological structures below 200nm in size. The Super-resolution optical microscope (SRM) technology inherits the advantages of non-contact and specificity of an optical microscope, and the resolution of the optical microscope is improved by 1-2 orders of magnitude by the physicochemical principle, so that the development and change rules of life can be understood at the molecular level, and the cell molecular mechanism of the anti-drug resistance and intervention treatment effect of organisms can be disclosed, which is one of the most important breakthroughs in the field of optical microscopy in the present century. In recent years, the rapid development of super-resolution optical imaging technology makes the connection between optical microscopes and the fields of biomedicine and the like more compact, but the existing technology has extremely strict requirements on samples (preparation) and fluorescent dyes, and the application of the technology in living body biological imaging is limited.
Hell, german scientist, stefanw, in 1994, proposed a Stimulated emission depletion (STED) microscopy technique according to einstein radiation theory. Based on the nonlinear relation between fluorescence saturation and excited state fluorescence excited radiation, the STED technology utilizes the second beam of laser with red shift of wavelength to selectively dissipate excited state molecules in advance, improves imaging resolution by compressing an effective point spread function of an excitation light spot, and theoretically can realize nanoscale resolution in a three-dimensional space. As a first theoretically proposed far-field super-resolution imaging method implemented in experiments, the STED technique has the advantages of fast imaging and no need for post-image reconstruction. However, to avoid the re-excitation effect affecting the super-resolution image quality, the evanescent laser wavelength is usually at the end of the emission spectrum of the fluorochromes. Because the stimulated emission cross section at the tail end of the emission spectrum is extremely small, the STED technology needs extremely high loss energy (generally, the loss energy is more than three orders of magnitude higher than that of the excitation light) to realize the improvement of the resolution. Too high a laser energy can cause photobleaching and phototoxicity, causing damage to fluorescent probes and biological tissues, thus limiting the application of this technique to live cell and tissue imaging. In 2013, the university of Zhejiang Confucian discipline teaches a Fluorescence Emission Difference (FED) super-resolution imaging method based on the idea of compressing the expansion function of the excitation light spot, and super-resolution imaging is realized at extremely low laser energy by subtracting a negative confocal image excited by an annular light spot from a confocal image excited by a Gaussian light spot. Because the stimulated radiation process is not involved, the method is simple and effective, and the imaging system is simple and low in cost. However, the fluorescence difference requires that two images are collected successively at the same position of the sample, and the switching between the imaging modes and the movement of the sample during imaging can cause pixel mismatch (i.e. the non-complete coincidence of the gaussian light spot and the annular light spot), thereby seriously reducing the quality of the super-resolution image, and therefore, the method is not suitable for long-time imaging research of biological samples such as living cells and the like, and cannot observe the biological samples for a long time and obtain high-resolution images.
Disclosure of Invention
The embodiment of the invention provides a fluorescence differential super-resolution imaging method and an imaging system, and aims to solve the problem that a biological sample cannot be observed for a long time and a high-quality super-resolution image cannot be obtained due to pixel mismatch in the conventional fluorescence differential super-resolution imaging method.
In a first aspect, an embodiment of the present invention provides a fluorescence differential super-resolution imaging method, which includes:
placing the sample stained with the fluorescent dye on an object stage and adjusting the position of the corner reflector in the first optical path;
emitting Gaussian pulse laser and splitting to obtain two beams of Gaussian pulse laser, wherein one beam of Gaussian pulse laser is transmitted along a second light path and then focuses and irradiates the sample, the other beam of Gaussian pulse laser is transmitted along the first light path and is converted into annular pulse laser and then focuses and irradiates the sample, and the pulse interval between the annular pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is longer than the fluorescence life of the fluorescent dye;
simultaneously collecting an excitation light pulse signal of the Gaussian pulse laser and a fluorescence signal generated after the sample is irradiated, wherein the fluorescence signal comprises time information and spatial information of fluorescence photons;
separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;
and analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image.
In a second aspect, an embodiment of the present invention provides a fluorescence differential super-resolution imaging system, which includes:
the system comprises a signal acquisition device and an imaging processing terminal;
the signal acquisition device is used for acquiring an excitation light pulse signal of the Gaussian pulse laser as a reference signal and acquiring a fluorescence signal generated after the sample is irradiated;
the imaging processing terminal is used for processing the excitation light pulse signal and the fluorescence signal acquired by the signal acquisition device to obtain the target super-resolution image.
The embodiment of the invention provides a fluorescence differential super-resolution imaging method and an imaging system. The method comprises the steps of extending or shortening the optical path of a Gaussian pulse laser beam transmitted in a first optical path by adjusting the position of a corner reflector arranged in the first optical path, converting the Gaussian pulse laser beam transmitted along the first optical path into annular pulse laser and then focusing to irradiate a sample, transmitting another Gaussian pulse laser beam along a second optical path to focus to irradiate the sample, wherein the pulse interval between the annular pulse laser beam for irradiating the sample and the Gaussian pulse laser beam for irradiating the sample is longer than the fluorescence life of a fluorescent dye, and the acquired fluorescence signal contains time information and space information of fluorescence photons. And separating a first image and a second image from the fluorescence signal through data processing, and analyzing and processing the first image and the second image according to an image processing rule to obtain a target super-resolution image with further improved resolution. By the method, the damage to the biological sample is reduced by adopting the low-power laser, the photobleaching effect of the fluorescent dye is reduced, the effective time of super-resolution imaging is prolonged, the high-resolution image containing the fine structure characteristics is obtained by combining image enhancement treatment, the method is particularly suitable for observing the biological sample for a long time and obtaining the high-resolution image, and a good technical effect is obtained in the practical application process.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic flow chart of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 2 is a schematic view of a sub-flow of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 3 is a schematic view of a sub-flow of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 4 is a schematic view of a sub-flow chart of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 5 is a schematic view of a sub-flow chart of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 6 is a schematic view of a sub-flow chart of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 7 is a schematic view of a sub-flow chart of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention;
FIG. 8 is a schematic diagram of a fluorescence differential super-resolution imaging system provided in an embodiment of the present invention;
fig. 9 is a schematic block diagram of an imaging processing terminal provided by an embodiment of the present invention;
FIG. 10 is a schematic diagram illustrating the effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention;
FIG. 11 is a schematic diagram illustrating the effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention;
FIG. 12 is a schematic diagram illustrating the effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention;
FIG. 13 is a schematic diagram illustrating the effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention;
fig. 14 is a schematic diagram illustrating an effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
Referring to fig. 1 and 8, fig. 1 is a schematic flow chart of a fluorescence differential super-resolution imaging method according to an embodiment of the present invention, and fig. 8 is a schematic view of a fluorescence differential super-resolution imaging system according to an embodiment of the present invention. The fluorescence differential super-resolution imaging method is applied to an imaging system, the imaging system comprises a signal acquisition device 10 and an imaging processing terminal 20, the method is executed by combining the signal acquisition device 10 with application software installed in the imaging processing terminal 20, the imaging system is a system device for executing the fluorescence differential super-resolution imaging method to realize high-resolution imaging on a sample, the signal acquisition device 10 is a device for emitting Gaussian pulse laser to detect the sample and acquiring an excitation light pulse signal and a fluorescence signal, and the imaging processing terminal 20 is terminal equipment, such as a workstation, a desktop computer, a notebook computer, a tablet computer or a mobile phone, for acquiring the excitation light pulse signal and the fluorescence signal acquired by the signal acquisition device and then performing imaging processing to obtain a target super-resolution image.
As shown in fig. 1, the method includes steps S110 to S150.
S110, placing the sample dyed by the fluorescent dye on a stage and adjusting the position of the corner reflector in the first light path.
A sample stained with a fluorescent dye is placed on the stage and the position of the corner reflector in the first optical path is adjusted. Firstly, a sample is dyed by adopting a fluorescent dye, specifically, the sample can be biological materials such as living cells, viruses or tissues, the fluorescent dye is a material which generates an autofluorescence signal after being irradiated by laser, a corner reflector is arranged in a first light path, Gaussian pulse laser can be transmitted along the first light path and a second light path respectively, and the position of the corner reflector can be adjusted to prolong or shorten the optical path of the Gaussian pulse laser in the first light path so as to change the optical path interval of the Gaussian pulse laser in the first light path and the second light path.
Fig. 14 is a schematic diagram illustrating the usage effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention, specifically, as shown in fig. 14, when the corner reflector is not adjusted, the corner reflector is located at position (i) in fig. 14, and the optical path of the gaussian pulse laser propagating along the first optical path (the time required for the light to propagate along a certain path for a certain distance) is τ1When the optical path interval between the first optical path and the second optical path is delta tau1(optical path interval is equal to the pulse interval time between the ring-shaped pulsed laser irradiating the sample and the gaussian pulsed laser irradiating the sample); adjusting the position of the corner reflector to the position II in the figure 14, wherein the distance between the position I and the position II is S, the optical path of the Gaussian pulse laser propagating along the first optical path is tau1+2S/c, where c is the speed of light, the optical path distance between the first and second optical paths is Δ τ2＝Δτ1+2S/c。
And S120, emitting Gaussian pulse laser and splitting light to obtain two beams of Gaussian pulse laser, wherein one beam of Gaussian pulse laser is transmitted along a second light path and then focuses and irradiates the sample, and the other beam of Gaussian pulse laser is transmitted along the first light path and is converted into annular pulse laser and then focuses and irradiates the sample.
And emitting Gaussian pulse laser and splitting to obtain two beams of Gaussian pulse laser, wherein one beam of Gaussian pulse laser is transmitted along a second light path and then focuses and irradiates the sample, the other beam of Gaussian pulse laser is transmitted along the first light path and is converted into annular pulse laser and then focuses and irradiates the sample, and the pulse interval between the annular pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is longer than the fluorescence life of the fluorescent dye.
Specifically, a spiral phase plate may be disposed in front of the corner reflector of the first optical path, the gaussian pulse laser propagated along the first optical path is converted into an annular pulse laser by the spiral phase plate, the annular pulse laser propagated along the first optical path and a beam of the gaussian pulse laser propagated along the second optical path are focused at different times and irradiate the sample, the dyed sample generates a fluorescent signal after being irradiated by the fluorescent dye, and the annular pulse laser may irradiate the sample before or after the gaussian pulse laser. The emitted laser is gaussian pulsed (e.g. at a pulse frequency of 80MHz), the frequency of the laser is inversely proportional to the pulse period of the laser, and the pulse period should at least include one complete autofluorescence process (usually in the order of nanoseconds and more). The longer the fluorescence lifetime, the larger the pulse period of the laser light and the smaller the frequency of the laser light. Wherein the power of the Gaussian pulse laser is related to the spectral characteristics of the luminescent material, and is usually 0.1-100 μ W. The larger the power of the gaussian pulse laser is, the smaller the central zero-intensity region of the annular light spot in the obtained annular image is, and the larger the peak intensity is, the larger the pulse interval between the annular pulse laser irradiating the sample and the gaussian pulse laser irradiating the sample needs to be larger than the fluorescence lifetime of the fluorescent dye. The pulse width of the gaussian pulse laser is in the order of hundred picoseconds, for example, the value range of the gaussian pulse laser may be 0.1-1 nanosecond, and in order to realize super-resolution imaging of a sample, the gaussian pulse laser needs to be controlled in the order of hundred picoseconds (100 picoseconds — 0.1 nanosecond).
S130, collecting an excitation light pulse signal of the Gaussian pulse laser and a fluorescence signal generated after the sample is irradiated, wherein the fluorescence signal comprises time information and spatial information of fluorescence photons.
Simultaneously acquiring an excitation light pulse signal of the Gaussian pulse laser and a fluorescence signal generated after a sample is irradiated, wherein the acquired excitation light pulse signal of the Gaussian pulse laser is used as a starting point of fluorescence life detection; the fluorescent dye generates a fluorescent photon signal through spontaneous radiation after being irradiated, the obtained fluorescent photon signal forms the fluorescent signal, the fluorescent signal comprises time information and space information of a fluorescent photon, the space information of the fluorescent photon is specific position information of the emitted fluorescent photon on a two-dimensional plane, the intensity of the fluorescent photon emitted by a fluorescent molecule is gradually reduced along with time in a single pulse period, and the time information of the fluorescent photon is the time information of the collected fluorescent photon reaching a detector relative to a reference signal.
And S140, separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule.
And the excitation light pulse signal and the fluorescence signal are respectively transmitted to an imaging processing terminal, and the excitation light pulse signal and the fluorescence signal are analyzed and processed by the imaging processing terminal to obtain a super-resolution image for high-resolution imaging of the sample. Specifically, the fluorescence signal is first divided according to a division rule and the excitation light pulse signal to obtain a first image and a second image. If the annular pulse laser irradiates the sample with the Gaussian pulse laser, the obtained first image is a confocal image, the second image is an annular image, the confocal image is a fluorescence life image generated by irradiating the sample with the Gaussian pulse laser, and the annular image is a fluorescence signal image generated by irradiating the sample with the Gaussian pulse laser and then irradiating the sample with the annular pulse laser; if the annular pulse laser irradiates the sample before the Gaussian pulse laser, the obtained first image is an annular image, the annular image is an annular fluorescence lifetime image generated by irradiating the sample through the annular pulse laser, and the second image is a confocal image.
In an embodiment, as shown in fig. 2, step S140 includes sub-steps S141, S142, S143, and S144.
In this embodiment, the annular pulse laser irradiates the sample with the gaussian pulse laser, the obtained first image is a confocal image, the second image is an annular image, and the segmentation rule includes a fluorescence intensity interval, a fluorescence intensity threshold, and a time threshold.
S141, taking the time point of the collected excitation light pulse signal as the initial time of fluorescence lifetime detection, and obtaining the intensity change of the fluorescence photon on a time channel to obtain a fluorescence attenuation curve of the fluorescence signal.
The time when the excitation light pulse signal is detected is used as the starting time of fluorescence lifetime detection, namely, the time is used as a zero point of a time channel, the intensity change of the fluorescence photon on the time channel is obtained according to the starting time, namely, the time is used as an abscissa, the intensity change of the fluorescence photon is obtained through photon number accumulation, one time channel is a period of unit time (for example, one time channel can be set to be 0.05 nanosecond), the ordinate is the intensity value of the fluorescence photon, the intensity of the fluorescence photon can be reflected through the number of the fluorescence photons collected through time accumulation in each time channel, the intensity of the fluorescence photon is higher when the number of the fluorescence photons in one time channel is larger, and the fluorescence attenuation curve of the fluorescence signal is obtained.
Fig. 10 is a schematic diagram illustrating an effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention. The experiment is carried out by adopting a fluorescent bead sample with the diameter of 23nm, the wavelength of Gaussian pulse laser is 635nm, the power is 35 mu W, the pulse frequency of the laser is 40MHz, the pulse width is 0.3 nanosecond (ns), and the optical path interval delta tau between the first optical path and the second optical path2At 12.5ns, the fluorescence decay curve of the obtained fluorescence signal is shown in FIG. 10 (a).
And S142, determining the segmentation point of the fluorescence attenuation curve according to the segmentation rule.
And determining the segmentation point of the fluorescence attenuation curve according to the segmentation rule. Specifically, the cutoff time of fluorescence lifetime detection can be determined according to a fluorescence attenuation curve and a division rule, a time channel position corresponding to a middle point of the start time and the end time is used as a division point, and a fluorescence signal is divided according to the division point.
In one embodiment, as shown in fig. 3, step S142 includes sub-steps S1421, S1422, S1423, and S1424.
S1421, judging whether the fluorescence intensity of each time channel of the fluorescence attenuation curve is within the fluorescence intensity interval, and acquiring the time channel with the fluorescence intensity within the fluorescence intensity interval as a first time channel; s1422, judging whether the fluorescence intensity of a time channel separated from the first time channel by the time threshold in the fluorescence attenuation curve is smaller 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 smaller than the fluorescence intensity threshold, taking the first time channel as the cutoff time of the fluorescence lifetime detection; s1424, taking the time channel position corresponding to the middle point of the starting time and the ending time as the dividing point.
Specifically, the fluorescence decay curve is composed of a plurality of points, each point is located in a time channel, and each point corresponds to a fluorescence intensity value. Whether the fluorescence intensity value of each time channel in the fluorescence attenuation curve is in the fluorescence intensity interval or not can be judged, the time channel with the fluorescence intensity in the fluorescence intensity interval is taken as a first time channel, the fluorescence intensity of the time channel with the time threshold value separated from the first time channel is obtained, whether the fluorescence intensity is smaller than the fluorescence intensity threshold value or not is judged, if the fluorescence intensity is smaller than the fluorescence intensity threshold value, the first time channel is taken as the cut-off time of fluorescence life detection, only one cut-off time is obtained according to the judgment method, and the position of the time channel corresponding to the middle point of the starting time and the ending time is taken as a dividing point.
For example, the fluorescence decay curve shown in FIG. 10 is determined to have a cutoff time of 25ns and an initial time of 0 using the method described above, and the midpoint between the initial time and the final time isτx12.5ns, a time-channel position corresponding to 12.5ns is taken as the division point.
S143, forming the confocal image according to the space information of the fluorescence photons in the fluorescence signals, wherein the fluorescence photons are located before the segmentation point.
And S144, forming the annular image according to the spatial information of the fluorescence photons behind the division point in the fluorescence signal.
And acquiring the spatial information of the fluorescence photons from the starting time to the division point in the fluorescence signal to form a confocal image, and acquiring the spatial information of the fluorescence photons from the time after the division point to the cut-off time in the fluorescence signal to form an annular image by taking the division point as a reference.
For example, as shown in fig. 10, after the obtained fluorescence signal is segmented, one confocal image of the sample is obtained as shown in fig. 10(B), and the corresponding one annular image is obtained as shown in fig. 10(c), wherein the confocal image can be named as image a, and the annular image can be named as image B.
S150, analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image.
The first image and the second image are analyzed and processed through the image processing rule, so that the resolution of imaging the sample can be greatly improved, and a target super-resolution image of the sample is obtained. The field of view of the confocal image and the annular image are the same (the image size is the same).
In one embodiment, as shown in FIG. 4, step S150 includes sub-steps S151 and S152.
And S151, multiplying the annular image by the enhancement coefficient in the image processing rule to obtain an enhanced annular image.
Specifically, the pixel value of each pixel in the ring image is multiplied by the enhancement coefficient to obtain a corresponding enhanced ring image. The enhancement coefficient is a coefficient value preset by a user, the value of the enhancement coefficient is greater than 1, and the enhancement coefficient can be an integer or a decimal.
Fig. 11 is a schematic diagram illustrating an effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention. For example, assuming that the enhancement coefficient is 1, the ring image is as shown in fig. 11(a), and the enhanced ring image (which may be expressed as 1 × B) obtained at this time is the same as the ring image B; taking the enhancement coefficient as 2, the enhanced ring image (which can be expressed as 2 × B) obtained at this time is shown in fig. 11 (B); taking the enhancement coefficient to be 4, the enhanced ring image (which can be expressed as 4 × B) obtained at this time is shown in fig. 11 (c).
S152, subtracting 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 has the same field of view (same image size) 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 annular image to obtain the pixel difference value of the pixel, and the pixel difference values of each pixel in the confocal image are obtained and combined to obtain a corresponding target super-resolution image.
For example, taking the enhancement coefficient as 1, the obtained enhanced ring image can be represented as 1 × B, and the target super-resolution image can be represented as a-1 × B, and the obtained target super-resolution image is shown in fig. 11 (d); taking the enhancement coefficient as 2, the obtained enhanced annular image can be expressed as 2 × B, the target super-resolution image can be expressed as a-2 × B, and the obtained target super-resolution image is shown in fig. 11 (e); taking the enhancement coefficient as 4, the resulting enhanced ring image can be represented as 4 × B, and the target super-resolution image can be represented as a-4 × B, and the resulting target super-resolution image is shown in fig. 11 (f).
In another embodiment, as shown in fig. 5, step S140 includes sub-steps S1401, S1402, S1403, and S1404.
In this embodiment, the annular pulse laser irradiates the sample before the gaussian pulse laser, the obtained first image is an annular image, the second image is a confocal image, and the segmentation rule includes a fluorescence intensity threshold and an intensity difference threshold.
S1401, the collected time point of the excitation light pulse signal is used as the initial time of fluorescence lifetime detection, and the intensity change of the fluorescence photon on a time channel is obtained, so as to obtain the fluorescence attenuation curve of the fluorescence signal.
This process is the same as S141 and will not be described herein.
Fig. 12 is a schematic diagram illustrating an effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention. The experiment is carried out by adopting a fluorescent bead sample with the diameter of 23nm, the wavelength of Gaussian pulse laser is 635nm, the power is 35 mu W, the pulse frequency of the laser is 80MHz, the pulse width is 0.3 nanosecond (ns), and the optical path interval delta tau between the first optical path and the second optical path2At 3 ns, the fluorescence decay curve of the obtained fluorescence signal is shown in FIG. 12 (a).
And S1402, determining the segmentation point of the fluorescence attenuation curve according to the segmentation rule.
And determining the segmentation point of the fluorescence attenuation curve according to the segmentation rule. Specifically, a corresponding one of the fluorescence attenuation curves may be determined as a segmentation point according to a segmentation rule.
In one embodiment, as shown in fig. 6, step S1402 includes sub-steps S14021, S14022 and S14023.
S14021, judging whether the fluorescence intensity of each time channel of the fluorescence attenuation curve is not less than the fluorescence intensity threshold value, and acquiring a curve segment not less than the fluorescence intensity threshold value in the fluorescence attenuation curve as a target curve segment; s14022, 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 larger than the intensity difference threshold value; s14023, if the absolute value of the fluorescence intensity difference between the target time channel and the two adjacent time channels is greater than the intensity difference threshold, taking the position of the target time channel as the dividing point.
Specifically, the fluorescence decay curve is composed of a plurality of points, each point is located in a time channel, and each point corresponds to a fluorescence intensity value. Whether the fluorescence intensity value of each time channel in the fluorescence attenuation curve is not less than the fluorescence intensity threshold value or not can be judged, the curve segment with the fluorescence intensity not less than the fluorescence intensity threshold value is used as a target curve segment, whether the absolute value of the fluorescence intensity difference value between each target time channel and two adjacent time channels in the target curve segment is larger than the intensity difference threshold value or not is judged, if so, the target time channel is used as a dividing point, and only one dividing point is obtained according to the judging method.
For example, the fluorescence decay curve shown in FIG. 12 is determined by the above method to correspond to a target time channel with a time τxIf' 4ns, the target time channel position is taken as the dividing point.
And S1403, forming the annular image according to the spatial information of the fluorescence photons positioned before the division point in the fluorescence signal.
And S1404, forming the confocal image according to the spatial information of the fluorescence photons behind the segmentation point in the fluorescence signal.
And taking the division point as a reference, acquiring the spatial information of the fluorescence photons from the starting time to the division point in the fluorescence signal to form an annular image, and acquiring the spatial information of the fluorescence photons from the time after the division point to the cut-off time in the fluorescence signal to form a confocal image.
For example, as shown in fig. 12, the obtained fluorescence signal is divided, and then one ring image of the sample is obtained as shown in fig. 12(b), and the corresponding one confocal image is obtained as shown in fig. 10 (c).
In this embodiment, as shown in fig. 7, step S150 includes sub-steps S1501 and S1502.
S1501, multiplying the annular image by the enhancement coefficient in the image processing rule to obtain an enhanced annular image.
Specifically, the pixel value of each pixel in the ring image is multiplied by the enhancement coefficient to obtain a corresponding enhanced ring image. The enhancement coefficient is a coefficient value preset by a user, the value of the enhancement coefficient is greater than 1, and the enhancement coefficient can be an integer or a decimal.
Fig. 13 is a schematic diagram illustrating an effect of the fluorescence differential super-resolution imaging method according to the embodiment of the present invention. For example, assuming that the enhancement coefficient is 1, the ring image is as shown in fig. 13(a), and the enhanced ring image (which may be expressed as 1 × B) obtained at this time is the same as the ring image B; taking the enhancement coefficient to be 1.25, the enhanced ring image (which can be expressed as 1.25 × B) obtained at this time is shown in fig. 13 (B); the enhanced ring image (which can be expressed as 1.5 × B) obtained at this time is shown in fig. 13(c) with an enhancement coefficient of 1.5.
S1502, subtracting 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 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 annular image to obtain the pixel difference value of the pixel, and the pixel difference values of each pixel in the confocal image are obtained and combined to obtain a corresponding target super-resolution image.
For example, taking the enhancement coefficient as 1, the target super-resolution image can be represented as a-1 × B, and the obtained target super-resolution image is shown in fig. 13 (d); taking the enhancement coefficient as 1.25, and representing the target super-resolution image as A-1.25 multiplied by B, wherein the obtained target super-resolution image is shown in FIG. 13 (e); taking the enhancement coefficient as 1.5, the target super-resolution image can be represented as A-1.5 × B, and the target super-resolution image obtained at this time is shown in FIG. 13 (f).
The fluorescence differential super-resolution imaging method provided by the embodiment of the invention is characterized in that the optical path of a Gaussian pulse laser beam propagating in a first optical path is prolonged or shortened by adjusting the position of a corner reflector arranged in the first optical path, the Gaussian pulse laser beam propagating along the first optical path is converted into annular pulse laser and then focused to irradiate a sample, the other Gaussian pulse laser beam propagates along a second optical path and focuses to irradiate the sample, the pulse interval between the annular pulse laser beam irradiating the sample and the Gaussian pulse laser beam irradiating the sample is longer than the fluorescence life of a fluorescent dye, and the acquired fluorescence signal comprises time information and space information of fluorescence photons. And separating a first image and a second image from the fluorescence signal through data processing, and analyzing and processing the first image and the second image according to an image processing rule to obtain a target super-resolution image with further improved resolution. By the method, the problem of pixel mismatch when the intensity is different is solved, the damage to the biological sample is reduced by adopting low-power Gaussian pulse laser, the photobleaching effect of fluorescent dye is reduced, the effective time of super-resolution imaging is prolonged, the long-time living cell dynamic super-resolution imaging research is facilitated, a high-resolution image containing fine structure characteristics is obtained by combining image enhancement processing, the imaging quality of a target super-resolution image is greatly improved, the method is particularly suitable for observing the biological sample for a long time and obtaining the high-resolution image, and a good technical effect is achieved in the practical application process.
The embodiment of the invention also provides a fluorescence differential super-resolution imaging system which can be used for realizing any embodiment of the fluorescence differential super-resolution imaging method. Specifically, referring to fig. 8 to 9, fig. 8 is a schematic diagram of a fluorescence differential super-resolution imaging system according to an embodiment of the present invention, and fig. 9 is a schematic block diagram of an imaging processing terminal according to an embodiment of the present invention, where the imaging system includes a signal acquisition device 10 and an imaging processing terminal 20.
The signal collecting device 10 is configured to collect an excitation light pulse signal of the gaussian pulse laser as a reference signal, and collect a fluorescence signal generated after the sample is irradiated.
Specifically, the signal acquisition device includes a laser 101, a first spectroscope 102, a second spectroscope 103, a third spectroscope 104, a dichroic mirror 105, the corner reflector 106, a spiral phase plate 107, a scanning galvanometer 108, an objective lens 109, the stage 110, a preamplifier 111, a first detector 112, a second detector 113, and a time-dependent single photon counter 114.
Wherein, the laser 101 is used for emitting Gaussian pulse laser; the first beam splitter 102 is configured to split the gaussian pulse laser to obtain two beams of gaussian pulse laser, where one beam of gaussian pulse laser propagates along the second optical path, and another beam of gaussian pulse laser propagates along the first optical path; the second beam splitter 103 is configured to split the gaussian pulse laser beam propagating along the second optical path, so that a part of the gaussian pulse laser beam enters the second detector, and another part of the gaussian pulse laser beam is reflected and propagates to the third beam splitter; the spiral phase plate 107 is configured to convert the gaussian pulse laser propagating along the first optical path into a ring pulse laser and propagate the ring pulse laser to the corner reflector; the corner reflector 106 is used for reflecting the incident annular pulse laser so as to enable the annular pulse laser to propagate to the third beam splitter; the third beam splitter 104 is configured to reflect the annular pulse laser and transmit the gaussian pulse laser propagating along the second optical path, so that two beams of laser may propagate along the same path; the dichroic mirror 105 is configured to reflect the annular pulse laser and the gaussian pulse laser propagating along the second optical path, so that two laser beams can propagate to the scanning galvanometer along the same path, and transmit the fluorescent signal; the scanning galvanometer 108 is used for synchronously scanning incident Gaussian pulse laser and annular pulse laser to realize area array imaging of a sample; the objective lens 109 is used for focusing the incident laser and then irradiating the sample; the stage 110 for placing and fixing a sample and moving the sample in three dimensions; the first detector 112 is used for detecting and collecting fluorescence photon signals emitted by the fluorescent dye after being irradiated by laser; the second detector 113 is configured to detect incident gaussian pulse laser to obtain the excitation light pulse signal; the preamplifier 111 is used for amplifying and filtering the fluorescence photon signal from the first detector; 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 includes time information and spatial information of fluorescence photons.
The imaging processing terminal 20 is configured to process the excitation light pulse signal and the fluorescence signal acquired by the signal acquisition device to obtain the target super-resolution image.
The imaging processing terminal 20 is a terminal device, such as a workstation, a desktop computer, a notebook computer, a tablet computer, or a mobile phone, for obtaining the excitation light pulse signal and the fluorescence signal acquired by the signal acquisition device and then performing imaging processing to obtain the super-resolution image of the target.
The imaging processing terminal 20 may perform the following steps: separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule; and analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image.
In one embodiment, as shown in fig. 6, the imaging processing terminal 20 includes a fluorescence signal dividing unit 210 and an image processing unit 220.
A fluorescence signal dividing unit 210 for separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset dividing rule; the image processing unit 220 is configured to analyze and process the first image and the second image according to a preset image processing rule, so as to obtain a high-resolution target super-resolution image.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (5)
1. A fluorescence differential super-resolution imaging method is applied to an imaging system and is characterized by comprising the following steps:
placing the sample stained with the fluorescent dye on an object stage and adjusting the position of the corner reflector in the first optical path;
emitting Gaussian pulse laser and splitting to obtain two beams of Gaussian pulse laser, wherein one beam of Gaussian pulse laser is transmitted along a second light path and then focuses and irradiates the sample, the other beam of Gaussian pulse laser is transmitted along the first light path and is converted into annular pulse laser and then focuses and irradiates the sample, and the pulse interval between the annular pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is longer than the fluorescence life of the fluorescent dye;
simultaneously collecting an excitation light pulse signal of the Gaussian pulse laser and a fluorescence signal generated after the sample is irradiated, wherein the fluorescence signal comprises time information and spatial information of fluorescence photons;
separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;
analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image;
the gaussian pulse laser propagating along the second optical path irradiates the sample before the annular pulse laser, the first image is a confocal image, the second image is an annular image, and the first image and the second image are separated from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule, including:
taking the time point of the collected excitation light pulse signal as the initial time of fluorescence lifetime detection, and obtaining the intensity change of the fluorescence photon on a time channel to obtain a fluorescence attenuation curve of the fluorescence signal; determining a segmentation point of the fluorescence attenuation curve according to the segmentation rule; forming the confocal image according to the spatial information of the fluorescence photons in the fluorescence signal before the division point; forming the annular image according to the spatial information of the fluorescence photons behind the division point in the fluorescence signal;
the analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image comprises the following steps:
multiplying the annular image by an enhancement coefficient in the image processing rule to obtain an enhanced annular image; subtracting the intensity value of the enhanced annular image from the intensity value of the confocal image to obtain a target super-resolution image;
the segmentation rule comprises a fluorescence intensity interval, a fluorescence intensity threshold and a time threshold, and the determination of the segmentation point of the fluorescence attenuation curve according to the segmentation rule comprises the following steps:
judging whether the fluorescence intensity of each time channel of the fluorescence attenuation curve is in the fluorescence intensity interval or not, and acquiring the time channel with the fluorescence intensity in the fluorescence intensity interval as a first time channel; judging whether the fluorescence intensity of a time channel separated from the first time channel by the time threshold value in the fluorescence attenuation curve is smaller than the fluorescence intensity threshold value; if the fluorescence intensity of the time channel separated from the first time channel by the time threshold is smaller than the fluorescence intensity threshold, taking the first time channel as the cutoff time of the fluorescence lifetime detection; and taking the time channel position corresponding to the middle point of the starting time and the ending time as the dividing point.
2. A fluorescence differential super-resolution imaging method is applied to an imaging system and is characterized by comprising the following steps:
placing the sample stained with the fluorescent dye on an object stage and adjusting the position of the corner reflector in the first optical path;
emitting Gaussian pulse laser and splitting to obtain two beams of Gaussian pulse laser, wherein one beam of Gaussian pulse laser is transmitted along a second light path and then focuses and irradiates the sample, the other beam of Gaussian pulse laser is transmitted along the first light path and is converted into annular pulse laser and then focuses and irradiates the sample, and the pulse interval between the annular pulse laser irradiating the sample and the Gaussian pulse laser irradiating the sample is longer than the fluorescence life of the fluorescent dye;
simultaneously collecting an excitation light pulse signal of the Gaussian pulse laser and a fluorescence signal generated after the sample is irradiated, wherein the fluorescence signal comprises time information and spatial information of fluorescence photons;
separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule;
analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image;
the gaussian pulse laser propagating along the second optical path irradiates the sample before the annular pulse laser, the first image is an annular image, the second image is a confocal image, and the first image and the second image are separated from the fluorescence signal according to the excitation light pulse signal and a preset segmentation rule, including:
taking the time point of the collected excitation light pulse signal as the initial time of fluorescence lifetime detection, and obtaining the intensity change of the fluorescence photon on a time channel to obtain a fluorescence attenuation curve of the fluorescence signal; determining a segmentation point of the fluorescence attenuation curve according to the segmentation rule; forming the annular image according to the spatial information of the fluorescence photons in the fluorescence signal before the division point; forming the confocal image according to the spatial information of the fluorescence photons behind the segmentation point in the fluorescence signal;
the analyzing and processing the first image and the second image according to a preset image processing rule to obtain a high-resolution target super-resolution image comprises the following steps:
multiplying the annular image by an enhancement coefficient in the image processing rule to obtain an enhanced annular image; subtracting the intensity value of the enhanced annular image from the intensity value of the confocal image to obtain a target super-resolution image;
the segmentation rule comprises a fluorescence intensity threshold value and an intensity difference threshold value, and the segmentation point of the fluorescence attenuation curve is determined according to the segmentation rule, and the method comprises the following steps:
judging whether the fluorescence intensity of each time channel of the fluorescence attenuation curve is not less than the fluorescence intensity threshold value or not, and acquiring a curve segment which is not less than the fluorescence intensity threshold value in the fluorescence attenuation curve 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 larger than the intensity difference threshold value or not; and if the absolute value of the fluorescence intensity difference between the target time channel and the two adjacent time channels is greater than the intensity difference threshold, taking the position of the target time channel as the dividing point.
3. A fluorescence differential super-resolution imaging system, which is used for implementing the fluorescence differential super-resolution imaging method according to any one of claims 1-2, wherein the imaging system comprises a signal acquisition device and an imaging processing terminal;
the signal acquisition device is used for acquiring an excitation light pulse signal of the Gaussian pulse laser as a reference signal and acquiring a fluorescence signal generated after the sample is irradiated;
the imaging processing terminal is used for processing the excitation light pulse signal and the fluorescence signal acquired by the signal acquisition device to obtain the target super-resolution image.
4. The fluorescence differential super-resolution imaging system according to claim 3, wherein the imaging processing terminal comprises:
a fluorescence signal dividing unit for separating a first image and a second image from the fluorescence signal according to the excitation light pulse signal and a preset dividing rule;
and the image processing unit is used for analyzing and processing the first image and the second image according to a preset image processing rule so as to obtain a high-resolution target super-resolution image.
5. The fluorescence differential super-resolution imaging system according to claim 3, wherein the signal acquisition device comprises a laser, a first spectroscope, a second spectroscope, a third spectroscope, a dichroic mirror, the corner reflector, a spiral phase plate, a scanning galvanometer, an objective lens, the objective table, a preamplifier, a first detector, a second detector, a time-dependent single photon counter;
the laser is used for emitting Gaussian pulse laser;
the first spectroscope is used for splitting the Gaussian pulse laser to obtain two beams of Gaussian pulse laser, wherein one beam of Gaussian pulse laser propagates along the second light path, and the other beam of Gaussian pulse laser propagates along the first light path;
the second beam splitter is configured to split the gaussian pulse laser beam propagating along the second optical path, so that a part of the gaussian pulse laser beam is incident to the second detector, and another part of the gaussian pulse laser beam is reflected and propagates to the third beam splitter;
the spiral phase plate is used for converting the Gaussian pulse laser propagating along the first optical path into annular pulse laser and propagating the annular pulse laser to the corner reflector;
the corner reflector is used for reflecting the incident annular pulse laser so as to enable the annular pulse laser to propagate to the third beam splitter;
the third beam splitter is used for reflecting the annular pulse laser and transmitting the Gaussian pulse laser transmitted along the second light path so that two beams of laser can be transmitted along the same path;
the dichroic mirror is used for reflecting the annular pulse laser and the Gaussian pulse laser transmitted along the second optical path so that two beams of laser can be transmitted to the scanning galvanometer along the same path and transmitting the fluorescent signal;
the scanning galvanometer is used for synchronously scanning incident Gaussian pulse laser and annular pulse laser to realize area array imaging of the sample;
the objective lens is used for focusing the incident laser and then irradiating the sample;
the object stage is used for placing and fixing a sample and three-dimensionally moving the sample;
the first detector is used for detecting and collecting fluorescence photon signals emitted by the fluorescent dye after being irradiated by laser;
the second detector is used for detecting the incident Gaussian pulse laser to obtain the excitation light pulse signal;
the preamplifier is used for amplifying and filtering the fluorescence photon signal from the first detector;
the time correlation single photon counter is used for signal storage and fluorescence lifetime imaging to obtain the fluorescence signal, wherein the fluorescence signal comprises time information and spatial information of fluorescence photons.
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