CN108982460B - Super-resolution imaging method and device and terminal equipment - Google Patents
Super-resolution imaging method and device and terminal equipment Download PDFInfo
- Publication number
- CN108982460B CN108982460B CN201810958217.1A CN201810958217A CN108982460B CN 108982460 B CN108982460 B CN 108982460B CN 201810958217 A CN201810958217 A CN 201810958217A CN 108982460 B CN108982460 B CN 108982460B
- Authority
- CN
- China
- Prior art keywords
- fluorescence intensity
- wavelength signal
- short
- super
- long
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention is suitable for the technical field of image processing, and provides a super-resolution imaging method, a super-resolution imaging device and terminal equipment, wherein the method comprises the following steps: exciting a fluorescent probe with double emission peaks on a biological sample by exciting light, and acting an affinity reagent on the excited fluorescent probe; irradiating the fluorescent probe acted by the affinity reagent by activated light to obtain a two-channel fluorescence intensity image; respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in a dual-channel fluorescence intensity image; selecting N frames of double-channel fluorescence intensity images, and respectively calculating the fluorescence intensity ratio of the short-wavelength signal and the long-wavelength signal to obtain N proportional fluorescence images; obtaining a super-resolution image by a STORM super-resolution imaging method; and coloring the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image. The invention can obtain a proportional super-resolution image and reflect the sample parameters of the fluorescent probe mark position in the biological sample.
Description
Technical Field
The invention relates to the technical field of image processing, in particular to a super-resolution imaging method, a super-resolution imaging device and terminal equipment.
Background
Fluorescence microscopy is widely used for cellular microbial imaging, with super-resolution localized imaging being one representative super-resolution fluorescence imaging technique. The technology combines single molecule imaging with a high-precision molecular positioning algorithm on the basis of an optical reconstruction microscope, and realizes the ultrahigh spatial resolution of 20-30 nm so as to observe the ultrastructure in cells. The key point of the technology is that the fluorescence probe in an excited state is randomly combined with the affinity reagent in the surrounding environment to cause the fluorescence to be attenuated to enter a dark state, the affinity reagent is randomly dropped off from the fluorescent molecule by using the activation light with another wavelength or the same wavelength to have the luminous capability again, so that the fluorescent molecule is randomly changed between the luminous state and the dark state, then multi-frame images of the fluorescent molecule are continuously recorded, a single-molecule positioning algorithm is carried out to determine the central position, and finally the super-resolution fluorescence image is reconstructed through the images.
However, the current optical reconstruction microscope can only perform super-resolution imaging on the structure of a biological sample, and cannot perform functional super-resolution imaging or super-resolution imaging research on sample parameter quantification.
Disclosure of Invention
The invention mainly aims to provide a super-resolution imaging method, a super-resolution imaging device and terminal equipment, which are used for solving the problems that in the prior art, only super-resolution imaging can be carried out on the structure of a biological sample, and functional super-resolution imaging or sample parameter quantitative super-resolution imaging research cannot be carried out.
In order to achieve the above object, a first aspect of embodiments of the present invention provides a super-resolution imaging method, including:
exciting a fluorescent probe with double emission peaks on a biological sample by exciting light, and acting an affinity reagent on the excited fluorescent probe;
irradiating the fluorescent probe acted by the affinity reagent by activated light to form a two-channel fluorescence intensity image;
respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image;
selecting N frames of double-channel fluorescence intensity images, selecting the fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal from the double-channel fluorescence intensity images of the same frame according to the respectively collected short-wavelength signal and long-wavelength signal, and respectively calculating fluorescence intensity ratios of the fluorescence intensity images to obtain N proportional fluorescence images, wherein N is a positive integer;
reconstructing the proportional fluorescence image by a STORM (storage Optical Reconstruction microscope) super-resolution imaging method to obtain a super-resolution image;
and coloring the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image.
With reference to the first aspect of the present invention, in a first implementation manner of the first aspect of the present invention, the super-resolution imaging method further includes:
establishing a function model of the ratio of the environmental parameter of the fluorescent probe to the fluorescence intensity;
the establishing of the function model of the ratio of the environmental parameter of the fluorescent probe to the fluorescence intensity comprises the following steps:
placing the fluorescent probe with the double emission peaks in a probe solution test environment, and exciting the fluorescent probe by exciting light;
changing a first parameter of the probe solution testing environment to obtain a steady-state fluorescence emission spectrum of the fluorescent probe under different first parameters;
and calculating a fluorescence intensity test ratio according to the double emission peaks in the steady-state fluorescence emission spectrum, and establishing a function model of the first parameter and the fluorescence intensity test ratio.
In combination with the first aspect of the present invention, in a second embodiment of the first aspect of the present invention, the irradiating the fluorescent probes acted on by the affinity reagent with the activating light to form a two-channel fluorescence intensity image includes:
obtaining the short wavelength signal according to a first structure in the fluorescent probe;
obtaining the long wavelength signal according to a second structure in the fluorescent probe;
when the exciting light irradiates the fluorescent probe, the first structure or the second structure emits fluorescence;
when the activation light acts on the fluorescent probe acted by the affinity reagent, the first structure and the second structure alternately emit light according to the binding state of the first structure and the affinity reagent to form the dual-channel fluorescence intensity image.
In combination with the second embodiment of the first aspect of the present invention, in the third embodiment of the first aspect of the present invention, the two-channel fluorescence intensity image comprises a fluorescence intensity image of a short wavelength signal and a fluorescence intensity image of a long wavelength signal;
the fluorescence intensity image of the short-wavelength signal corresponds to the luminescence process of the first structure in the fluorescent probe;
the fluorescence intensity image of the long wavelength signal corresponds to a luminescence process of the second structure in the fluorescent probe.
In a fourth embodiment of the first aspect of the present invention, in combination with the first aspect of the present invention, the separately acquiring the short-wavelength signal of the short-wavelength emission peak and the long-wavelength signal of the long-wavelength emission peak in the two-channel fluorescence intensity image includes:
separating the excitation light from the short wavelength signal and the long wavelength signal by a first dichroic mirror;
after the short-wavelength signal and the long-wavelength signal are separated through a second dichroic mirror, the short-wavelength signal is sent to a reflector through a first filter, and the reflector transmits the filtered short-wavelength signal to a first EMCCD; the long wavelength signal passes through a second filter, and the filtered long wavelength signal is transmitted to the first EMCCD, thereby collecting the two-channel fluorescence intensity image.
Different regions of the first EMCCD receive the filtered short wavelength signal and the filtered long wavelength signal, respectively.
With reference to the first aspect of the present invention, in a fifth embodiment of the first aspect of the present invention, the separately collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the fluorescence emission spectrum includes:
separating the excitation light from the short wavelength signal and the long wavelength signal by a first dichroic mirror;
after the short-wavelength signal and the long-wavelength signal are separated through a second dichroic mirror, the short-wavelength signal is sent to a reflector through a first filter, and the reflector transmits the filtered short-wavelength signal to a first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to a second EMCCD;
the first and second EMCCDs receive the filtered short wavelength signal and the filtered long wavelength signal, respectively, to collect the two-channel fluorescence intensity image.
With reference to the first to fifth embodiments of the first aspect of the present invention, in a sixth embodiment of the first aspect of the present invention, the selecting N frames of dual-channel fluorescence intensity images, selecting a fluorescence intensity image of the short-wavelength signal and a fluorescence intensity image of the long-wavelength signal from the separately acquired short-wavelength signal and long-wavelength signal in the dual-channel fluorescence intensity image of the same frame, and separately calculating fluorescence intensity ratios thereof to obtain N proportional fluorescence images, where N is a positive integer includes:
selecting the fluorescence intensity image of the Nth short-wavelength signal according to the two-channel fluorescence intensity image and the respectively collected short-wavelength signals, and calculating the fluorescence intensity of an emission peak of the Nth short-wavelength signal;
selecting a fluorescence intensity image of the Nth long-wavelength signal according to the two-channel fluorescence intensity image and the respectively collected long-wavelength signals, and calculating the fluorescence intensity of an emission peak of the fluorescence intensity image;
the calculation formula for calculating the fluorescence intensity ratio is as follows:
wherein, IBNIs the emission peak fluorescence intensity of the long wavelength signal, IANIs the emission peak fluorescence intensity of the short wavelength signal.
A second aspect of the present invention provides a super-resolution imaging apparatus, comprising:
the excitation module is used for exciting the fluorescent probe with double emission peaks on the biological sample by exciting light and enabling the affinity reagent to act on the excited fluorescent probe;
the fluorescence intensity image acquisition module is used for irradiating the fluorescent probe acted by the affinity reagent by activated light to obtain a two-channel fluorescence intensity image;
the signal acquisition module is used for respectively acquiring a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image;
the proportional fluorescence image calculation module is used for selecting N frames of double-channel fluorescence intensity images, selecting the fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal from the double-channel fluorescence intensity images of the same frame according to the respectively collected short-wavelength signal and long-wavelength signal, and respectively calculating the fluorescence intensity ratios of the fluorescence intensity images to obtain N proportional fluorescence images, wherein N is a positive integer, and N is a positive integer;
the image reconstruction module is used for reconstructing the proportional fluorescence image by a STORM super-resolution imaging method to obtain a super-resolution image;
and the image acquisition module is used for coloring the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image.
A third aspect of the embodiments of the present invention provides a terminal device for propagating source selection in a complex network, including a memory, a processor, and a computer program stored in the memory and executable on the processor, where the processor implements the steps of the method provided in the first aspect when executing the computer program.
A fourth aspect of embodiments of the present invention provides a computer-readable storage medium storing a computer program which, when executed by a processor, implements the steps of the method as provided in the first aspect above.
The super-resolution imaging method provided by the embodiment of the invention uses the fluorescent probe with double emission peaks to mark a biological sample, and reconstructs a super-resolution image by a STORM super-resolution imaging method after the fluorescent probe is excited, because the fluorescent probe can generate fluorescent signals with double emission peaks after being excited, namely a short-wavelength signal with a short-wavelength emission peak and a long-wavelength signal with a long-wavelength emission peak, a dual-channel fluorescence intensity image can be formed when the fluorescent probe acts through a nucleophilic agent and activation light, and then the fluorescent signals are respectively collected by an optical signal collecting device, the short-wavelength signal with the short-wavelength emission peak and the long-wavelength signal with the long-wavelength emission peak, and the ratio of the fluorescence intensity of the short-wavelength signal and the fluorescence intensity of the long-wavelength signal emission peak can be calculated according to the dual-channel fluorescence intensity image at the same time, so, and finally, coloring the super-resolution image according to the proportional fluorescent image to obtain a proportional super-resolution image. The proportional super-resolution image obtained by the super-resolution imaging method provided by the embodiment of the invention not only can reflect the structure of the biological sample, but also can reflect the sample parameters at the fluorescent probe mark position in the biological sample through the fluorescence intensity ratio, thereby analyzing the functions of the biological sample according to the sample parameters.
Drawings
Fig. 1 is a schematic flow chart of an implementation of a super-resolution imaging method according to an embodiment of the present invention;
fig. 2 is a schematic diagram illustrating a detailed implementation flow of S103 in fig. 1 according to a second embodiment of the present invention;
FIG. 3 is a schematic diagram of the alternative luminescence of the fluorescent probe according to the second embodiment of the present invention;
fig. 4 is a schematic flowchart illustrating a detailed implementation process of S104 in fig. 1 according to a third embodiment of the present invention;
FIG. 5 is a schematic diagram of an apparatus for carrying out the steps of FIG. 2;
fig. 6 is a diagram illustrating an effect of the super-resolution imaging method according to the fourth embodiment of the present invention;
fig. 7 is a schematic structural diagram of a super-resolution imaging apparatus according to a sixth embodiment of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Suffixes such as "module", "part", or "unit" used to denote elements are used herein only for the convenience of description of the present invention, and have no specific meaning in themselves. Thus, "module" and "component" may be used in a mixture.
In the following description, the serial numbers of the embodiments of the invention are merely for description and do not represent the merits of the embodiments.
Example one
As shown in fig. 1, an embodiment of the present invention provides a super-resolution imaging method, which can be applied to super-resolution positioning imaging of an optical reconstruction microscope, and includes:
s101, exciting the fluorescent probe with double emission peaks on the biological sample by exciting light, and acting the affinity reagent on the excited fluorescent probe.
In the above step S101, after the fluorescent probe having the dual emission peaks is excited, the fluorescent emission spectrum of the fluorescent probe will show two emission peaks, i.e. a short wavelength emission peak and a long wavelength emission peak.
In a specific application, before the excitation light acts on the biological sample, the excitation light should also pass through an optical processing element, such as a lens, a field stop, a tube lens, an objective lens, etc., so that the excitation light can uniformly and intensively irradiate on the biological sample, thereby exciting the fluorescent probe.
S102, irradiating the fluorescent probe acted by the affinity reagent by activated light to form a two-channel fluorescence intensity image.
In step S102, the affinity reagent does not bind to the probe, and when the structure in the fluorescent probe is covered with the affinity reagent, the structure does not emit light, and when the affinity reagent falls off from the structure, the structure has a light-emitting capability again.
In embodiments of the invention, the affinity reagent is reversible, i.e., under certain conditions, the affinity reagent is capable of inter-structural transfer in the fluorescent probe.
In a specific application, the activating light and the exciting light may have the same frequency or different frequencies, and are not specifically limited in the embodiment of the present invention.
S103, respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image.
In the above step S103, since the fluorescent probe after the action of the reagent is still a fluorescent probe having a double emission peak, the fluorescence emission spectrum obtained in the step S103 still has a double emission peak. When the activated light irradiates the fluorescent probe acted by the affinity reagent, the affinity reagent can be transferred from one structure in the fluorescent probe to the other structure in the fluorescent probe, and the two structures emit light alternately; therefore, from the structure of alternately emitting light, a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak can be obtained separately in the resulting fluorescence emission spectrum.
In a specific application, the signal in the fluorescence emission spectrum may be collected by any spectrum analyzer or combination of optical components, such as an EMCCD, which is not specifically limited in the embodiments of the present invention.
S104, selecting N frames of double-channel fluorescence intensity images, selecting the fluorescence intensity images of the short-wavelength signals and the fluorescence intensity images of the long-wavelength signals from the double-channel fluorescence intensity images of the same frame according to the respectively collected short-wavelength signals and long-wavelength signals, and respectively calculating fluorescence intensity ratios of the fluorescence intensity images to obtain N proportional fluorescence images, wherein N is a positive integer.
In the step S104, the fluorescence intensity images corresponding to the short wavelength signal and the long wavelength signal in the two-channel fluorescence intensity image of the same frame are selected, and the fluorescence intensity ratio is calculated respectively to obtain N proportional fluorescence images, which are equivalent to multi-frame images in which the fluorescence probe flickers are continuously recorded within a period of time, where the nth proportional fluorescence image is an nth two-channel fluorescence intensity image, and can reflect the fluorescence intensity images corresponding to the short wavelength signal and the long wavelength signal with the same time, and the nth proportional fluorescence image and the (N + 1) th proportional fluorescence image may be two adjacent frames of images or two non-adjacent frames of images.
The embodiment of the present invention further provides a detailed implementation step of calculating the fluorescence intensity ratio in step S104, which includes:
selecting the fluorescence intensity image of the Nth short-wavelength signal according to the two-channel fluorescence intensity image and the respectively collected short-wavelength signals, and calculating the fluorescence intensity of an emission peak of the Nth short-wavelength signal;
selecting a fluorescence intensity image of the Nth long-wavelength signal according to the two-channel fluorescence intensity image and the respectively collected long-wavelength signals, and calculating the fluorescence intensity of an emission peak of the fluorescence intensity image;
the calculation formula for calculating the fluorescence intensity ratio is as follows:
wherein, IBNIs the emission peak fluorescence intensity of the long wavelength signal, IANIs the emission peak fluorescence intensity of the short wavelength signal.
In a particular application, a larger number of scaled fluorescence images (e.g., 1000 scaled fluorescence images) may be needed as a basis for image reconstruction.
The embodiment of the invention also provides a super-resolution imaging method, which is used for establishing a function model of the ratio of the environmental parameter of the fluorescent probe to the fluorescence intensity;
the establishing of the function model of the ratio of the environmental parameter of the fluorescent probe to the fluorescence intensity comprises the following steps:
placing the fluorescent probe with the double emission peaks in a probe solution test environment, and exciting the fluorescent probe by exciting light;
changing a first parameter of the probe solution testing environment to obtain a steady-state fluorescence emission spectrum of the fluorescent probe under different first parameters;
and calculating a fluorescence intensity test ratio according to the double emission peaks in the steady-state fluorescence emission spectrum, and establishing a function model of the first parameter and the fluorescence intensity test ratio.
In specific application, the function model of the first parameter and the fluorescence intensity test ratio is a corresponding relation between the fluorescence intensity ratio and a sample parameter at a fluorescent probe mark position in a biological sample; for example, if the first parameter is solution concentration, the fluorescence intensity ratio can reflect the solution concentration at the probe label of the biological sample when the probe is applied to the biological sample including the solution.
S105, reconstructing the proportional fluorescence image by a STORM (storage Optical Reconstruction microscope) super-resolution imaging method to obtain a super-resolution image.
In specific application, the STORM super-resolution imaging method is to continuously record multi-frame images of fluorescent molecules through a random optical reconstruction microscope, determine a central position through a single-molecule positioning algorithm, and finally reconstruct a super-resolution fluorescent image through the images.
And S106, coloring the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image.
The super-resolution imaging method provided by the embodiment of the invention uses the fluorescent probe with double emission peaks to mark a biological sample, and reconstructs a super-resolution image by a STORM super-resolution imaging method after the fluorescent probe is excited, because the fluorescent probe can generate fluorescent signals with double emission peaks after being excited, namely a short-wavelength signal with a short-wavelength emission peak and a long-wavelength signal with a long-wavelength emission peak, a double-channel fluorescence intensity image can be formed when the fluorescent probe acts through a nucleophilic agent and activation light, and then the fluorescent signals are respectively collected by an optical signal collecting device, the short-wavelength signal with the short-wavelength emission peak and the long-wavelength signal with the long-wavelength emission peak, and the ratio of the fluorescence intensity of the short-wavelength signal and the fluorescence intensity of the long-wavelength signal emission peak can be calculated according to the double-channel fluorescence intensity image at the same time, so, and finally, coloring the super-resolution image according to the proportional fluorescent image to obtain a proportional super-resolution image. The proportional super-resolution image obtained by the super-resolution imaging method provided by the embodiment of the invention not only can reflect the structure of the biological sample, but also can reflect the sample parameters at the fluorescent probe mark position in the biological sample through the fluorescence intensity ratio, thereby analyzing the functions of the biological sample according to the sample parameters.
Example two
As shown in fig. 2, the embodiment of the present invention exemplarily shows a detailed implementation flow of S102 in the first embodiment, where step S102 is:
s102, irradiating the fluorescent probe acted by the affinity reagent by activated light to obtain a two-channel fluorescence intensity image.
The detailed implementation process comprises the following steps:
and S1021, obtaining the short-wavelength signal according to the first structure in the fluorescent probe.
S1022, obtaining the long wavelength signal according to the second structure in the fluorescent probe.
In the above steps S1021 and S1022, the wavelength of the activation light is the same as the wavelength of the excitation light, wherein the short wavelength emission peak is caused by the first structure in the probe, the long wavelength emission peak is caused by the second structure in the probe, and the fluorescence emission spectra of the two emission peaks do not completely overlap.
And S1023, when the exciting light irradiates the fluorescent probe, the first structure or the second structure emits fluorescent light.
S1024, when the activation light acts on the fluorescent probe acted by the affinity reagent, the first structure and the second structure alternately emit light according to the binding state of the first structure and the affinity reagent, and the two-channel fluorescence intensity image is formed.
In the above steps S1023 and S1024, when the laser irradiates the fluorescent probe, the first structure and the second structure in the fluorescent probe may absorb the energy of the excitation light to transition to the excited state and emit the fluorescent light, that is, when the laser irradiates the fluorescent probe, the first structure may emit the fluorescent light, and the second structure may emit the fluorescent light.
In the embodiment of the invention, if the first structure of one of the fluorescent probes emits fluorescence after being irradiated by the excitation light, the first structure is an energy acceptor, and the second structure is an energy donor; at the moment, the energy donor provides energy to the energy acceptor, so that the energy acceptor emits fluorescence, and the energy donor emits weak fluorescence due to energy loss; when the fluorescent probe acted by the affinity reagent is irradiated by the activating light, the energy donor does not provide energy to the energy acceptor any more, so that the energy acceptor does not emit fluorescence, and the energy donor does not lose energy to emit fluorescence. Due to the randomness of the binding of the affinity reagent to the fluorescent probes, the first structure and the second structure of each fluorescent probe in the biological sample emit fluorescence with randomness.
As shown in fig. 3, which is a schematic diagram of the alternative luminescence of the fluorescent probe provided in the embodiment of the present invention, first, the energy receptor emits light after the excitation light is irradiated; when the affinity reagent is not combined with the fluorescent probe, the energy does not emit fluorescence, and due to the reversibility of the affinity reagent, the energy donor reversely absorbs the energy of the activated light to emit fluorescence under the irradiation of the activated light, and then the fluorescence is randomly circulated on the basis, so that the aim of alternately emitting light by the fluorescent probe is fulfilled.
In an embodiment of the present invention, the two-channel fluorescence intensity images of steps S1021 to S1024 include a fluorescence intensity image of a short wavelength signal and a fluorescence intensity image of a long wavelength signal; the fluorescence intensity image of the short-wavelength signal corresponds to the luminescence process of the first structure in the fluorescent probe; the fluorescence intensity image of the long wavelength signal corresponds to a luminescence process of the second structure in the fluorescent probe.
The super-resolution imaging method provided by the embodiment of the invention utilizes the fluorescent probe with double emission peaks to mark the biological sample, and enables a single fluorescent probe molecule in the biological sample to randomly and alternately emit fluorescence with two different wavelengths under the irradiation of the active light and the reversible action of the affinity reagent.
EXAMPLE III
As shown in fig. 4, the embodiment of the present invention exemplarily shows a detailed implementation flow of step S103 in the first embodiment, where step S103 is:
s103, respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image.
The detailed implementation process comprises the following steps:
s1031, separating the excitation light from the short-wavelength signal and the long-wavelength signal by a first dichroic mirror.
In step S1031, the dichroic mirror transmits light of a certain wavelength almost completely, and reflects light of other wavelengths almost completely.
In a specific application, after the excitation light acts on the biological sample, the fluorescent probe on the biological sample emits light including a short-wavelength signal, a long-wavelength signal and a small number of excitation lights, so that the excitation light is separated from the short-wavelength signal and the long-wavelength signal by the first dichroic mirror.
S1032, after the short wavelength signal and the long wavelength signal are separated by the second dichroic mirror, the short wavelength signal is sent to a mirror through a first filter, and the mirror transmits the filtered short wavelength signal to a first EMCCD (electro-multiplex CCD, electron multiplying CCD); the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to the first EMCCD.
In step S1032, the light wave signal is separated by the dichroic mirror, and the separated short wavelength signal and long wavelength signal are filtered by the filter and transmitted to the same EMCCD.
In specific application, a single emission wavelength is easily interfered by environmental noise, the system noise proportion in an imaging result is increased, a reconstructed image is fuzzy and other results are caused, and algorithm noise reduction is carried out during later result processing to avoid or reduce the influence, so that the later algorithm processing in the super-resolution imaging method based on the optical reconstruction microscope can be simplified by carrying out filtering processing on the image before image reconstruction.
S1033, different regions of the first EMCCD receive the filtered short wavelength signal and the filtered long wavelength signal, respectively, thereby collecting the two-channel fluorescence intensity image.
In step S1033, the size of the short wavelength signal or the long wavelength signal should be smaller than the maximum pixel size of the EMCCD; for example, if the maximum pixel size of the EMCCD is 512 × 512, the pixel size of the collected short wavelength signal or long wavelength signal should not be larger than 256 × 256.
In a specific application, the different areas of one EMCCD are used for respectively receiving the optical path signals, and synchronous control is not needed.
As shown in fig. 5, an apparatus structure schematic diagram for implementing steps S1031 to S1032 is further provided in the embodiment of the present invention.
The reference numbers are as follows: 51. a laser; 52. a lens group; 53. a field stop; 54. a tube mirror; 55. a biological sample; 56. an objective lens; 57. a first dichroic mirror; 58. a mirror; 59. a second dichroic mirror; 510. a first filter; 511. a second filter; EMCCD 512.
In the embodiment of the present invention, the lens group is used for converging or diverging the light beam; the field stop is used to make appropriate adjustments to the beam size.
In the embodiment of the present invention, two identical EMCCDs may also be used to receive the short wavelength signal and the long wavelength signal, respectively, and the detailed implementation flow is as follows:
separating the excitation light from the short wavelength signal and the long wavelength signal by a first dichroic mirror;
after the short-wavelength signal and the long-wavelength signal are separated through a second dichroic mirror, the short-wavelength signal is sent to a reflector through a first filter, and the reflector transmits the filtered short-wavelength signal to a first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to a second EMCCD;
the first and second EMCCDs receive the filtered short wavelength signal and the filtered long wavelength signal, respectively, to collect the two-channel fluorescence intensity image.
In specific application, two identical EMCCDs are used for respectively acquiring two paths of signal light under the same condition, data acquisition of a larger pixel area can be realized, but requirements on high synchronization of the two EMCCDs are provided, and synchronous control is required.
According to the super-resolution imaging method provided by the embodiment of the invention, after the dichroic mirror separates the signal light into two paths, partial stray light is filtered by the filter, and the two paths of signal light are collected by the EMCCD, so that the noise resistance, the environment medium non-uniformity resistance and the system instability resistance of the biological sample are improved, the imaging signal-to-noise ratio is improved, and the later data processing process is simplified.
ExamplesFourthly
The embodiment of the invention exemplarily illustrates the implementation flow of the super-resolution imaging method provided in the first to third embodiments.
As shown in fig. 6, after the short wavelength signal of the short wavelength emission peak and the long wavelength signal of the long wavelength emission peak are collected, data processing is performed according to the two signals to obtain a proportional fluorescence image. The data processing process can be shown in fig. 6, where IB + IA is represented as an unseparated long-wavelength signal and short-wavelength signal, which are separated into an optical long-wavelength signal IB and short-wavelength signal IA after being respectively collected by the super-resolution imaging method provided in the third embodiment; after the collection is finished, when the ratio of the fluorescence intensity of the emission peak is calculated, the fluorescence intensity maps of the two paths of signals at the same moment are taken as the ratio, namely
Obtaining a proportional fluorescence image, performing algorithm reconstruction on a large number of ratio maps to obtain a super-resolution image, coloring the mark points with pseudo colors to obtain a proportional super-resolution image, wherein different colors represent ratios
The magnitude of the value.
EXAMPLE five
The embodiment of the invention exemplarily illustrates the practical application of the super-resolution imaging method provided in the first to third embodiments. How to reflect the sample parameters of the fluorescent probe mark position in the biological sample in practical application by the expression scale type super-resolution image, thereby analyzing the functions of the biological sample according to the sample parameters.
The embodiment of the invention provides an example of super-resolution imaging for realizing quantitative research on the viscosity value of the microenvironment of a marked sample by using the super-resolution imaging method provided by the first embodiment to the fourth embodiment.
In the embodiment of the invention, the provided fluorescent probe is a viscosity-sensitive fluorescent probe, and when the environmental viscosity changes, the steady-state emission spectrum of the fluorescent probe correspondingly changes. The short wavelength emission peak is caused by a structure A in the probe, the long wavelength emission peak is caused by a structure B in the probe, and the two fluorescence emission spectrums do not have an overlapping region, wherein IB is the fluorescence intensity of the emission peak of the structure B, and IA is the fluorescence intensity of the emission peak of the structure A.
In specific application, a function relation of the IB/IA value relative to the viscosity is constructed, namely steady-state emission spectra of the fluorescent probe in solutions with different viscosities are measured, IB/IA processing is carried out on the steady-state spectra under different viscosities respectively to obtain the IB/IA values under different viscosities, then the proportional point values are plotted and linear or nonlinear function fitting is carried out to obtain a fitting function relation of the IB/IA relative to the viscosity.
In practical applications, a biological sample is labeled with a fluorescent probe, excited with a laser, and imaged in a super-resolution imaging system to obtain a large number of proportional fluorescent images. During post-stage data processing, a ratio of each frame of two-channel proportional fluorescence image IB and IA is firstly made, then a large number of fluorescence intensity ratio images are subjected to algorithm reconstruction to obtain a super-resolution image of a marked organism structure, each pixel of the marked organism structure is colored by a pseudo-color, the value of each pixel point in the image is the value of IB/IA, different colors represent the value of the IB/IA ratio, the value of the IB/IA ratio can correspondingly find viscosity values under different ratios in a fitting function relation formula of a solution test result, namely the difference of the pseudo-color represents the distribution difference of the viscosity values in a microenvironment, meanwhile, the reconstructed super-resolution image shows a super-fine structure diagram of the marked organism structure, and finally super-resolution imaging of quantitative research of the viscosity values of the microenvironment is realized.
The embodiment of the invention also provides an example of super-resolution imaging for realizing quantitative research on the mitochondrial membrane protein of the marked sample by using the super-resolution imaging method provided by the first embodiment to the third embodiment.
In the embodiment of the invention, the provided fluorescent probe is a fluorescent probe capable of specifically labeling mitochondrial membrane protein, and when a solution or biological test is carried out, the steady-state emission spectrum of the probe is correspondingly changed after the membrane protein in the environment is specifically combined with the probe. The short wavelength emission peak is caused by a structure A in the probe, the long wavelength emission peak is caused by a structure B in the probe, and the two fluorescence emission spectra do not have a serious overlapping region, so that the two fluorescence emissions are prevented from generating crosstalk, IB is the fluorescence intensity of the emission peak of the structure B, and IA is the fluorescence intensity of the emission peak of the structure A.
In the specific application, a function relation of the IB/IA value relative to the mitochondrial membrane protein content or concentration is constructed, namely steady-state emission spectra of the fluorescent probe 2 in solutions with different mitochondrial membrane protein contents or concentrations are measured, IB/IA processing is respectively carried out on the steady-state spectra with different contents or concentrations to obtain the IB/IA values with different mitochondrial membrane protein contents or concentrations, then the proportional values are plotted against the contents or concentrations, and linear or nonlinear function fitting is carried out to obtain a fitting function relation of the IB/IA relative to the mitochondrial membrane protein contents or concentrations.
In practical application, a fluorescence probe is used for marking mitochondrial membrane proteins of organisms, excitation is carried out by using exciting light, and imaging is carried out in a super-resolution imaging system, so that a large quantity of proportional fluorescence images are obtained. During post-stage data processing, a ratio of each frame of dual-channel fluorescence intensity image IB and IA is firstly made, then a large number of proportion fluorescence images are subjected to algorithm reconstruction to obtain a super-resolution image of a marked organism structure, each pixel of the marked organism structure is colored by a pseudo-color, the value of each pixel point in the image is the value of IB/IA, different colors represent the size of the IB/IA ratio, the size of the IB/IA ratio can be used for correspondingly finding protein content values under different ratios in a fitting function relation formula of a solution test result, namely the difference of the pseudo-color represents the distribution difference of the protein content values in a microenvironment, meanwhile, the reconstructed super-resolution image shows a super-fine structure diagram of the marked organism structure, and finally, the super-resolution imaging of quantitative research of the mitochondrial membrane protein content values is realized.
EXAMPLE six
As shown in fig. 7, an embodiment of the present invention provides a super-resolution imaging apparatus 70, including:
the excitation module 71 is configured to excite, by excitation light, a fluorescent probe with two emission peaks on the biological sample, and apply the affinity reagent to the excited fluorescent probe;
a fluorescence intensity image acquisition module 72 for irradiating the fluorescent probe acted by the affinity reagent with activated light to obtain a two-channel fluorescence intensity image;
a signal collecting module 73, configured to collect a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image, respectively;
a proportional fluorescence image calculation module 74, configured to select N frames of two-channel fluorescence intensity images, select a fluorescence intensity image of the short-wavelength signal and a fluorescence intensity image of the long-wavelength signal from the two-channel fluorescence intensity images of the same frame according to the short-wavelength signal and the long-wavelength signal respectively collected, and calculate fluorescence intensity ratios thereof, respectively, to obtain N proportional fluorescence images, where N is a positive integer, and N is a positive integer;
an image reconstruction module 75, configured to reconstruct the proportional fluorescence image by a STORM super-resolution imaging method to obtain a super-resolution image;
and an image obtaining module 76, configured to color the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image.
In a specific application, the fluorescence intensity image obtaining module 72 includes:
a short wavelength emission peak obtaining unit for obtaining the short wavelength emission peak according to a first structure in the fluorescent probe;
a long wavelength emission peak obtaining unit for obtaining the long wavelength emission peak according to a second structure in the fluorescent probe;
wherein the first structure is an energy acceptor and the second structure is an energy donor;
and the double-channel fluorescence intensity image acquisition unit is used for acting the activation light on the processed fluorescent probe to enable the first structure and the second structure to alternately emit light so as to obtain a double-channel fluorescence intensity image.
The embodiment of the present invention further provides a terminal device, which includes a memory, a processor, and a computer program stored in the memory and capable of running on the processor, and when the processor executes the computer program, the processor implements each step in the super-resolution imaging method as described in the first to third embodiments.
An embodiment of the present invention further provides a storage medium, which is a computer-readable storage medium, and a computer program is stored on the storage medium, and when the computer program is executed by a processor, the computer program implements the steps in the super-resolution imaging method according to the first to third embodiments.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the foregoing embodiments illustrate the present invention in detail, those of ordinary skill in the art will understand that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the spirit and scope of the embodiments of the present invention, and are intended to be included within the scope of the present invention.
Claims (10)
1. A method of super-resolution imaging, comprising:
exciting a fluorescent probe with double emission peaks on a biological sample by exciting light, and acting an affinity reagent on the excited fluorescent probe;
irradiating the fluorescent probe acted by the affinity reagent by activated light to form a two-channel fluorescence intensity image;
respectively collecting a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image;
selecting N frames of double-channel fluorescence intensity images, selecting the fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal from the double-channel fluorescence intensity images of the same frame according to the respectively collected short-wavelength signal and long-wavelength signal, respectively calculating fluorescence intensity ratios of the two images, and obtaining N proportional fluorescence images, wherein N is a positive integer, and the calculation formula for calculating the fluorescence intensity ratio is as follows:
wherein, IBNIs the emission peak fluorescence intensity of the long wavelength signal, IANIs the emission peak fluorescence intensity of the short wavelength signal;
reconstructing the proportional fluorescence image by a STORM super-resolution imaging method to obtain a super-resolution image, wherein the super-resolution imaging method comprises the following steps: after the collection, the signals are separated into a light long-wavelength signal IB and a light short-wavelength signal IA, and when the ratio of the fluorescence intensity of the emission peak is calculated after the collection is finished, the fluorescence intensity maps of the two signals at the same moment are used as the ratio, namely
Obtaining a proportional fluorescence image, performing algorithm reconstruction on a large number of ratio maps to obtain a super-resolution image, coloring the mark points with pseudo colors to obtain a proportional super-resolution image, wherein different colors represent ratios
The magnitude of the value;
and coloring the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image.
2. The super-resolution imaging method according to claim 1, further comprising:
establishing a function model of the ratio of the environmental parameter of the fluorescent probe to the fluorescence intensity;
the establishing of the function model of the ratio of the environmental parameter of the fluorescent probe to the fluorescence intensity comprises the following steps:
placing the fluorescent probe with the double emission peaks in a probe solution test environment, and exciting the fluorescent probe by exciting light;
changing a first parameter of the probe solution testing environment to obtain a steady-state fluorescence emission spectrum of the fluorescent probe under different first parameters;
and calculating a fluorescence intensity ratio according to the double emission peaks in the steady-state fluorescence emission spectrum, and establishing a function model of the first parameter and the fluorescence intensity ratio.
3. The super-resolution imaging method according to claim 1, wherein the obtaining of the two-channel fluorescence intensity image by irradiating the fluorescence probe after the affinity reagent by the activating light comprises:
obtaining the short wavelength signal according to a first structure in the fluorescent probe;
obtaining the long wavelength signal according to a second structure in the fluorescent probe;
when the exciting light irradiates the fluorescent probe, the first structure or the second structure emits fluorescence;
when the activation light acts on the fluorescent probe acted by the affinity reagent, the first structure and the second structure alternately emit light according to the binding state of the first structure and the affinity reagent to form the dual-channel fluorescence intensity image.
4. The super-resolution imaging method according to claim 3, wherein the two-channel fluorescence intensity image includes a fluorescence intensity image of a short wavelength signal and a fluorescence intensity image of a long wavelength signal;
the fluorescence intensity image of the short-wavelength signal corresponds to the luminescence process of the first structure in the fluorescent probe;
the fluorescence intensity image of the long wavelength signal corresponds to a luminescence process of the second structure in the fluorescent probe.
5. The super-resolution imaging method according to claim 1, wherein the separately acquiring the short wavelength signal of the short wavelength emission peak and the long wavelength signal of the long wavelength emission peak in the two-channel fluorescence intensity image comprises:
separating the excitation light from the short wavelength signal and the long wavelength signal by a first dichroic mirror;
after the short-wavelength signal and the long-wavelength signal are separated through a second dichroic mirror, the short-wavelength signal is sent to a reflector through a first filter, and the reflector transmits the filtered short-wavelength signal to a first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to the first EMCCD;
different regions of the first EMCCD receive the filtered short wavelength signal and the filtered long wavelength signal, respectively, thereby collecting the dual channel fluorescence intensity image.
6. The super-resolution imaging method according to claim 1, wherein the acquiring the short wavelength signal of the short wavelength emission peak and the long wavelength signal of the long wavelength emission peak in the two-channel fluorescence intensity image respectively comprises:
separating the excitation light from the short wavelength signal and the long wavelength signal by a first dichroic mirror;
after the short-wavelength signal and the long-wavelength signal are separated through a second dichroic mirror, the short-wavelength signal is sent to a reflector through a first filter, and the reflector transmits the filtered short-wavelength signal to a first EMCCD; the long-wavelength signal passes through a second filter, and the filtered long-wavelength signal is transmitted to a second EMCCD;
the first and second EMCCDs receive the filtered short wavelength signal and the filtered long wavelength signal, respectively, to collect the two-channel fluorescence intensity image.
7. The super-resolution imaging method according to any one of claims 1 to 6, wherein the selecting N frames of the dual-channel fluorescence intensity images, selecting the fluorescence intensity image of the short wavelength signal and the fluorescence intensity image of the long wavelength signal in the dual-channel fluorescence intensity image of the same frame according to the separately acquired short wavelength signal and long wavelength signal, and separately calculating fluorescence intensity ratios thereof to obtain N proportional fluorescence images, where N is a positive integer includes:
selecting the fluorescence intensity image of the Nth short-wavelength signal according to the two-channel fluorescence intensity image and the respectively collected short-wavelength signals, and calculating the fluorescence intensity of an emission peak of the Nth short-wavelength signal;
selecting the fluorescence intensity image of the Nth long-wavelength signal according to the two-channel fluorescence intensity image and the respectively collected long-wavelength signals, and calculating the fluorescence intensity of an emission peak of the fluorescence intensity image;
the calculation formula for calculating the fluorescence intensity ratio is as follows:
wherein, IBNIs the emission peak fluorescence intensity of the long wavelength signal, IANIs the emission peak fluorescence intensity of the short wavelength signal.
8. A super-resolution imaging apparatus, comprising:
the excitation module is used for exciting the fluorescent probe with double emission peaks on the biological sample by exciting light and enabling the affinity reagent to act on the excited fluorescent probe;
the fluorescence intensity image acquisition module is used for irradiating the fluorescent probe acted by the affinity reagent by activated light to obtain a two-channel fluorescence intensity image;
the signal acquisition module is used for respectively acquiring a short-wavelength signal of a short-wavelength emission peak and a long-wavelength signal of a long-wavelength emission peak in the two-channel fluorescence intensity image;
the proportional fluorescence image calculation module is used for selecting N frames of double-channel fluorescence intensity images, selecting the fluorescence intensity image of the short-wavelength signal and the fluorescence intensity image of the long-wavelength signal from the double-channel fluorescence intensity images of the same frame according to the respectively collected short-wavelength signal and long-wavelength signal, and respectively calculating the fluorescence intensity ratios of the fluorescence intensity images to obtain N proportional fluorescence images, wherein N is a positive integer, and N is a positive integer;
the image reconstruction module is used for reconstructing the proportional fluorescence image by a STORM super-resolution imaging method to obtain a super-resolution image;
and the image acquisition module is used for coloring the super-resolution image according to the N proportional fluorescent images to obtain a proportional super-resolution image.
9. A terminal device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor implements the steps of the super-resolution imaging method according to any one of claims 1 to 7 when executing the computer program.
10. A storage medium being a computer readable storage medium having a computer program stored thereon, wherein the computer program, when executed by a processor, performs the steps of the super resolution imaging method according to any of claims 1 to 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810958217.1A CN108982460B (en) | 2018-08-22 | 2018-08-22 | Super-resolution imaging method and device and terminal equipment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810958217.1A CN108982460B (en) | 2018-08-22 | 2018-08-22 | Super-resolution imaging method and device and terminal equipment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108982460A CN108982460A (en) | 2018-12-11 |
| CN108982460B true CN108982460B (en) | 2021-03-30 |
Family
ID=64554253
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810958217.1A Active CN108982460B (en) | 2018-08-22 | 2018-08-22 | Super-resolution imaging method and device and terminal equipment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108982460B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7838302B2 (en) * | 2006-08-07 | 2010-11-23 | President And Fellows Of Harvard College | Sub-diffraction limit image resolution and other imaging techniques |
| WO2011112634A2 (en) * | 2010-03-08 | 2011-09-15 | California Institute Of Technology | Molecular indicia of cellular constituents and resolving the same by super-resolution technologies in single cells |
| WO2016070854A1 (en) * | 2014-11-05 | 2016-05-12 | Benzhong Tang | Photoactivatable bioprobes: design, method of preparation and applications |
| US9835870B2 (en) * | 2015-06-05 | 2017-12-05 | Vasily N. Astratov | Super-resolution microscopy methods and systems enhanced by dielectric microspheres or microcylinders used in combination with metallic nanostructures |
-
2018
- 2018-08-22 CN CN201810958217.1A patent/CN108982460B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108982460A (en) | 2018-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE112009005521B4 (en) | Method and device for the localization of individual dye molecules in fluorescence microscopy | |
| Kumar et al. | FLIM FRET technology for drug discovery: Automated multiwell‐plate high‐content analysis, multiplexed readouts and application in situ | |
| Grecco et al. | Global analysis of time correlated single photon counting FRET-FLIM data | |
| Thorley et al. | Super-resolution microscopy: a comparison of commercially available options | |
| EP1291627B1 (en) | Method and Apparatus for multiparameter acquisition of single photons to produce simultaneously time and space as well as time and wavelength resolved fluorescence images | |
| JP2006242899A (en) | Method for estimating concentration of fluorescent dye from multiple fluorescence, and method for estimating fluorescence intensity from multiple fluorescence | |
| US20110293154A1 (en) | Method and system for characterizing a sample by imaging fluorescence microscopy | |
| JP2012530947A (en) | Method for evaluating fluorescence events in microscopic images | |
| DE102004039035A1 (en) | Method and apparatus for fluorescence lifetime imaging nanoscopy | |
| US7280203B2 (en) | Method for setting a fluorescence spectrum measurement system for microscopy | |
| Sahl et al. | High‐Resolution Tracking of Single‐Molecule Diffusion in Membranes by Confocalized and Spatially Differentiated Fluorescence Photon Stream Recording | |
| Pawlowska et al. | Embracing the uncertainty: the evolution of SOFI into a diverse family of fluctuation-based super-resolution microscopy methods | |
| CN105044066B (en) | A kind of nanometer OCT image method and system based on broadband stimulated radiation | |
| Descloux et al. | Experimental combination of super-resolution optical fluctuation imaging with structured illumination microscopy for large fields-of-view | |
| CN108982460B (en) | Super-resolution imaging method and device and terminal equipment | |
| Ivanchenko et al. | Fluorescence correlation spectroscopy: principles and developments | |
| Li et al. | Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples | |
| WO2020037527A1 (en) | Super-resolution imaging method and apparatus, and terminal device | |
| US11768363B2 (en) | Method and apparatus for microscopy imaging with resolution correction | |
| Elson et al. | Wide-field fluorescence lifetime imaging with optical sectioning and spectral resolution applied to biological samples | |
| Zeng et al. | Simultaneous multicolour imaging using quantum dot structured illumination microscopy | |
| KR20210110997A (en) | Device and method for measuring fluorescence lifetime | |
| Alvelid et al. | Far Red‐Shifted CdTe Quantum Dots for Multicolour Stimulated Emission Depletion Nanoscopy | |
| Premadasa et al. | Spatially co-registered wide-field nonlinear optical imaging of living and complex biosystems in a total internal reflection geometry | |
| Rüttinger | Confocal microscopy and quantitative single molecule techniques for metrology in molecular medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |