CN108982460A - A kind of super-resolution imaging method, device and terminal device - Google Patents
A kind of super-resolution imaging method, device and terminal device Download PDFInfo
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Abstract
The present invention is suitable for technical field of image processing, provide a kind of super-resolution imaging method, device and terminal device, method includes: to excite the fluorescence probe on biological sample with double emission peaks, and affinity reagent is acted on the fluorescence probe after excitation by exciting light；By activated light, the fluorescence probe after affinity reagent acts on is irradiated, binary channels fluorescence intensity image is obtained；It acquires in binary channels fluorescence intensity image respectively, short wavelength's signal of short wavelength's emission peak and the long wavelength signals at long wavelength emission peak；N frame binary channels fluorescence intensity image is selected, the fluorescence intensity ratio of short wavelength's signal and long wavelength signals is calculated separately, obtains N number of scale fluorescence images；By STORM super-resolution imaging method, super resolution image is obtained；Super resolution image is coloured according to N number of scale fluorescence images, obtains proportional-type super resolution image.Proportional-type super resolution image can be obtained through the invention, reflect the sample parameters of fluorescence probe mark in biological sample.
Description
Technical field
The present invention relates to technical field of image processing more particularly to a kind of super-resolution imaging methods, device and terminal device.
Background technique
Fluorescence microscope is widely used in cell microorganism imaging, wherein super-resolution is positioned to seem a kind of representativeness
Super-resolution fluorescence imaging technique.The technology is on the basis of optical reconstruction is microscopical, by single molecular imaging and high-precision molecule
Location algorithm combines, and realizes the superelevation spatial resolution of 20~30nm, to observe the ultra microstructure in cell.This skill
The key of art is the affinity reagent random incorporation in the fluorescence probe and ambient enviroment using excitation state, leads to its fluorescence decay
Into dark-state, recycles the activated light of another wavelength or Same Wavelength that affinity reagent is made to fall off at random from fluorescent molecule and have again
Then preparation light ability continuously records the more of fluorescent molecule so that fluorescent molecule changes between dark-state at random luminous
Frame image carries out unimolecule location algorithm and determines center, goes out super-resolution fluorescence image finally by image reconstruction.
However, optical reconstruction microscope only can carry out super-resolution imaging to the structure of biological sample at present, can not do work
Can property super-resolution imaging or sample parameters it is quantitative super-resolution imaging research.
Summary of the invention
It is existing to solve it is a primary object of the present invention to propose a kind of super-resolution imaging method, device and terminal device
Super-resolution imaging can only be carried out to the structure of biological sample in technology, cannot do functional super-resolution imaging or sample parameters are quantitative
Super-resolution imaging study a question.
To achieve the above object, first aspect of the embodiment of the present invention provides a kind of super-resolution imaging method, comprising:
By exciting light, the fluorescence probe on biological sample with double emission peaks is excited, affinity reagent is acted on into excitation
On fluorescence probe afterwards；
By activated light, the fluorescence probe after affinity reagent acts on is irradiated, binary channels fluorescence intensity image is formed；
It is acquired in the binary channels fluorescence intensity image respectively, the short wavelength's signal and long wavelength emission of short wavelength's emission peak
The long wavelength signals at peak；
N frame binary channels fluorescence intensity image is selected, according to the short wavelength's signal and the long wave acquired respectively
Long signal, in the binary channels fluorescence intensity image of same frame, select short wavelength's signal fluorescence intensity image and
The fluorescence intensity image of the long wavelength signals, and its fluorescence intensity ratio is calculated separately, N number of scale fluorescence images are obtained,
Middle N is positive integer；
By STORM, (Stochastic Optical Reconstruction Microscopy, random optical reconstruct are aobvious
Micro mirror) super-resolution imaging method, the scale fluorescence images are reconstructed, super resolution image is obtained；
The super resolution image is coloured according to N number of scale fluorescence images, obtains proportional-type super resolution image.
In conjunction with first aspect present invention, in the first embodiment of first aspect present invention, the super-resolution imaging method
Further include:
Establish the environmental parameter of the fluorescence probe and the function model of the fluorescence intensity ratio；
It is described to establish the fluorescence probe environmental parameter and the function model of the fluorescence intensity ratio includes:
The fluorescence probe for having double emission peaks is placed in probe solution test environment, by exciting light, described in excitation
Fluorescence probe；
The first parameter for changing the probe solution test environment, obtains under different first parameters, described glimmering
The steady-state fluorescence emission spectrum of light probe；
It according to double emission peaks in the steady-state fluorescence emission spectrum, calculates fluorescence intensity and tests ratio, and described in foundation
The function model of first parameter and fluorescence intensity test ratio.
It is described to pass through activated light, irradiation in the second embodiment of first aspect present invention in conjunction with first aspect present invention
Fluorescence probe after affinity reagent effect, forming binary channels fluorescence intensity image includes:
According to the first structure in the fluorescence probe, short wavelength's signal is obtained；
According to the second structure in the fluorescence probe, the long wavelength signals are obtained；
When the exciting light irradiates the fluorescence probe, the first structure or the second structure emit fluorescence；
When the activated light acts on the fluorescence probe after the affinity reagent effect, the first structure and described second
Structure is luminous according to replacing with the bonding state of the affinity reagent, forms the binary channels fluorescence intensity image.
It is described in the third embodiment of first aspect present invention in conjunction with the second embodiment of first aspect present invention
Binary channels fluorescence intensity image, the fluorescence intensity image of fluorescence intensity image and long wavelength signals including short wavelength's signal；
The fluorescence intensity image of short wavelength's signal corresponds to the luminescence process of the first structure in the fluorescence probe；
The fluorescence intensity image of the long wavelength signals corresponds to the luminescence process of the second structure in the fluorescence probe.
In conjunction with first aspect present invention, in the 4th embodiment of first aspect present invention, it is described acquire respectively it is described double
In channel fluorescence intensity image, short wavelength's signal of short wavelength's emission peak and the long wavelength signals at long wavelength emission peak include:
The exciting light is separated with short wavelength's signal and the long wavelength signals by the first dichroic mirror；
By the second dichroic mirror by after short wavelength's signal and long wavelength signals separation, short wavelength's signal is passed through
It crosses first filter and is sent to reflecting mirror, filtered short wavelength's signal is transmitted to the first EMCCD by the reflecting mirror；Institute
Long wavelength signals are stated by second filter, the filtered long wavelength signals are transmitted to the first EMCCD, to collect
The binary channels fluorescence intensity image.；
The different zones of first EMCCD receive filtered short wavelength's signal and the filtered length respectively
Wavelength signals.
In conjunction with first aspect present invention, in the 5th embodiment of first aspect present invention, it is described acquire respectively it is described glimmering
In optical emission spectroscopy, short wavelength's signal of short wavelength's emission peak and the long wavelength signals at long wavelength emission peak, comprising:
The exciting light is separated with short wavelength's signal and the long wavelength signals by the first dichroic mirror；
By the second dichroic mirror by after short wavelength's signal and long wavelength signals separation, short wavelength's signal is passed through
It crosses first filter and is sent to reflecting mirror, filtered short wavelength's signal is transmitted to the first EMCCD by the reflecting mirror；Institute
Long wavelength signals are stated by second filter, the filtered long wavelength signals are transmitted to the 2nd EMCCD；
First EMCCD and the 2nd EMCCD receive filtered short wavelength's signal and the filtering respectively
Long wavelength signals afterwards, to collect the binary channels fluorescence intensity image.
In conjunction with the first to the 5th embodiment of first aspect present invention, the sixth embodiment of first aspect present invention
In, the selection N frame binary channels fluorescence intensity image, according to the short wavelength's signal acquired respectively and the long wavelength
Signal selects fluorescence intensity image and the institute of short wavelength's signal in the binary channels fluorescence intensity image of same frame
The fluorescence intensity image of long wavelength signals is stated, and calculates separately its fluorescence intensity ratio, obtains N number of scale fluorescence images, wherein N
Include: for positive integer
According to the binary channels fluorescence intensity image and the short wavelength's signal acquired respectively, the n-th institute is selected
The fluorescence intensity image for stating short wavelength's signal calculates its emission peak fluorescence intensity；
According to the binary channels fluorescence intensity image and the long wavelength signals acquired respectively, the n-th institute is selected
The fluorescence intensity image for stating long wavelength signals calculates its emission peak fluorescence intensity；
Calculate the calculation formula of the fluorescence intensity ratio are as follows:
Wherein, IBNFor the emission peak fluorescence intensity of the long wavelength signals, IANEmission peak for short wavelength's signal is glimmering
Luminous intensity.
Second aspect of the present invention provides a kind of super-resolution imaging device, comprising:
Excitation module, for exciting the fluorescence probe on biological sample with double emission peaks, and will be affine by exciting light
Reagent acts on the fluorescence probe after excitation；
Fluorescence intensity image collection module, for irradiating the fluorescence probe after affinity reagent acts on, obtaining by activated light
Binary channels fluorescence intensity image；
Signal acquisition module, for being acquired in the binary channels fluorescence intensity image respectively, the shortwave of short wavelength's emission peak
The long wavelength signals of long signal and long wavelength emission peak；
Scale fluorescence images computing module is acquired according to described respectively for selecting N frame binary channels fluorescence intensity image
Short wavelength's signal and the long wavelength signals select described short in the binary channels fluorescence intensity image of same frame
The fluorescence intensity image of wavelength signals and the fluorescence intensity image of the long wavelength signals, and calculate separately its fluorescence intensity ratio
Value, obtains N number of scale fluorescence images, and wherein N is positive integer, and wherein N is positive integer；
Image reconstruction module, for by STORM super-resolution imaging method, the scale fluorescence images to be reconstructed,
Obtain super resolution image；
Image collection module obtains ratio for colouring according to N number of scale fluorescence images to the super resolution image
Type super resolution image.
The third aspect of the embodiment of the present invention provides a kind of terminal device of propagating source selection in complex network, including deposits
Reservoir, processor and it is stored in the computer program that can be run in above-mentioned memory and on above-mentioned processor, above-mentioned processing
The step of device realizes method provided by first aspect as above when executing above-mentioned computer program.
The fourth aspect of the embodiment of the present invention provides a kind of computer readable storage medium, above-mentioned computer-readable storage
Media storage has computer program, and above-mentioned computer program realizes method provided by first aspect as above when being executed by processor
The step of.
The super-resolution imaging method that the embodiment of the present invention proposes, using the fluorescence probe with double emission peaks, to biological sample
Product are marked, and super resolution image are reconstructed by STORM super-resolution imaging method after excitation fluorescence probe, due to fluorescence probe
The fluorescence signal with double emission peaks can be generated after excitation, i.e. short wavelength's signal with short wavelength's emission peak and having is grown
The long wavelength signals of peak emission wavelength, therefore while acting on this fluorescence probe by nucleophilic reagent and activated light, is capable of forming binary channels
Then fluorescence intensity image is acquired respectively by light signal acquisition device, short wavelength's signal and tool with short wavelength's emission peak
There are the long wavelength signals at long wavelength emission peak, according to the binary channels fluorescence intensity image of synchronization, shortwave long letter can be calculated
Number and the fluorescence intensity ratio with long wavelength signals emission peak, obtain scale fluorescence images, finally according to scale fluorescence images
Super resolution image is coloured, proportional-type super resolution image is obtained.The super-resolution imaging method that its is proposed through the embodiment of the present invention and
The proportional-type super resolution image of acquisition, not only can reflect the structure of biological sample, can also be reflected by fluorescence intensity ratio
The sample parameters of fluorescence probe mark in biological sample, so as to analyze the function of biological sample according to sample parameters.
Detailed description of the invention
Fig. 1 is the implementation process schematic diagram for the super-resolution imaging method that the embodiment of the present invention one provides；
Fig. 2 is the detailed implementation process schematic diagram of S103 in Fig. 1 provided by Embodiment 2 of the present invention；
Fig. 3 is the schematic diagram that fluorescence probe provided by Embodiment 2 of the present invention alternately shines；
The detailed implementation process schematic diagram of S104 in Fig. 1 that Fig. 4 provides for the embodiment of the present invention three；
Fig. 5 is the schematic device for realizing step in Fig. 2；
Fig. 6 is the realization effect picture for the super-resolution imaging method that the embodiment of the present invention four provides；
Fig. 7 is the structural schematic diagram for the super-resolution imaging device that the embodiment of the present invention six provides.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not intended to limit the present invention.
It should be noted that, in this document, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or the device that include a series of elements not only include those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or device institute it is intrinsic
Element.In the absence of more restrictions, the element limited by sentence "including a ...", it is not excluded that including being somebody's turn to do
There is also other identical elements in the process, method of element, article or device.
Herein, using the suffix for indicating such as " module ", " component " or " unit " of element only for advantageous
In explanation of the invention, there is no specific meanings for itself.Therefore, " module " can be used mixedly with " component ".
In subsequent description, inventive embodiments serial number is for illustration only, does not represent the advantages or disadvantages of the embodiments.
Embodiment one
As shown in Figure 1, it is micro- to can be applied to optical reconstruction the embodiment of the invention provides a kind of super-resolution imaging method
In the super-resolution positioning imaging of mirror comprising:
S101, pass through exciting light, excite the fluorescence probe on biological sample with double emission peaks, and affinity reagent is acted on
On fluorescence probe after excitation.
In above-mentioned steps S101, after there is the fluorescence probe of double emission peaks to be excited, the fluorescence emission spectrum of fluorescence probe
In will show two emission peaks, i.e. short wavelength's emission peak and long wavelength emission peak.
In a particular application, exciting light acts on before biological sample, should also pass through optical processing elements, such as lens, view
Field diaphragm, Guan Jing, object lens etc., so that exciting light can equably, be intensively radiated on biological sample, so that fluorescence be excited to visit
Needle.
S102, pass through activated light, the fluorescence probe after irradiation affinity reagent effect forms binary channels fluorescence intensity image.
In above-mentioned steps S102, affinity reagent is not in conjunction with probe, when the structure in affinity reagent covering fluorescence probe,
This structure does not shine, and when affinity reagent falls off from this structure, this structure has luminous power again.
In embodiments of the present invention, affinity reagent has invertibity, i.e., under certain condition, affinity reagent can be in fluorescence
It is shifted between structure in probe.
In a particular application, activated light and exciting light can have identical frequency, it is possible to have different frequencies,
It is not limited specifically in the embodiment of the present invention.
S103, it is acquired in the binary channels fluorescence intensity image respectively, the short wavelength's signal and long wave of short wavelength's emission peak
The long wavelength signals of long emission peak.
In above-mentioned steps S103, since the fluorescence probe after reagent effect is still the fluorescence probe with double emission peaks, because
Fluorescence emission spectrum obtained in this step S103 still has double emission peaks.And after activated light irradiation affinity reagent effect
When fluorescence probe, affinity reagent can be made to be transferred to another structure in fluorescence probe from a structure in fluorescence probe,
Two structures will alternately shine；Therefore it from alternately luminous structure, obtained fluorescence emission spectrum, can obtain respectively short
Short wavelength's signal of peak emission wavelength and the long wavelength signals at long wavelength emission peak.
In a particular application, the signal acquired in fluorescence emission spectrum can pass through arbitrary spectrometric instrument or optics
Component combination is realized, such as EMCCD, is not specifically limited to it in the embodiment of the present invention.
S104, selection N frame binary channels fluorescence intensity image, according to the short wavelength's signal acquired respectively and described
Long wavelength signals select the fluorescence intensity figure of short wavelength's signal in the binary channels fluorescence intensity image of same frame
The fluorescence intensity image of picture and the long wavelength signals, and its fluorescence intensity ratio is calculated separately, obtain N number of scale fluorescence figure
Picture, wherein N is positive integer.
It in above-mentioned steps S104, selects in the binary channels fluorescence intensity image of same frame, short wavelength's signal and long wave long letter
Fluorescence intensity image corresponding to number, and its fluorescence intensity ratio is calculated separately, N number of scale fluorescence images are obtained, are equivalent to
The multiple image of fluorescence probe flashing is continuously had recorded in a period of time, n-th scale fluorescence images are nth frame binary channels fluorescence
Intensity image, can reflect fluorescence intensity image corresponding to time identical short wavelength's signal and long wavelength signals, and N
A scale fluorescence images and the N+1 scale fluorescence images can be adjacent two field pictures, can also be non-conterminous two frames figure
Picture.
The embodiment of the present invention also proposed the detailed realization step that fluorescence intensity ratio value is calculated in above-mentioned steps S104, packet
It includes:
According to the binary channels fluorescence intensity image and the short wavelength's signal acquired respectively, the n-th institute is selected
The fluorescence intensity image for stating short wavelength's signal calculates its emission peak fluorescence intensity；
According to the binary channels fluorescence intensity image and the long wavelength signals acquired respectively, the n-th institute is selected
The fluorescence intensity image for stating long wavelength signals calculates its emission peak fluorescence intensity；
Calculate the calculation formula of the fluorescence intensity ratio are as follows:
Wherein, IBNFor the emission peak fluorescence intensity of the long wavelength signals, IANEmission peak for short wavelength's signal is glimmering
Luminous intensity.
In a particular application, it needs with large number of scale fluorescence images (such as 1000 scale fluorescence images),
Basis as image reconstruction.
The embodiment of the present invention also provides a kind of super-resolution imaging method, for establish the environmental parameter of the fluorescence probe with
The function model of the fluorescence intensity ratio；
It is described to establish the fluorescence probe environmental parameter and the function model of the fluorescence intensity ratio includes:
The fluorescence probe for having double emission peaks is placed in probe solution test environment, by exciting light, described in excitation
Fluorescence probe；
The first parameter for changing the probe solution test environment, obtains under different first parameters, described glimmering
The steady-state fluorescence emission spectrum of light probe；
It according to double emission peaks in the steady-state fluorescence emission spectrum, calculates fluorescence intensity and tests ratio, and described in foundation
The function model of first parameter and fluorescence intensity test ratio.
In a particular application, the function model of the first parameter and fluorescence intensity test ratio, i.e. fluorescence intensity ratio
With the corresponding relationship of the sample parameters of fluorescence probe mark in biological sample；For example, if the first parameter is solution concentration, it will
When this probe is acted in the biological sample including solution, fluorescence intensity ratio can reflect the probe mark of biological sample
Solution concentration.
S105, pass through STORM (Stochastic Optical Reconstruction Microscopy, random optical
Reconstruct microscope) super-resolution imaging method, the scale fluorescence images are reconstructed, super resolution image is obtained.
In a particular application, STORM super-resolution imaging method reconstructs microscope by random optical, continuously records fluorescence
The multiple image of molecule carries out unimolecule location algorithm and determines center, goes out super-resolution fluorescence figure finally by image reconstruction
Picture.
S106, the super resolution image is coloured according to N number of scale fluorescence images, obtains proportional-type super-resolution figure
Picture.
The super-resolution imaging method that the embodiment of the present invention proposes, using the fluorescence probe with double emission peaks, to biological sample
Product are marked, and super resolution image are reconstructed by STORM super-resolution imaging method after excitation fluorescence probe, due to fluorescence probe
The fluorescence signal with double emission peaks can be generated after excitation, i.e. short wavelength's signal with short wavelength's emission peak and having is grown
The long wavelength signals of peak emission wavelength, therefore while acting on this fluorescence probe by nucleophilic reagent and activated light, is capable of forming binary channels
Then fluorescence intensity image is collected respectively by light signal acquisition device, short wavelength's signal and tool with short wavelength's emission peak
There are the long wavelength signals at long wavelength emission peak, according to the binary channels fluorescence intensity image of synchronization, shortwave long letter can be calculated
Number and the fluorescence intensity ratio with long wavelength signals emission peak, obtain scale fluorescence images, finally according to scale fluorescence images
Super resolution image is coloured, proportional-type super resolution image is obtained.The super-resolution imaging method that its is proposed through the embodiment of the present invention and
The proportional-type super resolution image of acquisition, not only can reflect the structure of biological sample, can also be reflected by fluorescence intensity ratio
The sample parameters of fluorescence probe mark in biological sample, so as to analyze the function of biological sample according to sample parameters.
Embodiment two
As shown in Fig. 2, one kind that the embodiment of the present invention illustratively shows S102 in above-described embodiment one is realized in detail
Process, wherein step S102 are as follows:
S102, pass through activated light, the fluorescence probe after irradiation affinity reagent effect obtains binary channels fluorescence intensity image.
Its detailed implementation process includes:
S1021, according to the first structure in the fluorescence probe, obtain short wavelength's signal.
S1022, according to the second structure in the fluorescence probe, obtain the long wavelength signals.
It is identical by the wavelength of activated light and the wavelength of exciting light, wherein shortwave in above-mentioned steps S1021 and step S1022
Long emission peak is caused by first structure in probe, and long wavelength emission peak is caused by the second structure in probe, and two emission peaks
Fluorescence emission spectrum is not exclusively overlapped.
When S1023, the exciting light irradiate the fluorescence probe, the first structure or the second structure emit fluorescence.
When S1024, the activated light act on the fluorescence probe after the affinity reagent effect, the first structure and institute
It is luminous according to replacing with the bonding state of the affinity reagent to state the second structure, forms the binary channels fluorescence intensity image.
In above-mentioned steps S1023 and step S1024, in the case where laser irradiation fluorescence probe, in fluorescence probe
One structure and the second structure are possible to absorption exciting light energy and transit to excitation state in transmitting fluorescence, i.e. laser irradiation fluorescence is visited
When needle, first structure transmitting fluorescence can be, be also possible to the second structure transmitting fluorescence.
In embodiments of the present invention, if after exciting light irradiation, the first structure of one of fluorescence probe emits fluorescence, then
First structure is energy acceptor, and the second structure is energy donor；Energy donor provides energy to energy acceptor at this time, so that energy
Acceptor emission fluorescence, energy donor is because losing energy hair week fluorescent；And by activated light, after irradiating the effect of this affinity reagent
When fluorescence probe, energy donor no longer provides energy to energy acceptor, so that energy acceptor does not emit fluorescence, energy donor is not because
Lose energy transmitting fluorescence.Due to randomness of the affinity reagent in conjunction with fluorescence probe, so that in biological sample, each fluorescence probe
First structure and the second structure transmitting fluorescence also have randomness.
As shown in figure 3, being the schematic diagram that fluorescence probe provided in an embodiment of the present invention alternately shines.Firstly, exciting light shines
Rear energy acceptor is penetrated to shine；When affinity reagent is not in conjunction with fluorescence probe, energy acceptor does not reabsorb energy donor and excitation
The energy of light, no longer transmitting fluorescence, due to the invertibity of affinity reagent, and under the irradiation of activated light, energy donor is reverse
It absorbs activating light energy and emits fluorescence, then on this basis, random rotation achievees the purpose that fluorescence probe alternately shines.
In embodiments of the present invention, binary channels fluorescence intensity image described in above-mentioned steps S1021 to step S1024, packet
Include the fluorescence intensity image of short wavelength's signal and the fluorescence intensity image of long wavelength signals；The fluorescence intensity of short wavelength's signal
Image corresponds to the luminescence process of the first structure in the fluorescence probe；The fluorescence intensity image of the long wavelength signals, it is corresponding
The luminescence process of the second structure in the fluorescence probe.
The super-resolution imaging method that the embodiment of the present invention proposes, using the fluorescence probe with double emission peaks to biological sample
It is marked, under activated light irradiation and affinity reagent reversible action, hands over single fluorescent probe molecule in biological sample at random
For the fluorescence of two different wave lengths of transmitting.
Embodiment three
As shown in figure 4, the embodiment of the present invention illustratively shows one kind of step S103 in above-described embodiment one in detail
Implementation process, wherein step S103 are as follows:
S103, it is acquired in the binary channels fluorescence intensity image respectively, the short wavelength's signal and long wave of short wavelength's emission peak
The long wavelength signals of long emission peak.
Its detailed implementation process includes:
S1031, the exciting light is separated with short wavelength's signal and the long wavelength signals by the first dichroic mirror.
In above-mentioned steps S1031, dichroic mirror almost penetrates the light of certain wavelength, and several to the light of other wavelength
It is fully reflective.
In a particular application, after exciting light acts on biological sample, light that the fluorescence probe on biological sample is emitted
In include short wavelength's signal, long wavelength signals and minority exciting light, therefore by the first dichroic mirror by exciting light and shortwave
Long signal and long wavelength signals separation.
S1032, by the second dichroic mirror by short wavelength's signal and the long wavelength signals separation after, the short wavelength
Signal is sent to reflecting mirror by first filter, and filtered short wavelength's signal is transmitted to first by the reflecting mirror
EMCCD (ElectBon-MultiplyinA CCD, electron multiplication CCD)；The long wavelength signals pass through second filter, described
Filtered long wavelength signals are transmitted to the first EMCCD.
In above-mentioned steps S1032, equally by dichroic mirror separate lightwave signal, while by after separation short wavelength's signal with
Long wavelength signals are sent in same EMCCD after filter filtering.
In a particular application, single launch wavelength is easy to will cause system noise ratio in imaging results by ambient noise interference
Example increases, reconstructed image is fuzzy etc. as a result, to avoid or mitigating this influence, and when anaphase processing will carry out algorithm noise reduction,
Therefore before image reconstruction, filtering processing is done to it can simplify based in the microscopical super-resolution imaging method of optical reconstruction
Later period algorithm process.
S1033, the first EMCCD different zones receive filtered short wavelength's signal and the filtering respectively
Long wavelength signals afterwards, to collect the binary channels fluorescence intensity image.
In above-mentioned steps S1033, the size of short wavelength's signal or long wavelength signals should be less than the maximum pixel ruler of EMCCD
It is very little；If such as the maximum pixel of EMCCD is having a size of 512*512, then the picture of short wavelength's signal or long wavelength signals collected
Plain size should not exceed 256*256.
In a particular application, using the different zones of an EMCCD, receiving light path signal does not need synchronously control then respectively.
As shown in figure 5, the embodiment of the present invention gives the apparatus structure for realizing above-mentioned steps S1031 to step S1032
Schematic diagram.
Its appended drawing reference is as follows: 51. lasers；52. lens group；53. field stop；54. pipe mirror；55. biological sample；
56. object lens；57. the first dichroic mirror；58. reflecting mirror；59. the second dichroic mirror；510. first filter；511. second filter；
512.EMCCD。
In embodiments of the present invention, lens group is for convergence or divergent beams；Field stop is used to carry out beam size
It is appropriate to adjust.
In embodiments of the present invention, two identical EMCCD can also be used to receive short wavelength's signal and long wavelength respectively
Signal, detailed implementation process are as follows:
The exciting light is separated with short wavelength's signal and the long wavelength signals by the first dichroic mirror；
By the second dichroic mirror by after short wavelength's signal and long wavelength signals separation, short wavelength's signal is passed through
It crosses first filter and is sent to reflecting mirror, filtered short wavelength's signal is transmitted to the first EMCCD by the reflecting mirror；Institute
Long wavelength signals are stated by second filter, the filtered long wavelength signals are transmitted to the 2nd EMCCD；
First EMCCD and the 2nd EMCCD receive filtered short wavelength's signal and the filtering respectively
Long wavelength signals afterwards, to collect the binary channels fluorescence intensity image.
In a particular application, two paths of signals light is acquired respectively under the same conditions using two identical EMCCD,
It is able to achieve the data acquisition of bigger pixel region, but requirement is proposed to the high level of synchronization of two EMCCD, needs to synchronize control
System.
The super-resolution imaging method that the embodiment of the present invention proposes, after signal light is separated into two-way by dichroic mirror, respectively
Part stray light is filtered out through wave filter, then two paths of signals light is acquired by EMCCD, to improve biological sample anti-noise
Sound, environment resistant medium be uneven and the ability of anti-systematic jitters, improves imaging signal to noise ratio, simplifies later data processing
Process.
Example IV
The embodiment of the present invention carries out the implementation process of super-resolution imaging method provided by embodiment one to embodiment three
Illustrative explanation.
As shown in fig. 6, in the long wave long letter for the short wavelength's signal and long wavelength emission peak for acquiring short wavelength's emission peak respectively
After number, data processing need to be carried out according to this two paths of signals, obtain scale fluorescence images.Its data handling procedure can pass through Fig. 6
It indicates, wherein IB+IA is expressed as unsegregated long wavelength signals and short wavelength's signal, by super-resolution provided by embodiment three
Imaging method is separated into light long wavelength signals IB and short wavelength's signal IA after acquiring respectively；After the completion of acquisition, it is glimmering to carry out emission peak
When intensity ratio calculates, then the two paths of signals fluorescence intensity figure under synchronization is made into ratio, i.e.,Obtain scale fluorescence
A large amount of ratio chart progress algorithm is finally reconstructed to obtain super resolution image and with pseudo-colours to label Point Coloring, be obtained by image
Proportional-type super resolution image, wherein different colours represent ratioThe size of value.
Embodiment five
The embodiment of the present invention carries out the practical application of super-resolution imaging method provided by embodiment one to embodiment three
Illustrative explanation.To show how proportional-type super resolution image reflects that fluorescence probe marks in biological sample in practical applications
The sample parameters at place, to analyze the function of biological sample according to sample parameters.
The embodiment of the present invention proposes one kind, uses above-described embodiment one to super-resolution imaging side provided by example IV
Method, the example for realizing the super-resolution imaging of labeled sample microenvironment viscosity number quantitative study.
In the embodiment of the present invention, provided fluorescence probe is the fluorescence probe to viscosity-sensitive, when environment viscosity occurs
When change, steady state emission spectrum can also change accordingly.Short wavelength's emission peak is caused by structure A in probe, long wavelength
Emission peak is caused by structure B in probe, and overlapping region is not present in two fluorescence emission spectrums, and IB is structure B emission peak fluorescence
Intensity, IA are structure A emission peak fluorescence intensity.
In a particular application, functional relation of the value of IB/IA relative to viscosity is first constructed, i.e. measurement fluorescence probe is not
With the steady state emission spectrum in viscosity solution, the stable state spectrum under different viscosities is subjected to IB/IA processing respectively, obtained different viscous
It spends the value of lower IB/IA, then the mapping of each ratio point value is gone forward side by side line or nonlinear function approximation, obtain IB/IA relative to viscous
The fitting function relational expression of degree.
In practical applications, biological sample is marked using fluorescence probe, is excited using laser, and in oversubscription
It distinguishes in imaging system and is imaged, obtain a large amount of scale fluorescence images.When later data processing, first by each frame binary channels ratio
Fluorescent image IB and IA do ratio, and a large amount of fluorescence intensity ratio images are then carried out algorithm reconstruct, obtain labeled organism
The super resolution image of structure, and with pseudo-colours to each pixel shader of labeled structure, value, that is, IB/ of each pixel in image
The value of IA, different colours represent the size of IB/IA ratio, and the size of IB/IA ratio can be in the fitting of solution testing result
The corresponding viscosity number found under different ratios in functional relation, the i.e. difference of pseudo-colours represent point of microenvironment medium viscosity value
Cloth difference, while being the hyperfine structure figure for being labeled structure shown by the super resolution image reconstructed, finally realize micro-loop
The super-resolution imaging of border viscosity number quantitative study.
The embodiment of the present invention also proposes one kind, uses super-resolution imaging side provided by above-described embodiment one to embodiment three
Method, the example for realizing the super-resolution imaging of labeled sample mitochondrial membrane protein quantification research.
In the embodiment of the present invention, provided fluorescence probe be can specific labeled mitochondria memebrane protein fluorescence probe,
When carrying out solution or biological test, with after probe specific bond, steady state emission spectrum can also be sent out memebrane protein accordingly in environment
It is raw to change.Short wavelength's emission peak is caused by structure A in probe, and long wavelength emission peak is caused by structure B in probe, and two fluorescence
Overlapping region is not present in emission spectrum, and the two fluorescent emission generation is avoided to harass, and IB is structure B emission peak fluorescence intensity,
IA is structure A emission peak fluorescence intensity.
It is being particularly applicable in, is first constructing functional relation of the value of IB/IA relative to mitochondrial membrance protein content or concentration,
That is steady state emission spectrum of the measurement fluorescence probe 2 in the solution of different mitochondrial membrance protein contents or concentration, by different content
Or the stable state spectrum under concentration carries out IB/IA processing respectively, obtains IB/IA under different mitochondrial membrance protein contents or concentration
Value, then ratio value goes forward side by side line or nonlinear function approximation to content or concentration mapping, IB/IA is obtained relative to mitochondria
The fitting function relational expression of Membrane protein's amount or concentration.
In practical applications, organism mitochondrial membrance protein is marked using fluorescence probe, is carried out using exciting light
Excitation, and be imaged in super-resolution imaging system, obtain a large amount of scale fluorescence images.It, first will be every when later data processing
One frame binary channels fluorescence intensity image IB and IA do ratio, and a large amount of scale fluorescence images are then carried out algorithm reconstruct, obtain by
The super resolution image of organism structure is marked, and with pseudo-colours to each pixel shader of labeled structure, each pixel in image
Value, that is, IB/IA value of point, different colours represent the size of IB/IA ratio, and the size of IB/IA ratio can be in solution testing
As a result the corresponding protein content value found under different ratios in fitting function relational expression, i.e. the difference of pseudo-colours represents micro-
The distributional difference of protein content value in environment, while being the superfinishing for being labeled structure shown by the super resolution image reconstructed
Fine texture figure, the final super-resolution imaging for realizing the quantitative study of mitochondrial membrance protein content value.
Embodiment six
As shown in fig. 7, the embodiment of the invention provides a kind of super-resolution imaging devices 70, comprising:
Excitation module 71, for exciting the fluorescence probe on biological sample with double emission peaks, and will be close by exciting light
It is acted on the fluorescence probe after excitation with reagent；
Fluorescence intensity image collection module 72, for irradiating the fluorescence probe after affinity reagent acts on, obtaining by activated light
Obtain binary channels fluorescence intensity image；
Signal acquisition module 73, for being acquired in the binary channels fluorescence intensity image respectively, short wavelength's emission peak it is short
The long wavelength signals of wavelength signals and long wavelength emission peak；
Scale fluorescence images computing module 74 is acquired for selecting N frame binary channels fluorescence intensity image according to described respectively
Short wavelength's signal and the long wavelength signals, in the binary channels fluorescence intensity image of same frame, described in selection
The fluorescence intensity image of short wavelength's signal and the fluorescence intensity image of the long wavelength signals, and calculate separately its fluorescence intensity ratio
Value, obtains N number of scale fluorescence images, and wherein N is positive integer, and wherein N is positive integer；
Image reconstruction module 75, for carrying out weight to the scale fluorescence images by STORM super-resolution imaging method
Structure obtains super resolution image；
Image collection module 76 obtains ratio for colouring according to N number of scale fluorescence images to the super resolution image
Example type super resolution image.
In a particular application, above-mentioned fluorescence intensity image collection module 73 includes:
Short wavelength's emission peak acquiring unit, for obtaining the short wavelength according to the first structure in the fluorescence probe
Emission peak；
Long wavelength emission peak acquiring unit, for obtaining the long wavelength according to the second structure in the fluorescence probe
Emission peak；
Wherein, the first structure is energy acceptor, and second structure is energy donor；
Binary channels fluorescence intensity image acquisition unit, for acting on the activated light, described treated that fluorescence is visited
Needle makes the first structure and second structure alternately shine, obtains the binary channels fluorescence intensity image.
The embodiment of the present invention also provide a kind of terminal device include memory, processor and storage on a memory and can be
The computer program run on processor when the processor executes the computer program, is realized such as embodiment one to implementation
Each step in super-resolution imaging method described in example three.
The embodiment of the present invention also provides a kind of storage medium, and the storage medium is computer readable storage medium, thereon
It is stored with computer program, when the computer program is executed by processor, is realized as described in embodiment one to embodiment three
Super-resolution imaging method in each step.
Embodiment described above is merely illustrative of the technical solution of the present invention, rather than its limitations；Although previous embodiment
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of super-resolution imaging method characterized by comprising
By exciting light, the fluorescence probe on biological sample with double emission peaks is excited, and after affinity reagent is acted on excitation
Fluorescence probe on；
By activated light, the fluorescence probe after affinity reagent acts on is irradiated, binary channels fluorescence intensity image is formed；
It is acquired in the binary channels fluorescence intensity image respectively, short wavelength's signal of short wavelength's emission peak and long wavelength emission peak
Long wavelength signals；
N frame binary channels fluorescence intensity image is selected, according to the short wavelength's signal and the long wave long letter acquired respectively
Number, in the binary channels fluorescence intensity image of same frame, select the fluorescence intensity image of short wavelength's signal and described
The fluorescence intensity image of long wavelength signals, and its fluorescence intensity ratio is calculated separately, N number of scale fluorescence images are obtained, wherein N is
Positive integer；
By STORM super-resolution imaging method, the scale fluorescence images are reconstructed, obtain super resolution image；
The super resolution image is coloured according to N number of scale fluorescence images, obtains proportional-type super resolution image.
2. super-resolution imaging method as described in claim 1, which is characterized in that the super-resolution imaging method further include:
Establish the environmental parameter of the fluorescence probe and the function model of the fluorescence intensity ratio；
It is described to establish the fluorescence probe environmental parameter and the function model of the fluorescence intensity ratio includes:
The fluorescence probe for having double emission peaks is placed in probe solution test environment, the fluorescence is excited by exciting light
Probe；
The first parameter for changing the probe solution test environment, obtains under different first parameters, the fluorescence is visited
The steady-state fluorescence emission spectrum of needle；
According to double emission peaks in the steady-state fluorescence emission spectrum, fluorescence intensity ratio is calculated, and establishes first parameter
With the function model of the fluorescence intensity ratio.
3. super-resolution imaging method as described in claim 1, which is characterized in that it is described by activated light, irradiate affinity reagent
Fluorescence probe after effect, obtaining binary channels fluorescence intensity image includes:
According to the first structure in the fluorescence probe, short wavelength's signal is obtained；
According to the second structure in the fluorescence probe, the long wavelength signals are obtained；
When the exciting light irradiates the fluorescence probe, the first structure or the second structure emit fluorescence；
When the activated light acts on the fluorescence probe after the affinity reagent effect, the first structure and second structure
It shines according to replacing with the bonding state of the affinity reagent, forms the binary channels fluorescence intensity image.
4. super-resolution imaging method as claimed in claim 3, which is characterized in that the binary channels fluorescence intensity image, including
The fluorescence intensity image of short wavelength's signal and the fluorescence intensity image of long wavelength signals；
The fluorescence intensity image of short wavelength's signal corresponds to the luminescence process of the first structure in the fluorescence probe；
The fluorescence intensity image of the long wavelength signals corresponds to the luminescence process of the second structure in the fluorescence probe.
5. super-resolution imaging method as described in claim 1, which is characterized in that described to acquire the binary channels fluorescence respectively strong
It spends in image, short wavelength's signal of short wavelength's emission peak and the long wavelength signals at long wavelength emission peak include:
The exciting light is separated with short wavelength's signal and the long wavelength signals by the first dichroic mirror；
By the second dichroic mirror by after short wavelength's signal and long wavelength signals separation, short wavelength's signal is by the
One filter is sent to reflecting mirror, and filtered short wavelength's signal is transmitted to the first EMCCD by the reflecting mirror；The length
Wavelength signals pass through second filter, and the filtered long wavelength signals are transmitted to the first EMCCD；
The different zones of first EMCCD receive filtered short wavelength's signal and the filtered long wavelength respectively
Signal, to collect the binary channels fluorescence intensity image.
6. super-resolution imaging method as described in claim 1, which is characterized in that described to acquire the fluorescence emission spectrum respectively
In, short wavelength's signal of short wavelength's emission peak and the long wavelength signals at long wavelength emission peak, comprising:
The exciting light is separated with short wavelength's signal and the long wavelength signals by the first dichroic mirror；
By the second dichroic mirror by after short wavelength's signal and long wavelength signals separation, short wavelength's signal is by the
One filter is sent to reflecting mirror, and filtered short wavelength's signal is transmitted to the first EMCCD by the reflecting mirror；The length
Wavelength signals pass through second filter, and the filtered long wavelength signals are transmitted to the 2nd EMCCD；
First EMCCD and the 2nd EMCCD receives filtered short wavelength's signal and described filtered respectively
Long wavelength signals, to collect the binary channels fluorescence intensity image.
7. such as super-resolution imaging method as claimed in any one of claims 1 to 6, which is characterized in that the selection N frame binary channels is glimmering
Light intensity image, according to the short wavelength's signal and the long wavelength signals acquired respectively, in the described double of same frame
In channel fluorescence intensity image, the fluorescence intensity image of short wavelength's signal and the fluorescence intensity of the long wavelength signals are selected
Image, and its fluorescence intensity ratio is calculated separately, N number of scale fluorescence images are obtained, wherein N includes: for positive integer
According to the binary channels fluorescence intensity image and the short wavelength's signal acquired respectively, select short described in the n-th
The fluorescence intensity image of wavelength signals calculates its emission peak fluorescence intensity；
According to the binary channels fluorescence intensity image and the long wavelength signals acquired respectively, select long described in the n-th
The fluorescence intensity image of wavelength signals calculates its emission peak fluorescence intensity；
Calculate the calculation formula of the fluorescence intensity ratio are as follows:
Wherein, IBNFor the emission peak fluorescence intensity of the long wavelength signals, IANEmission peak fluorescence for short wavelength's signal is strong
Degree.
8. a kind of super-resolution imaging device characterized by comprising
Excitation module, for by exciting light, exciting the fluorescence probe on biological sample with double emission peaks, and by affinity reagent
On fluorescence probe after acting on excitation；
Fluorescence intensity image collection module, for irradiating the fluorescence probe after affinity reagent acts on, obtaining bilateral by activated light
Road fluorescence intensity image；
Signal acquisition module, for being acquired in the binary channels fluorescence intensity image respectively, the shortwave long letter of short wavelength's emission peak
Number and long wavelength emission peak long wavelength signals；
Scale fluorescence images computing module, for selecting N frame binary channels fluorescence intensity image, according to it is described acquire respectively described in
Short wavelength's signal and the long wavelength signals select the short wavelength in the binary channels fluorescence intensity image of same frame
The fluorescence intensity image of signal and the fluorescence intensity image of the long wavelength signals, and its fluorescence intensity ratio is calculated separately, it obtains
N number of scale fluorescence images are obtained, wherein N is positive integer, and wherein N is positive integer；
Image reconstruction module is obtained for by STORM super-resolution imaging method, the scale fluorescence images to be reconstructed
Super resolution image；
It is super to obtain proportional-type for colouring according to N number of scale fluorescence images to the super resolution image for image collection module
Resolution image.
9. a kind of terminal device, which is characterized in that on a memory and can be on a processor including memory, processor and storage
The computer program of operation, which is characterized in that when the processor executes the computer program, realize such as claim 1 to 7
Each step in described in any item super-resolution imaging methods.
10. a kind of storage medium, the storage medium is computer readable storage medium, is stored thereon with computer program,
Be characterized in that, when the computer program is executed by processor, realize super-resolution as described in any one of claim 1 to 7 at
Each step in image space method.
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