阅读说明：本技术 一种实现波长复用的超分辨多维的光学存储方法 (Super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing ) 是由 张静宇 颜志 于 2020-04-01 设计创作，主要内容包括：本发明公开了一种实现波长复用的超分辨多维的光学存储方法,步骤包括：取第一可逆开关荧光蛋白和第二可逆开关荧光蛋白混合；采用第一光束和第二光束将目标点处激发至荧光态；采用第三光束和第四光束对目标点处的环形区域进行辐照,使其由荧光态转换为非荧光态,且中央区域为荧光态；控制所述第五光束和第六光束的辐照时间对所述中央区域进行辐照,使所述中央区域内形成四种荧光混合态；采用所述第一光束和第二光束将除所述中央区域以外的区域转换为荧光态。本发明通过引入两种不同的可逆开关荧光蛋白,对目标点进行标记时,将不同的可逆开关荧光蛋白进行漂白并产生四种荧光混合态,实现波长复用的超分辨多维光学存储,提高了光学存储维度。(The invention discloses a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing, which comprises the following steps: mixing the first reversible switch fluorescent protein and the second reversible switch fluorescent protein; exciting the target point to a fluorescent state using the first and second light beams; irradiating the annular region at the target point by using a third light beam and a fourth light beam to convert the annular region from a fluorescent state to a non-fluorescent state, wherein the central region is in a fluorescent state; controlling the irradiation time of the fifth light beam and the sixth light beam to irradiate the central region, so that four fluorescence mixed states are formed in the central region; converting regions other than the central region into a fluorescent state using the first and second light beams. According to the invention, two different reversible switch fluorescent proteins are introduced, when a target point is marked, the different reversible switch fluorescent proteins are bleached and four fluorescence mixed states are generated, so that the wavelength-multiplexed super-resolution multi-dimensional optical storage is realized, and the optical storage dimension is improved.)

1. A super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing is characterized by comprising the following steps:

taking and uniformly mixing the first reversible switch fluorescent protein and the second reversible switch fluorescent protein;

irradiating a target point by adopting a first light beam and a second light beam in sequence, and exciting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein to respective fluorescence states respectively;

irradiating an annular region at a target point by adopting a third light beam and a fourth light beam in sequence, respectively converting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein in the annular region from a fluorescent state to a non-fluorescent state, wherein a central region surrounded by the annular region shows the fluorescent states of the two reversible switch fluorescent proteins;

irradiating the central region by using a fifth light beam and a sixth light beam, controlling the irradiation time of the fifth light beam and the sixth light beam, respectively converting the respective fluorescence states of the first reversible switching fluorescent protein and the second reversible switching fluorescent protein in the central region into bleaching states of different degrees, and forming four fluorescence mixed states in the central region;

and irradiating the region except the central region by using the first light beam and the second light beam, and respectively converting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein from a non-fluorescent state to a fluorescent state for matching with the four fluorescent mixed states of the central region to realize wavelength multiplexing super-resolution multi-dimensional optical storage.

2. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 1, wherein the first reversibly switchable fluorescent protein and the second reversibly switchable fluorescent protein emit different fluorescence and have different permanently bleached wavelengths;

the wavelength and energy of the first reversible switch fluorescent protein during state switching do not affect the second reversible switch fluorescent protein.

3. The method according to claim 2, wherein the first light beam and the second light beam are two different gaussian light beams, the first light beam irradiates the first reversibly switched fluorescent protein and converts the first reversibly switched fluorescent protein into the fluorescent state, and the second light beam irradiates the second reversibly switched fluorescent protein and converts the second reversibly switched fluorescent protein into the fluorescent state.

4. The method according to claim 2, wherein the third beam and the fourth beam are two different laguerre gaussian beams, the third beam irradiates the first reversible switching fluorescent protein to convert it from a fluorescent state to a non-fluorescent state, and the fourth beam irradiates the second reversible switching fluorescent protein to convert it from a fluorescent state to a non-fluorescent state.

5. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 2, wherein the fifth beam and the sixth beam are two different gaussian beams, the fifth beam irradiates the first reversibly switched fluorescent protein to convert the first reversibly switched fluorescent protein from a fluorescent state to a bleached state, and the sixth beam irradiates the second reversibly switched fluorescent protein to convert the second reversibly switched fluorescent protein from the fluorescent state to the bleached state.

6. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 5, wherein the step of generating one of the four mixed fluorescence states comprises:

the irradiation time of the fifth light beam is not 0, and the irradiation time of the sixth light beam is 0, in the central region, the first reversible switch fluorescent protein is converted from a fluorescent state to a bleached state, and the second reversible switch fluorescent protein is in a fluorescent state.

7. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 5, wherein the step of generating one of the four mixed fluorescence states comprises:

the irradiation time of the fifth light beam is 0, and the irradiation time of the sixth light beam is not 0, in the central region, the first reversible switch fluorescent protein is in a fluorescent state, and the second reversible switch fluorescent protein is converted from the fluorescent state to a bleached state.

8. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 5, wherein the step of generating one of the four mixed fluorescence states comprises:

the irradiation time of the fifth light beam and the irradiation time of the sixth light beam are both not 0, and in the central area, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are converted from respective fluorescence states into a bleaching state.

9. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 5, wherein the step of generating one of the four mixed fluorescence states comprises:

the irradiation time of the fifth light beam and the irradiation time of the sixth light beam are both 0, and in the central region, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are in respective fluorescence states.

10. The method for realizing wavelength-multiplexed super-resolution multi-dimensional optical storage according to claim 2, wherein the central region is a region smaller than a diffraction limit.

Technical Field

The invention relates to the field of optical storage, in particular to a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing.

Background

In recent years, the development of world economy and society is greatly promoted by digital information technology, and with the development of artificial intelligence and big data, the requirements of various industries on information data storage are increasing day by day, and the amount of information data generated by various departments is estimated to almost double every year. Currently, optical data storage technology is mature day by day due to advantages of low energy consumption, high data security and the like, but the data storage capacity of the optical data storage technology is greatly restricted by the optical diffraction limit. Optical data storage has therefore developed primarily in both super-resolution and multi-dimensional aspects to increase the capacity of optical data storage.

In recent years, many super-resolution imaging technologies, including "stimulated radiation depletion microscopy" (STED) "and its derivative technology" reversible saturated optical fluorescence conversion microscopy "(RESOLFT)" utilize fluorophore labeled molecules to achieve imaging beyond the diffraction limit, and the RESOLFT technology can achieve data storage of super-resolution labels. The single STED technology in the prior art needs to apply high-power-loss light, the problem of light damage is inevitably brought in the super-resolution imaging process, meanwhile, the dimensionality of optical data storage is greatly limited, and the requirement of multi-dimensionality optical data storage is difficult to meet. It is therefore desirable to provide a new optical storage method for solving the problems of the prior art.

Disclosure of Invention

The invention aims to provide a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing, which is used for solving the problem that the optical data storage method in the prior art is difficult to meet the requirement of multi-dimensional optical data storage.

In order to solve the above technical problem, the present invention provides a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing, which is characterized in that the method comprises the following steps: taking and uniformly mixing the first reversible switch fluorescent protein and the second reversible switch fluorescent protein; irradiating the target point by adopting a first light beam and a second light beam in sequence, and exciting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein to respective fluorescent states respectively; irradiating the annular region at the target point by adopting a third light beam and a fourth light beam in sequence to respectively convert the first reversible switch fluorescent protein and the second reversible switch fluorescent protein in the annular region from a fluorescent state to a non-fluorescent state, wherein the central region surrounded by the annular region shows the fluorescent states of the two reversible switch fluorescent proteins; irradiating the central area by using a fifth light beam and a sixth light beam, controlling the irradiation time of the fifth light beam and the sixth light beam, respectively converting the respective fluorescence states of the first reversible switch fluorescent protein and the second reversible switch fluorescent protein in the central area into bleaching states of different degrees, and forming four fluorescence mixed states in the central area; and irradiating the region except the central region by adopting the first light beam and the second light beam, and respectively converting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein from a non-fluorescent state to a fluorescent state for matching with four fluorescent mixed states of the central region to realize wavelength multiplexing super-resolution multi-dimensional optical storage.

Wherein, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein emit different fluorescence, and the wavelength of the permanent bleaching is different; the wavelength and energy of the first reversible switch fluorescent protein during state switching do not affect the second reversible switch fluorescent protein.

The first light beam and the second light beam are two different Gaussian light beams, the first light beam irradiates the first reversible switch fluorescent protein and then converts the first reversible switch fluorescent protein into a fluorescent state, and the second light beam irradiates the second reversible switch fluorescent protein and then converts the second reversible switch fluorescent protein into the fluorescent state.

The third light beam and the fourth light beam are two different Laguerre Gaussian light beams, the third light beam irradiates the first reversible switch fluorescent protein to enable the first reversible switch fluorescent protein to be converted from a fluorescent state to a non-fluorescent state, and the fourth light beam irradiates the second reversible switch fluorescent protein to enable the second reversible switch fluorescent protein to be converted from the fluorescent state to the non-fluorescent state.

The fifth light beam and the sixth light beam are two different Gaussian light beams, the fifth light beam irradiates the first reversible switch fluorescent protein to enable the first reversible switch fluorescent protein to be converted from a fluorescent state to a bleached state, and the sixth light beam irradiates the second reversible switch fluorescent protein to enable the second reversible switch fluorescent protein to be converted from the fluorescent state to the bleached state.

Wherein, the generation step of one fluorescence mixed state in the four fluorescence mixed states is as follows: the irradiation time of the fifth light beam is not 0, and the irradiation time of the sixth light beam is 0, in the central region, the first reversible switch fluorescent protein is converted from a fluorescent state to a bleached state, and the second reversible switch fluorescent protein is in a fluorescent state.

Wherein, the generation step of one fluorescence mixed state in the four fluorescence mixed states is as follows: the irradiation time of the fifth light beam is 0, and the irradiation time of the sixth light beam is not 0, in the central region, the first reversible switch fluorescent protein is in a fluorescent state, and the second reversible switch fluorescent protein is converted from the fluorescent state to a bleached state.

Wherein, the generation step of one fluorescence mixed state in the four fluorescence mixed states is as follows: the irradiation time of the fifth light beam and the irradiation time of the sixth light beam are not 0, and in the central area, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are converted into a bleaching state from respective fluorescent states.

Wherein, the generation step of one fluorescence mixed state in the four fluorescence mixed states is as follows: the irradiation time of the fifth light beam and the irradiation time of the sixth light beam are both 0, and in the central area, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are in respective fluorescence states.

Wherein the central region is a region less than the diffraction limit.

The invention has the beneficial effects that: different from the situation of the prior art, the invention provides a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing, wherein two different reversible switch fluorescent proteins are introduced, in the process of marking a target point, the different reversible switch fluorescent proteins are bleached and four fluorescent mixed states are generated, different codes can be correspondingly realized by controlling different wavelengths, energies or irradiation time, the super-resolution multi-dimensional optical storage for wavelength multiplexing is realized, and the optical storage dimension is improved.

Drawings

FIG. 1 is a flow chart of an embodiment of a super-resolution multi-dimensional optical storage method for wavelength multiplexing according to the present invention;

FIG. 2 is a schematic diagram of an apparatus for implementing an embodiment of the super-resolution multi-dimensional optical storage method for wavelength multiplexing according to the present invention;

FIG. 3 is a schematic diagram of state transitions of two reversible fluorescence-emitting proteins in an embodiment of the super-resolution multi-dimensional optical storage method for wavelength multiplexing according to the present invention;

FIG. 4 is a diagram of a labeling process according to an embodiment of the super-resolution multi-dimensional optical storage method for wavelength multiplexing according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Referring to fig. 1-2, fig. 1 is a flowchart illustrating an embodiment of a super-resolution multi-dimensional optical storage method for implementing wavelength multiplexing according to the present invention, and fig. 2 is a schematic diagram illustrating an apparatus of an embodiment of a super-resolution multi-dimensional optical storage method for implementing wavelength multiplexing according to the present invention. The invention discloses a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing, which comprises the following steps:

s1: and (3) taking the first reversible switch fluorescent protein and the second reversible switch fluorescent protein, and uniformly mixing. In this embodiment, the first reversible switching fluorescent protein and the second reversible switching fluorescent protein that are selected emit different fluorescence, and the wavelength that is permanently bleached is also different, and the wavelength and energy of the first reversible switching fluorescent protein when the state is switched do not affect the second reversible switching fluorescent protein, that is, the two reversible switching fluorescent proteins that are selected are fluorescent proteins that do not affect each other, and both of the two reversible switching fluorescent proteins can be switched between a fluorescent state and a non-fluorescent state, and for the purpose of distinguishing, the fluorescence emitted by the first reversible switching fluorescent protein in the fluorescent state can be recorded as fluorescence 1, and the fluorescence emitted by the second reversible switching fluorescent protein in the fluorescent state can be recorded as fluorescence 2.

S2: and irradiating the target point by adopting the first light beam and the second light beam in sequence, and exciting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein to respective fluorescence states respectively. In this step, the first light beam and the second light beam are two different gaussian light beams, the first light beam irradiates the first reversible switch fluorescent protein and converts the first reversible switch fluorescent protein into a fluorescent state, the second light beam irradiates the second reversible switch fluorescent protein and converts the second reversible switch fluorescent protein into the fluorescent state, the first light beam and the second light beam respectively pass through the dichroic mirror and the objective mirror and reach target points of the two reversible switch fluorescent proteins, and the first reversible switch fluorescent protein and the second reversible switch fluorescent protein at the target points are excited to respective fluorescent states, namely, at this time, the first light beam and the second light beam are in a state of overlapping fluorescence 1 and fluorescence 2.

S3: and irradiating the annular region at the target point by adopting the third light beam and the fourth light beam in sequence to respectively convert the first reversible switch fluorescent protein and the second reversible switch fluorescent protein in the annular region from a fluorescent state to a non-fluorescent state, wherein the central region surrounded by the annular region shows the fluorescent states of the two reversible switch fluorescent proteins. In the step, the third light beam and the fourth light beam are two different Laguerre Gauss light beams, the third light beam irradiates the first reversible switch fluorescent protein to enable the first reversible switch fluorescent protein to be converted from a fluorescent state to a non-fluorescent state, the fourth light beam irradiates the second reversible switch fluorescent protein to enable the second reversible switch fluorescent protein to be converted from the fluorescent state to the non-fluorescent state, the third light beam and the fourth light beam respectively pass through a dichroic mirror and an objective mirror and then reach target points of the two reversible switch fluorescent proteins, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein in the annular region are converted from the fluorescent state to the non-fluorescent state, and the central region surrounded by the annular region still keeps a state of overlapping fluorescence 1 and fluorescence 2; thus, after the annular irradiation of the Laguerre Gaussian beam, a point smaller than the diffraction limit is formed in the central region, and the central region and the annular region show two states of a fluorescence state and a non-fluorescence state.

S4: and irradiating the central area by using the fifth light beam and the sixth light beam, controlling the irradiation time of the fifth light beam and the sixth light beam, respectively converting the respective fluorescence states of the first reversible switch fluorescent protein and the second reversible switch fluorescent protein in the central area into bleaching states with different degrees, and forming four fluorescence mixed states in the central area. The four fluorescence mixing states here respectively include four generation processes, which are as follows: (1) the irradiation time of the fifth light beam is not 0, and the irradiation time of the sixth light beam is 0, in the central area, the first reversible switch fluorescent protein is converted from a fluorescent state to a bleached state, and the second reversible switch fluorescent protein is in a fluorescent state, namely the central area shows fluorescence 2; (2) the irradiation time of the fifth light beam is 0, and the irradiation time of the sixth light beam is not 0, in the central area, the first reversible switch fluorescent protein is in a fluorescent state, the second reversible switch fluorescent protein is converted from the fluorescent state to a bleached state, namely, the central area shows fluorescence 1; (3) the irradiation time of the fifth light beam and the irradiation time of the sixth light beam are not 0, and in the central area, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are converted into a bleaching state from respective fluorescent states, namely the central area shows no fluorescence; (4) the irradiation time of the fifth light beam and the irradiation time of the sixth light beam are both 0, and in the central region, the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are in respective fluorescence states, that is, the central region shows a state of overlapping fluorescence 1 and fluorescence 2.

In this step, the fifth light beam and the sixth light beam are two different gaussian light beams, the fifth light beam irradiates the first reversible switch fluorescent protein to convert the fluorescent state into a bleached state, and the sixth light beam irradiates the second reversible switch fluorescent protein to convert the fluorescent state into the bleached state, namely the fifth light beam and the sixth light beam respectively play a role in bleaching the first reversible switch fluorescent protein and the second reversible switch fluorescent protein; the central area can present four different fluorescence mixing states by controlling the irradiation time of the fifth light beam and the sixth light beam, the four different fluorescence mixing states mainly represent different combinations of the types of the fluorescence proteins to be bleached and the bleaching degree, and corresponding combination modes can be selected according to actual coding requirements, so that light beam switches with different wavelengths or energies can be controlled to correspond to different codes, and a wavelength multiplexing mapping relation is established.

S5: and irradiating the region except the central region by adopting the first light beam and the second light beam, and respectively converting the first reversible switch fluorescent protein and the second reversible switch fluorescent protein from a non-fluorescent state to a fluorescent state for matching with four fluorescent mixed states of the central region to realize wavelength multiplexing super-resolution multi-dimensional optical storage. In this step, the point of the central region beyond the diffraction limit is still the fluorescence mixed state formed after the step S4, and the part outside the central region is converted from the non-fluorescence state to the fluorescence state, so as to complete the marking process for a certain target point, and when marking a plurality of target points, the multi-point marking process can be realized by repeating the operations of the steps S2 to S5, which is not described herein again.

Further, with reference to the above description of the super-resolution multi-dimensional optical storage method for implementing wavelength multiplexing according to the present invention, a principle of the super-resolution multi-dimensional optical storage method is described, please refer to fig. 3 to 4, fig. 3 is a schematic diagram of state transition of two types of reversible fluorescence proteins in an embodiment of the super-resolution multi-dimensional optical storage method for implementing wavelength multiplexing according to the present invention, and fig. 4 is a diagram of a labeling process in an embodiment of the super-resolution multi-dimensional optical storage method for implementing wavelength multiplexing according to the present invention. As shown in fig. 3, for the present embodiment, the first reversible switching fluorescent protein and the second reversible switching fluorescent protein both have three states, i.e., a non-fluorescent state, a fluorescent state, and a bleached state, and have reversibility when switching between the non-fluorescent state and the fluorescent state, but not reversibility when switching from the fluorescent state to the bleached state; however, for the first reversible switch fluorescent protein and the second reversible switch fluorescent protein, the expression of the first reversible switch fluorescent protein in the fluorescence state is marked as fluorescence 1, the expression of the second reversible switch fluorescent protein in the fluorescence state is marked as fluorescence 2, the wavelength and the energy during state switching are different and do not affect each other, in other words, when the first reversible switch fluorescent protein and the second reversible switch fluorescent protein are mixed for application, two sets of relatively independent state conversion mechanisms are formed, reasonable coding is performed through the arrangement and combination of the two independent mechanisms, and the dimension of optical storage can be improved.

As shown in fig. 4, the states of the marks at various times during the implementation of the super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing are shown, in the figure, the processes 1 to 4 respectively correspondingly show the steps S2 to S5, and in the step S4, the control of the irradiation time corresponding to the fifth light beam and the sixth light beam is selected according to the actual coding requirements, and four fluorescence mixed states of the superposition of fluorescence 1 and fluorescence 2, the fluorescence 1, the fluorescence 2, and the non-fluorescence shown in the figure can be shown in the central region, and because the central region is a region exceeding the diffraction limit, the fluorescence mixed state formed after the step S4 can be used to the step S5, so that the marks of the target points can be flexibly regulated, and the mapping relationship between the marks and the codes formed by the steps can well realize the super-resolution multi-dimensional optical storage for realizing wavelength multiplexing.

It should be noted that the selected reversible switch fluorescent protein, the light beam wavelength, and the conversion state have a close correspondence, and the reversible switch fluorescent protein, the light beam wavelength, and the conversion state can be selected according to the actual encoding requirement, which is not limited herein.

Different from the situation of the prior art, the invention provides a super-resolution multi-dimensional optical storage method for realizing wavelength multiplexing, wherein two different reversible switch fluorescent proteins are introduced, in the process of marking a target point, the different reversible switch fluorescent proteins are bleached and four fluorescent mixed states are generated, different codes can be correspondingly realized by controlling different wavelengths, energies or irradiation time, the super-resolution multi-dimensional optical storage for wavelength multiplexing is realized, and the optical storage dimension is improved.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.