CN111489768B - Optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing - Google Patents
Optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing Download PDFInfo
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- CN111489768B CN111489768B CN202010250891.1A CN202010250891A CN111489768B CN 111489768 B CN111489768 B CN 111489768B CN 202010250891 A CN202010250891 A CN 202010250891A CN 111489768 B CN111489768 B CN 111489768B
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- G11—INFORMATION STORAGE
- G11B—INFORMATION STORAGE BASED ON RELATIVE MOVEMENT BETWEEN RECORD CARRIER AND TRANSDUCER
- G11B7/00—Recording or reproducing by optical means, e.g. recording using a thermal beam of optical radiation by modifying optical properties or the physical structure, reproducing using an optical beam at lower power by sensing optical properties; Record carriers therefor
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- G11B—INFORMATION STORAGE BASED ON RELATIVE MOVEMENT BETWEEN RECORD CARRIER AND TRANSDUCER
- G11B7/00—Recording or reproducing by optical means, e.g. recording using a thermal beam of optical radiation by modifying optical properties or the physical structure, reproducing using an optical beam at lower power by sensing optical properties; Record carriers therefor
- G11B7/24—Record carriers characterised by shape, structure or physical properties, or by the selection of the material
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Abstract
The invention discloses an optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing, which comprises the following steps: exciting the reversibly switched fluorescent protein at the target point to a first state with a second light beam; irradiating an annular region at a target point by using a first light beam to convert the reversible switch fluorescent protein in the annular region from a first state to a second state; irradiating the central region by using a third light beam to convert the reversible switch fluorescent protein in the central region from a first state to a mixed state; and irradiating the region except the central region by using a second light beam, wherein the obtained reversible switch fluorescent protein is in a form that the first state surrounds and surrounds the mixed state. The invention converts the state of the reversible switch fluorescent protein through different light beams, so that the reversible switch fluorescent protein is in a surrounding form, realizes multidimensional marks of different codes, and achieves the aim of multi-order multiplexing optical storage of super-resolution fluorescence intensity.
Description
Technical Field
The invention relates to the field of optical storage, in particular to an optical storage method for realizing super-resolution fluorescence intensity multi-order 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 an optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing, which is used for solving the problem that the super-resolution 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 problems, the present invention provides an optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing, comprising the steps of: taking reversible switch fluorescent protein and crystallizing to form crystals; exciting the reversible switch fluorescent protein at the target point to a first state by adopting a second light beam; irradiating the annular region at the target point by using a first light beam to convert the reversible switch fluorescent protein in the annular region from a first state to a second state, wherein the central region surrounded by the annular region is in the first state; irradiating the central area by using a third light beam to convert the reversible switch fluorescent protein in the central area from the first state into a mixed state consisting of the first state and the third state; and irradiating the region except the central region by using a second light beam to convert the region from the second state to the first state, wherein the obtained reversible switch fluorescent protein is in a form of surrounding mixed state surrounded by the first state and is used for marking the target point in a multi-stage fluorescence intensity state.
The first state, the second state and the third state are three different fluorescent protein states, the reversible switch fluorescent protein has reversibility when being switched between the first state and the second state, and the reversible switch fluorescent protein also has the characteristic of being switched from the first state to the third state.
The reversible switch fluorescent protein is a fluorescent protein with the characteristic of switching among different states after being irradiated by light beams with different wavelengths or energies.
The second light beam is a Gaussian light beam and has the characteristic of converting the reversible switch fluorescent protein from the second state to the first state after the reversible switch fluorescent protein is irradiated.
The first light beam is a Laguerre Gaussian light beam and has the characteristic of converting the reversible switch fluorescent protein from a first state to a second state after the reversible switch fluorescent protein is irradiated.
The third light beam is a Gaussian light beam and has the characteristic of converting the reversible switch fluorescent protein from the first state to the third state after the reversible switch fluorescent protein is irradiated.
Wherein the central region is a region less than the diffraction limit.
In the step of irradiating the central area with the third light beam, the mixed state is made to present a characteristic of multi-order change by controlling the irradiation time of the central area with the third light beam.
The invention has the beneficial effects that: the invention provides an optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing, which is characterized in that a first light beam and a second light beam are used for carrying out state conversion on reversible switch fluorescent protein, so that the reversible switch fluorescent protein is in a surrounding form, and the central area is smaller than the diffraction limit; the central area is converted into a third state under the irradiation of a third light beam, and multidimensional marks with different codes can be realized by adjusting the exposure time of the third light beam, so that the aim of multi-order multiplexing optical storage of the super-resolution fluorescence intensity is fulfilled.
Drawings
FIG. 1 is a flow chart of an embodiment of an optical storage method for realizing multi-order multiplexing of super-resolution fluorescence intensity in the present invention;
FIG. 2 is a schematic diagram of an apparatus for implementing an embodiment of the optical storage method for multi-order multiplexing of super-resolution fluorescence intensity in the present invention;
FIG. 3 is a schematic diagram illustrating state transition of reversible fluorescence protein in an embodiment of the optical storage method for realizing multi-order multiplexing of super-resolution fluorescence intensity in the present invention;
FIG. 4 is a diagram of a labeling process of reversible fluorescence protein in an embodiment of the optical storage method for realizing multi-order multiplexing of super-resolution fluorescence intensity in 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 an optical storage method for achieving multi-order multiplexing of super-resolution fluorescence intensity according to the present invention, and fig. 2 is a schematic diagram illustrating an apparatus for implementing an embodiment of an optical storage method for achieving multi-order multiplexing of super-resolution fluorescence intensity according to the present invention. The optical storage method for realizing the multi-order multiplexing of the super-resolution fluorescence intensity comprises the following steps:
s1: taking the reversible switch fluorescent protein and crystallizing to form crystals. In this embodiment, the selected reversibly switchable fluorescent protein can be switched between different states after being irradiated by light beams with different wavelengths or energies, for example, the selected reversibly switchable fluorescent protein can be switched between a first state and a second state after being irradiated by the light beams, or can be switched between the first state and a third state, and the switching process may be a reversible process or an irreversible process, which is not limited herein.
S2: and exciting the reversibly switched fluorescent protein at the target point to a first state by using a second light beam. In this embodiment, the second light beam is a gaussian light beam and has a characteristic of converting the reversible switch fluorescent protein from the second state to the first state after being irradiated, and in this step, the second light beam sequentially passes through the dichroic mirror and the objective lens and reaches the target point of the reversible switch fluorescent protein, so that the reversible switch fluorescent protein at the target point is converted from the second state to the first state; the objective lens used may preferably be a high numerical aperture objective lens that allows tight focusing of the beam.
S3: and irradiating the annular region at the target point by using the first light beam to convert the reversible switch fluorescent protein in the annular region from the first state to the second state, wherein the central region surrounded by the annular region is in the first state. In this embodiment, the first light beam is a laguerre gaussian light beam and has a characteristic of converting the reversible switch fluorescent protein from the first state to the second state after being irradiated, and in this step, the first light beam sequentially passes through a dichroic mirror and an objective lens and reaches a target point of the reversible switch fluorescent protein, so that the reversible switch fluorescent protein at the target point is converted from the first state to the second state, and the central region is still in the first state; 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 assume two different states.
S4: and irradiating the central area by using a third light beam to convert the reversible switch fluorescent protein in the central area from the first state into a mixed state consisting of the first state and the third state. In this embodiment, the third light beam is a gaussian light beam and has a characteristic of converting the reversible switch fluorescent protein from the first state to the third state after being irradiated; in the step, the irradiation time of the third light beam on the reversible switch fluorescent protein can be controlled to convert the first state in the central area into the third state, and the irradiation time of the third light beam on the central area is controlled to generate a mixed state effect with different degrees and multi-step change, wherein the mixed state comprises the first state and the third state.
S5: and irradiating the region except the central region by using a second light beam to convert the region from the second state to the first state, wherein the obtained reversible switch fluorescent protein is in a form that the first state surrounds and surrounds the mixed state. In the step, the point of the central area exceeding the diffraction limit is still in a mixed state formed by the first state and the third state, and the part outside the central area is converted into the first state from the second state.
The following describes an implementation process of the above optical storage method for realizing multi-order multiplexing of super-resolution fluorescence intensity by using a specific embodiment.
Example 1
In this embodiment, IrisFP is selected as a reversible switch fluorescent protein, and the first state, the second state, and the third state correspond to a green fluorescent state, a non-fluorescent state, and a red fluorescent state, respectively; IrisFP can realize repeated reversible switching between a green fluorescence state and a non-fluorescence state for many times, and can realize unidirectional switching from the green fluorescence state to a red fluorescence state; in the green fluorescence state, IrisFP can have a wavelength of 488nm and an energy of 52W/cm2The light beam of (2) excites fluorescence with a wavelength of 516nm, and in a red fluorescence state, the fluorescence can be excited at a wavelength of 551nm and an energy of 125W/cm2The light beam is irradiated to excite the fluorescence with the wavelength of 580 nm; the first light beam is a Laguerre Gaussian beam, and the second light beam and the third light beam are Gaussian beams.
In this embodiment, when the above optical storage method for implementing multi-order multiplexing of super-resolution fluorescence intensity is executed, the specific steps are as follows: forming an IrisFP crystal by taking an IrisFP crystal; the adopted wavelength is 405nm, and the energy is 47W/cm2The second light beam excites the IrisFP crystal at the target point to a green fluorescence state; the adopted wavelength is 488nm, and the energy is 500W/cm2The first light beam irradiates an annular region at a target point, so that the IrisFP crystal in the annular region is converted from a green fluorescence state to a non-fluorescence state, and a central region surrounded by the annular region is in the green fluorescence state; the adopted wavelength is 405nm, and the energy is 263W/cm2The central area is irradiated by the third light beam, so that the IrisFP crystal in the central area is converted into a mixed state consisting of green fluorescence and red fluorescence from a green fluorescence state; adopting 405nm and 47W/cm of energy2The second light beam irradiates the region except the central region to convert the non-fluorescence state into the green fluorescence state, the obtained IrisFP crystal is in a form that the green fluorescence state surrounds and surrounds the mixed state, thereby completing the marking of the target point, and according to the code, the marking of various conditions exceeding the diffraction limit point in the central region can be realized, including the complete green fluorescence state,The complete red fluorescence state and the mixed state formed by the green fluorescence state and the red fluorescence state achieve the aim of multi-dimensional multiplexing.
Example 2
In this embodiment, the rsEGFP is selected as a reversible switch fluorescent protein, and the first state, the second state and the third state respectively correspond to a fluorescent state, a non-fluorescent state and a bleached state; the rsEGFP can realize repeated reversible switching between a fluorescence state and a non-fluorescence state, and can realize unidirectional switching from the fluorescence state to a bleaching state; the first light beam is a Laguerre Gaussian beam, and the second light beam and the third light beam are Gaussian beams.
In this embodiment, when the above optical storage method for implementing multi-order multiplexing of super-resolution fluorescence intensity is executed, the specific steps are as follows: adopting rsEGFP crystal to form rsEGFP crystal; exciting the rsEGFP crystal at the target point to a fluorescent state by adopting a second light beam; irradiating the annular region at the target point by using a first light beam to convert the rsEGFP crystal in the annular region from a fluorescent state to a non-fluorescent state, wherein the central region surrounded by the annular region is in the fluorescent state; irradiating the central region by using a third light beam to convert the fluorescence of the rsEGFP crystal in the central region into a mixed state consisting of a fluorescence state and a bleaching state; and irradiating the region except the central region by adopting a second light beam to convert the region except the central region from a non-fluorescence state into a fluorescence state, wherein the obtained rsEGFP crystal is in a form that the fluorescence state surrounds and surrounds a mixed state, so that the marking of a target point is completed, and according to coding, the marking of various conditions exceeding a diffraction limit point in the central region can be realized, including a complete fluorescence state, a complete bleaching state and a mixed state formed by the fluorescence state and the bleaching state, so that the aim of multi-dimensional multiplexing is fulfilled.
Further, with reference to the above-mentioned optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing and the specific embodiment thereof, the principle of the optical storage method is described, please refer to fig. 3 to 4, fig. 3 is a schematic diagram of state conversion of the reversible fluorescence protein in an embodiment of the optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing in the present invention, and fig. 4 is a diagram of a labeling process of the reversible fluorescence protein in an embodiment of the optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing in the present invention. As shown in fig. 3, for the present embodiment, the first state, the second state and the third state are three different fluorescent protein states, and the reversible switch fluorescent protein has reversibility when switching between the first state and the second state, but does not have reversibility when switching from the first state to the third state, so that target points to be labeled can be combined in the three states, and the dimension of optical storage is improved. As shown in fig. 4, a, b, c, d, and e represent the mark states at each time in the implementation process of the optical storage method for implementing super-resolution fluorescence intensity multi-order multiplexing, the steps S2 to S5 are respectively shown between two adjacent times, and since the final time e represents a mixed state in which the central region is the first state and the third state, and the portion outside the central region is the first state, only the exposure time of the third light beam needs to be adjusted, so that the mixed state in the central region can present different mixing degrees, thereby implementing the marks with different codes and achieving the purpose of super-resolution fluorescence intensity multi-order multiplexing optical storage.
It should be noted that the selected reversible switch fluorescent protein, the wavelength or energy of the light beam, and the conversion state have a close correspondence, and the reversible switch fluorescent protein, the wavelength or energy of the light beam, and the conversion state can be selected according to the actual encoding requirement, which is not limited herein. In addition, the above steps S2 to S5 only describe the method for marking a single target point, and when marking a plurality of target points, the multi-point marking process can be implemented by repeating the operations of the above steps S2 to S5, which is not described herein again.
The invention provides an optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing, which is characterized in that a first light beam and a second light beam are used for carrying out state conversion on reversible switch fluorescent protein, so that the reversible switch fluorescent protein is in a surrounding form, and the central area is smaller than the diffraction limit; the central area is converted into a third state under the irradiation of a third light beam, and multidimensional marks with different codes can be realized by adjusting the exposure time of the third light beam, so that the aim of multi-order multiplexing optical storage of the super-resolution fluorescence intensity is fulfilled.
It should be noted that the above embodiments belong to the same inventive concept, and the description of each embodiment has a different emphasis, and reference may be made to the description in other embodiments where the description in individual embodiments is not detailed.
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.
Claims (7)
1. An optical storage method for realizing super-resolution fluorescence intensity multi-order multiplexing is characterized by comprising the following steps:
taking reversible switch fluorescent protein and crystallizing to form crystals;
exciting the reversibly switched fluorescent protein at the target point to a first state with a second light beam;
irradiating an annular region at a target point by using a first light beam to convert the reversible switch fluorescent protein in the annular region from a first state to a second state, wherein a central region surrounded by the annular region is in the first state, and the central region is a region smaller than a diffraction limit;
irradiating the central region by using a third light beam to convert the reversible switch fluorescent protein in the central region from the first state into a mixed state consisting of the first state and the third state;
and irradiating the region except the central region by using a second light beam to convert the region from the second state to the first state, wherein the obtained reversible switch fluorescent protein is in a form that the first state surrounds the mixed state, and is used for marking the target point in a multi-stage fluorescence intensity state.
2. The method as claimed in claim 1, wherein the first state, the second state and the third state are three different states of the fluorescent protein, the reversibly switchable fluorescent protein is reversible when switched between the first state and the second state, and the reversibly switchable fluorescent protein further has a property of being switched from the first state to the third state.
3. The optical storage method for realizing multi-order multiplexing of super-resolution fluorescence intensity as claimed in claim 1, wherein the reversible switching fluorescent protein is a fluorescent protein having a characteristic of switching between different states after being irradiated by light beams with different wavelengths or energies.
4. The method as claimed in claim 1, wherein the second beam is a gaussian beam and has a property of transforming the reversibly switched fluorescent protein from the second state to the first state after being irradiated.
5. The method as claimed in claim 1, wherein the first beam is a laguerre gaussian beam and has a property of transforming the reversibly switched fluorescent protein from the first state to the second state after being irradiated.
6. The method as claimed in claim 1, wherein the third beam is a gaussian beam and has a property of transforming the reversibly switched fluorescent protein from the first state to the third state after being irradiated.
7. The method as claimed in claim 1, wherein in the step of irradiating the central region with the third beam, the mixed state is characterized by multi-step variation by controlling the irradiation time of the central region with the third beam.
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