CN111982812A - Method for realizing optical super-resolution imaging by utilizing micron-scale liquid drops generated in real time - Google Patents
Method for realizing optical super-resolution imaging by utilizing micron-scale liquid drops generated in real time Download PDFInfo
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- CN111982812A CN111982812A CN202010832944.0A CN202010832944A CN111982812A CN 111982812 A CN111982812 A CN 111982812A CN 202010832944 A CN202010832944 A CN 202010832944A CN 111982812 A CN111982812 A CN 111982812A
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- 239000007788 liquid Substances 0.000 title claims abstract description 49
- 238000003384 imaging method Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000003287 optical effect Effects 0.000 title claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000011259 mixed solution Substances 0.000 claims abstract description 25
- 238000006073 displacement reaction Methods 0.000 claims abstract description 17
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 14
- 238000012576 optical tweezer Methods 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000002245 particle Substances 0.000 claims abstract description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000000523 sample Substances 0.000 description 26
- 239000004005 microsphere Substances 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 4
- 229910052710 silicon Inorganic materials 0.000 description 4
- 239000010703 silicon Substances 0.000 description 4
- 238000005286 illumination Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 238000002474 experimental method Methods 0.000 description 2
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- 230000004913 activation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
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- 238000004140 cleaning Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
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- 238000007654 immersion Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- QWVYNEUUYROOSZ-UHFFFAOYSA-N trioxido(oxo)vanadium;yttrium(3+) Chemical compound [Y+3].[O-][V]([O-])([O-])=O QWVYNEUUYROOSZ-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0181—Memory or computer-assisted visual determination
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Abstract
The invention relates to a method for performing super-resolution imaging by utilizing micron-scale liquid drops generated in real time. The method is characterized by comprising the following steps: initializing an optical tweezers system, and adjusting the power of a laser to enable the power of a laser beam reaching a solution in a sample cell to be 1w for forming and operating liquid drops; preparing a mixed solution, uniformly mixing a phosphate buffer solution and ethanol according to the volume ratio of 1 (3-8), and injecting the mixed solution into a sample cell; generation of droplets useful for imaging: placing the sample pool on a three-dimensional displacement platform, adjusting the displacement platform to enable laser beams to be converged at the axial middle position of the sample pool, and controlling the laser intensity and the irradiation time to control the particle size of liquid drops; and (3) observation imaging: and after the liquid drop grows, turning down the laser of the laser to enable the liquid drop not to grow any more, simultaneously enabling the optical trap to still capture the liquid drop, adjusting the displacement platform to move upwards to enable the liquid drop to just contact with the designated area of the sample, and observing and imaging.
Description
Technical Field
The invention relates to the field of super-resolution, in particular to a super-resolution imaging method for generating liquid drops in real time.
Background
Due to the diffraction limit, the resolution of the microscope is proportional to the wavelength of the illumination light and inversely proportional to the numerical aperture of the objective lens, and therefore, conventional optical microscopes cannot resolve microstructures smaller than 200nm in the visible range. However, with the development of the field of biological science and micro-nano manufacturing technology, people have higher requirements on microscopic imaging. Thus, over the past few decades, many super-resolution technologies have been developed.
For example, a scanning electron microscope is a main method for observing a micro-nano structure at the present stage, but the technology needs to electrify a sample under a vacuum condition, and a conducting layer is plated on an insulated object, so that the surface structure of the sample is destructive. The atomic force microscope can observe a biological sample, but the principle is that a tiny probe is used for performing contact scanning measurement on the surface of the sample, the measurement range is small, so the measurement speed is slow, a physical profile can be reconstructed only according to a scanning result, real-time imaging cannot be achieved, and equipment is very complex and expensive. The existing emerging microsphere super-resolution technology can realize the relatively simple and real-time imaging of a device, but still has certain defects: generally use the arm to realize the fixed of microballon and remove at microballon super resolution in-process, but along with the improvement of precision, the device is more and more complicated, and the cost of system also can increase substantially to at the in-process of fixed and removal microballon, the deformation can take place for the arm extrusion microballon, thereby leads to measuring result to produce the error. In recent years, the method for controlling the microspheres by using optical tweezers can solve the problems, but the defects of observing and imaging by using the microspheres are still difficult to overcome. For example, microspheres are difficult to clean from the surface of a sample after observation, and the risk of irreversible damage to the microstructure on the surface of the sample caused by repeated cleaning is great; in the process of utilizing the microspheres for super-resolution, the size of the microspheres cannot be controlled at any time, so that imaging is limited by a microsphere manufacturing process; in order to enable the optical tweezers to capture and manipulate the microspheres, the sample surface is often required to be treated, the surface activation energy is reduced, and the adhesion between the microspheres and the sample is reduced, but some sample surface microstructures are fragile and fluorinated to damage the microstructures, so that the types of the samples to be observed are limited.
Aiming at the current research situation, the invention provides a novel method for performing super-resolution imaging by using micron-scale quasi-spherical liquid drops generated in a mixed solution. The liquid drop is combined with the optical tweezers system, and the super-resolution capability is improved by adjusting the composition and the proportion of the solution. The microstructure imaging can be observed in real time at low cost without damage because the sample is not required to be cleaned specially after being observed.
Disclosure of Invention
The invention aims to provide a novel super-resolution imaging method, which combines micron-scale spherical liquid drops generated by a mixed solution under a laser beam with optical tweezers, can control the liquid drops, observes a microstructure smaller than 200nm in real time at low cost and without damage, and breaks through the diffraction limit. The composition of the mixed solution is selected on the basis of the principle that the relative refractive indexes of the liquid drop and the medium solution are high, and the size of the liquid drop is controlled on the basis of the principle that super-resolution imaging is clear. The technical scheme is as follows:
a method for performing super-resolution imaging by using micron-scale liquid drops generated in real time adopts a device comprising an optical tweezers system, an imaging system and a sample cell. The method is characterized by comprising the following steps:
(1) the optical tweezers system was initialized and the laser power was adjusted to 1w laser beam power into the sample cell solution for droplet formation and manipulation.
(2) Preparing a mixed solution, uniformly mixing a phosphate buffer solution and ethanol according to the volume ratio of 1 (3-8), and injecting the mixed solution into a sample cell.
(3) Generation of droplets useful for imaging: and (3) placing the sample cell on a three-dimensional displacement platform, adjusting the displacement platform to enable the laser beam to be converged at the axial middle position of the sample cell, and controlling the laser intensity and the irradiation time to control the particle size of the liquid drop.
(4) And (3) observation imaging: and after the liquid drop grows, turning down the laser of the laser to enable the liquid drop not to grow, enabling the optical trap to still capture the liquid drop, adjusting the displacement table to move upwards to enable the liquid drop to just contact with the designated area of the sample, and observing and imaging on an imaging interface.
Preferably, the phosphate buffer and the ethanol are mixed uniformly according to the volume ratio of 1: 5. After the liquid drop grows to 3-7um, the laser power is reduced to 30% of the original power, so that the liquid drop does not grow any more. The particle size of the liquid drops is controlled between 3um and 7 um.
Compared with the existing super-resolution technology, the invention utilizes the optical tweezers system to generate liquid drops in real time and control imaging. The mixed solution adopted for generating the liquid drops is a mixed solution of phosphate buffer solution and ethanol, and the volume ratio is 1: 5. The composition of the mixed solution is selected on the basis of high relative refractive index of the liquid drop and the medium solution, and the proportion of different solutions in the mixed solution is controlled on the basis of controllable liquid drop growth. Because the refractive index of ethanol is lower than that of isopropanol, the relative refractive index of the liquid drop and the medium solution is large, and the super-resolution capability is stronger. This can be achieved by FDTD solutions simulation, as shown in FIG. 1. When the volume ratio of the phosphate buffer solution to the ethanol is 1:5, the growth speed of the liquid drop is moderate, and the size of the liquid drop can be controlled. The device is simple, is convenient to operate, provides a brand-new super-resolution method, and enlarges the application range of the mixed solution for generating the liquid drops.
Drawings
FIG. 1 is a graph showing the comparison of the super-resolution power of three different mixed solutions.
Fig. 2 is a device for generating liquid drops in real time to realize optical super-resolution imaging.
In the figure: 1. a 1064nm laser; 2. a half-wave plate; 3. a dichroic mirror; 4. soaking the objective lens in water; 5. a sample cell; 6. a three-dimensional displacement table; 7. a semi-transparent semi-reflective mirror; 8. a lens; 9. a CCD camera; 10. an illumination source.
Fig. 3 is a diagram of the effect of real-time generation of droplets to achieve super-resolution imaging. A is a scanning electron micrograph of the observed sample grating; b is a drop map generated in real time; c is a super-resolution image observed through the droplet.
Detailed Description
The method and the device for generating liquid drops in real time for super-resolution imaging according to the present invention will be described in detail with reference to the following embodiments and the accompanying drawings.
In the experiment, the mixed solution used for forming the droplets is phosphate buffer solution (commercially available, the main components are potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride) and ethanol (commercially available, the content of ethanol is not less than 99.8%, and the water content is 0.2%), fig. 1 shows a droplet super-resolution capability contrast diagram formed by the mixed solution of the phosphate buffer solution and isopropanol, the mixed solution of the phosphate buffer solution and propanol, and the mixed solution of the phosphate buffer solution and the propanol, according to the photon nano-flow effect, stronger electric fields are formed on the back surfaces (near x ═ 4 um) of the droplets, and it can be seen that the super-resolution capability is stronger when ethanol and propanol are used, but the difference between the two is not much, and the. However, since propanol has a certain toxicity and cannot be widely used, the present invention uses a mixed solution of a phosphate buffer and ethanol in consideration of the above. When the volume ratio of the two solutions is 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8, microspheres are generated, but the proportion of the droplets with higher growth speed is not suitable to be selected because the sizes of the droplets need to be controlled, and the invention selects 1:5 is taken as the optimal mixture ratio.
The invention adopts the optical tweezers platform shown in figure 2 as the super-resolution platform. The platform consists of a diode-pumped neodymium-doped yttrium vanadate optical fiber coupling solid-state laser with 1064nm wavelength continuous output, an optical tweezers system consisting of a water immersion objective with a numerical aperture of 1.2, a working distance of 0.28mm and a magnification of 63, a half-wave plate, a dichroic mirror, a semi-transparent semi-reflecting mirror and a three-dimensional displacement platform; FIG. 3A shows a sample cell with a 50% duty cycle, a 360um period of silicon grating templates and 18 x 0.17mm cover slips; phosphate buffer and ethanol organic solvent were used in the experiment to form droplets; and the CCD camera, a 570nm illumination light source, a dichroic mirror and a lens.
The laser is converged at the axial middle position of the sample cell by controlling the displacement table to form and control a spherical liquid drop, the CCD lighting system is used for observing and imaging, and when the liquid drop grows to the size capable of being observed and imaged, the laser power is properly reduced, so that the liquid drop stops growing and can still be captured by the optical trap. And (5) moving the fine adjustment displacement platform upwards, and observing the CCD imaging interface at the same time until the micro-nano structure can be clearly seen.
The specific operation steps of the embodiment are as follows:
(1) and (3) initializing an optical tweezers system, adjusting a laser to enable the power of the laser to the sample cell to be 1w, and enabling the diameter of a focusing light spot to be 1um, so as to form and operate liquid drops.
(2) Preparing a sample cell, fixing a silicon grating template on a glass slide by using a sealing film, heating, placing a cover glass on the upper part, heating for 2 minutes at 100 ℃ on an electric heating plate, and melting the sealing film to enable the silicon grating template and the cover glass to be tightly combined to form a sample cell structure.
(3) Preparing a solution, mixing a phosphate buffer solution and an ethanol organic solvent in a volume ratio of 1:5, namely adding 10ul of the phosphate buffer solution into 40ul of the ethanol organic solvent for mixing, injecting the mixture into a sample cell, and fixing the sample cell on a displacement table.
(4) And moving the displacement table in the Z direction, putting a laser convergence point at the axial middle position of the sample pool, and adjusting the laser to 30% when the liquid drops grow to about 3um as shown in figure 3B. At which point the droplet no longer grows and the optical trap can still manipulate the droplet movement.
(5) Moving the displacement table, moving the microstructure part of the silicon grating template to be observed to the liquid dripping direction, finely adjusting the three-dimensional displacement table to ensure that the generated micro liquid drops just contact with the surface of the microstructure, and observing the microstructure imaging through a CCD interface, as shown in FIG. 3C: the striped structure can be seen through the droplets, where there are no droplets.
(6) When the microstructure at other positions is observed, the processes (4) and (5) are repeated.
The mixed solution can be a mixed solution of phosphate buffer solution and ethanol, or a mixed solution of phosphate buffer solution and propanol, or a mixed solution of phosphate buffer solution and acetone, and the droplets generated by different mixed solutions with different proportions have different super-resolution imaging capabilities.
Claims (4)
1. A method for performing super-resolution imaging by using micron-scale liquid drops generated in real time adopts a device comprising an optical tweezers system, an imaging system and a sample cell. The method is characterized by comprising the following steps:
(1) initializing an optical tweezers system, and adjusting the power of a laser to enable the power of a laser beam reaching a solution in a sample cell to be 1w for forming and operating liquid drops;
(2) preparing a mixed solution, uniformly mixing a phosphate buffer solution and ethanol according to the volume ratio of 1 (3-8), and injecting the mixed solution into a sample cell;
(3) generation of droplets useful for imaging: placing the sample pool on a three-dimensional displacement platform, adjusting the displacement platform to enable laser beams to be converged at the axial middle position of the sample pool, and controlling the laser intensity and the irradiation time to control the particle size of liquid drops;
(4) and (3) observation imaging: and after the liquid drop grows, turning down the laser of the laser to enable the liquid drop not to grow, enabling the optical trap to still capture the liquid drop, adjusting the displacement table to move upwards to enable the liquid drop to just contact with the designated area of the sample, and observing and imaging on an imaging interface.
2. The method according to claim 1, wherein the phosphate buffer and the ethanol are mixed uniformly in a volume ratio of 1: 5.
3. The method of claim 1, wherein after the droplet grows to 3-7um, the laser power is reduced to 30% of the original power, so that the droplet does not grow any more.
4. The method of claim 1, wherein the droplet size is controlled to be 3um to 7 um.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113484322A (en) * | 2021-07-13 | 2021-10-08 | 天津大学 | Optical tweezers super-resolution imaging method and system capable of feeding back axial optical trap position in real time |
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| CN104852259A (en) * | 2015-05-22 | 2015-08-19 | 哈尔滨工程大学 | Liquid drop whispering gallery mode laser and manufacturing method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113484322A (en) * | 2021-07-13 | 2021-10-08 | 天津大学 | Optical tweezers super-resolution imaging method and system capable of feeding back axial optical trap position in real time |
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Application publication date: 20201124 |