Method for carrying out super-resolution imaging by utilizing micron-scale liquid drops generated in real time

Abstract

The invention relates to a method for carrying out super-resolution imaging by utilizing micron-scale liquid drops generated in real time. An adopted device comprises an optical tweezer system, an imaging system and a sample pool. The method is characterized by comprising the following steps: initializing an optical tweezer system, adjusting the power of a laser to enable the power of a laser beamreaching a sample pool solution to be 1w, and forming and operating liquid drops; preparing a mixed solution, uniformly mixing a phosphate buffer solution and ethanol according to a volume ratio of 1: (3-8), and injecting the mixed solution into a sample pool; generating droplets available for imaging: the sample pool is placed on a three-dimensional displacement table, the displacement table isadjusted to enable laser beams to converge at the axial middle position of the sample pool, and the particle size of the liquid drops is controlled by controlling the laser intensity and the irradiation time; observing and imaging: after the droplet growth is completed, reducing the laser of the laser so that the droplet does not grow any more, and meanwhile, the optical trap can still capture thedroplet and adjust the displacement table to move upwards, and thus the droplet is just in contact with a specified area of the sample for observing and imaging.

Description

technical field [0001] The invention relates to the field of super-resolution, in particular to a super-resolution imaging method for generating liquid droplets in real time. Background technique [0002] Due to the existence of 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. Therefore, in the visible range, the traditional optical microscope cannot resolve microstructures smaller than 200nm. However, with the development of biological sciences and micro-nano manufacturing technology, people have higher requirements for microscopic imaging. Therefore, in the past few decades, many super-resolution techniques have emerged. [0003] For example, scanning electron microscopy is the main method for observing micro-nano structures at this stage, but this technique must energize the sample under vacuum conditions, and a conductive layer m...

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Problems solved by technology

[0003] For example, scanning electron microscopy is the main method for observing micro-nano structures at this stage, but this technique must energize the sample under vacuum conditions, and a conductive layer must be plated on insulating objects, so it is destructive to the surface structure of the sample.

Atomic force microscopy can observe biological samples, but its principle is to use tiny probes to perform contact scanning measurement on the surface of the sample. The measurement range is very small, so the measurement speed is very slow and the physical outline can only be reconstructed based on the scanning results, and real-time imaging cannot be achieved. And the equipment is very complicated and expensive

The emerging microsphere super-resolution technology can achieve relatively simple device and real-time imaging, but there are still some defects: in the process of microsphere super-resolution, the mechanical arm is generally used to realize the fixation and movement of the microsphere, but with the improvement of precision As the device becomes more and more complex, the cost of the system will also increase significantly, and in the process of fixing and moving the microspheres, the mechanical arm will deform the microspheres, which will lead to errors in the measurement results

In recent years, the method of using optical tweezers to control microspheres can solve the above problems, but many defects of microsphere observation and imaging are still difficult to overcome

For example, it is difficult to clean the microspheres from the surface of the sample after observation, and the risk of irreversible damage to the microstructure of the sample surface is extremely high after repeated cleaning; in the process of super-resolution using microspheres, the size of the microspheres cannot be controlled at any time, so the imaging is limited by the microspheres. Manufacturing process limitations; in order to enable optical tweezers to capture and manipulate microspheres, it is often necessary to treat the surface of the sample to reduce the surface activation energy and reduce the adhesion between the microspheres and the sample, but the surface microstructure of some samples is relatively fragile. Potentially disrupts the microstructure, thus limiting the variety of observed samples

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