Random positioning super-resolution microscopy method and device based on fluorescence-emission kill mechanism

Abstract

The invention discloses a random positioning super-resolution microscopy method and device based on a fluorescence-emission kill mechanism. The method includes the following steps: coaxial and common-path exciting light and restraining light are simultaneously focused on a sample; the position on the sample, with the stimulated fluorescence-emission characteristic, randomly emits fluorescent light; fluorescent signals are collected to generated a sparse fluorescent distribution image; diffraction spots are positioned in a unimolecule manner, and the final product is obtained after different fluorescent light positioning images are synthesized. The device includes a first laser light source, a second laser light source, a reflector, a first dichroic mirror, a kohler mirror group, a second dichroic mirror, a microobjective, a sample, an optical filter, a field lens, an ocular, a wide field sensing element and a computer. The invention has the advantages that the resolution ratio and fineness are high, 20 nm crosswise super-resolution images can be obtained; the structure is simple, and the cost is low; irreversible damage of intensive laser or fluorescence photobleaching to samples is reduced, and repeating utilization ratio of samples is increased; the function extensibility is strong.

Description

technical field [0001] The invention relates to the field of microscopic observation methods and equipment for biological samples, in particular to a random positioning super-resolution microscopy method and device based on a fluorescence emission suppression mechanism. Background technique [0002] Nanotechnology and biotechnology are the fastest-growing and hottest scientific fields in the 21st century. Nanotechnology has a wide range of applications, including imaging, measurement, processing, manipulation, etc. within the scale of 1-100nm. Many important organisms such as glucose, antibodies, viruses, etc. are in this scale range, and the need to study these tiny objects has driven the development of high-resolution microscopy. In turn, the development of super-resolution microscopy imaging technology has also promoted the progress of the entire life sciences. A major advantage of light microscopy over other microscopy techniques is the ability to study living cells in...

AI Technical Summary

Problems solved by technology

The electron microscopic imaging technology developed in the 1930s and various non-optical probe scanning microscopic imaging technologies that emerged in the early 1980s have nanometer or even higher resolution capabilities, but they exist to varying degrees. The complex structure of the system, the harsh requirements of the imaging and detection environment, and the cumbersome sample processing are difficult, especially the important optical information of the sample (such as reflectivity, refractive index, polarization state, and spectrum, etc.) cannot be obtained, so it cannot completely replace optical microscopic imaging. status

Although the above two methods can achieve the purpose of super-resolution microscopy, they are not perfect. For example, the target switching and reading microscopy techniques need to apply strong suppression light to the sample (generally hundreds of megaW / cm 2 ); while random switch and readout microscopy requires pre-bleaching of fluorescent molecules in the observation area

Both of these situations are prone to irreversible damage to the observed sample

Images