Dynamic super-resolution fluorescence imaging technology

Abstract

The invention provides a dynamic super-resolution fluorescence imaging technology. The invention relates to the super-resolution fluorescence imaging technology capable of capturing dynamic images. (1) A fluorescence microscopic imaging device is capable of super-resolution imaging and low-resolution imaging, and the field of view regions and object planes observed by two imaging modes are the same. For the same observation object, an image acquired by convolution of a super-resolution image and a conversion function is equivalent to a low-resolution image, and the conversion function is measured. (2) A dynamic image capturing and image processing method corresponding to the device comprises the steps that super-resolution and low-resolution modules are alternately used for continuous capturing through sequential control, and images are recorded and saved as a video; image processing is carried out; a small proportion of super-resolution images in the original video are taken as priorknowledge; the measured conversion function is combined; and low-resolution images in the original video are converted into super-resolution images through speculative calculation. The dynamic super-resolution fluorescence imaging technology provided by the invention is used for acquiring dynamic super-resolution images with high time resolution and long time.

Description

technical field [0001] The invention relates to the technical field of super-resolution fluorescence imaging, in particular to a super-resolution fluorescence imaging method capable of shooting dynamic images and capable of time resolution. Background technique [0002] Fluorescence microscopy is very suitable for imaging biomolecules, cells, etc. due to its far-field, non-invasive characteristics. Traditional fluorescence microscopes (such as wide-field illumination, scanning confocal, total internal reflection illumination, etc.) are limited by the Abbe diffraction limit, and their spatial resolution cannot be better than 200nm, so they cannot distinguish finer structures. In the past ten years, a variety of fluorescence imaging methods that break through the diffraction limit have been developed, called super-resolution imaging, such as stimulated emission depletion imaging (STED), reversible saturation / switching optical transition imaging (RESOLFT), and photoactivated lo...

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Problems solved by technology

[0003] STED, SSIM / NSIM and other methods require higher intensity laser illumination than traditional fluorescence imaging methods, which seriously aggravate the fluorescence bleaching and phototoxicity of the sample compared with the weak light illumination of traditional fluorescence imaging; RESOLFT / PALM / FPALM / STORM Methods such as PALM / FPALM / STORM and other molecular positioning methods need to repeatedly turn on / off fluorescent molecules, and the fluorescent molecules will be bleached after multiple switching cycles; in addition, PALM / FPALM / STORM and other molecular positioning methods take a relatively long time to acquire a complete image

Therefore, when these imaging methods with spatial super-resolution capabilities are applied to the imaging of dynamic processes in living cells, the number of image frames that can be continuously captured is far less than that of traditional fluorescence imaging methods, which means that they cannot perform long-term imaging with high temporal resolution. question

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