Advanced DNA imaging: improving spatial resolution.. (ImaginDNA)
Advanced DNA imaging: improving spatial resolution and contrast through photoswitching
Date du début: 1 févr. 2013,
Date de fin: 31 janv. 2017
Fluorescence microscopy is one of the most convenient and widespread tools used in the life sciences. An important challenge, however, is to improve its spatial resolution, which is limited to about 200 nm. Recent “super-resolution” techniques such as photoactivation-localization microscopy (PALM) can provide images with a spatial resolution of tens of nm. Most studies performed with these techniques have imaged the nanoscale distribution of proteins. However, little progress has been seen on DNA super-resolution imaging due to challenges in labelling. My main research is aimed at exploring new ways to label DNA in high density with photoswitchable fluorophores and improving spatial resolution in fluorescence microscopy. This will allow opening up new opportunities to study a broad range of problems in Biology and Nanoscience. My recent work has shown that PALM-like imaging of DNA can achieve a spatial resolution below 40 nm by using intercalating cyanine dyes in combination with a buffer that promotes photoblinking. This proposal aims at optimizing this approach, and at finding new alternatives for DNA super-resolution imaging. I propose two research lines: 1) Use correlative atomic force microscopy and PALM-like imaging to optimize the above methodology. As first targets, DNA origami will be used. Correlative microscopy will then be applied to study chromosome structure; 2) Study the photophysical properties at the ensemble and single-molecule level of a new material, CyDNA (DNA highly substituted with cyanine dyes in a controllable way). Bringing together Cy3 and Cy5 dyes in the same CyDNA with high density results in a photoswitch with new properties. CyDNA photoswitching will be used for super-resolution imaging in combination with fluorescence in situ hybridization, and also to improve image contrast with optical lock-in detection imaging. Further opportunities to apply the developed methodology will also be identified throughout the fellowship
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