Fluorescence microscopy is a defining technology in cellular biology but the diffraction of light limits the resolution of standard microscopes to ~200 nm, thus preventing detailed analyses of molecular structures. We are actively interested in microscopy methods based on stochastic photoswitching and computational localization of single fluorophores (“pointillism”), which enable greatly improved resolution. We have implemented pointillist super-resolution microscopy hardware and reconstruction software (Figure 2) and applied this imaging technique to various studies of pathogens in their cellular hosts .
In addition to pursuing biological applications, we aim to push some of the limitations of existing super-resolution methods. One such limitation is the use of invasive fluorescent tags, which can perturb biological functions. We demonstrated FlAsH-PALM, which combines FlAsH-tetracysteine tagging with stochastic localization microscopy. FlAsH-PALM allows to obtain <30 nm resolution images of delicate microbial proteins such as the HIV integrase, without disrupting their function .
We currently explore different experimental and computational strategies to further improve the information content, spatio-temporal resolution and reliability of pointillist microscopy.
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