University of Minnesota
School of Physics & Astronomy

Physics and Astronomy Calendar

Thursday, May 9th 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Angel Mancebo, University of Minnesota
Subject: Mitigating phototoxicity in (super-resolution) fluorescence microscopy using precisely calibrated feedback illumination

Fluorescence microscopy is a powerful method for measuring spatial and dynamic information of specifically-labeled proteins in living cells. With the development of superresolution fluorescence microscopy methods diffraction-limited structures can be resolved and quantitative measurements of the spatio-temporal organization of proteins can be made. Because single-molecule localization-based superresolution microscopy methods rely on a spatio-temporally sparse distribution of fluorescent labels, long data acquisition times and high excitation powers are needed to localize a high fraction of molecules. Satisfying these two requirements results in a large amount of energy delivered to the cells by the lasers which is detrimental to cell health. The unfavorable implications are two-fold: unintended cell stress may compromise an experiment; and dying cells become autofluorescent making it impossible to detect single molecules. We developed a technique which makes use of a digital mirror array for accurately and precisely patterning the short-wavelength activation laser. This technique can be applied to any conventional or superresolution fluorescence microscopy experiment that requires spatial patterning as well as optogenetic experiments. We demonstrate this technique on budding yeast for confining the activation laser to only the plasma membrane where proteins with the plextrin homology domain are labeled with the photoactivatable protein mEos2. Patterned photoactivation mitigates cell death which improves single-molecule statistics by enabling longer acquisition times and an increase in the number of single-molecule localizations.

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