Cell biophysics of fluorescent probes for super-resolution optical microscopy
published: July 9, 2018, recorded: May 2018, views: 371
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Several light microscopy methods how to break the classical Abbe’s optical resolution limit exist nowadays. The most developed and at the same time providing the best theoretical resolution of 20 nm is the PhotoActivation Localization Microscopy (PALM)/STochastic Optical Reconstruction Microscopy (STORM). For PALM, unique optical property of photoswitchable/photoactivatable fluorescent proteins, namely irreversible photoconversion is principal factor to achieve desired resolution. These proteins were originally found in reef corals and their optical and physico-chemical properties, as excitation-emission spectra, quantum yield and monomeric forms were improved by genetic technologies and molecular modelling. In case of STORM photoswitching is facilitated by chemically synthetized chromophores. Due to high resolution demands, PALM/STORM work with fixated samples only. To be possible to acquire living cells, although by reduction in resolution, several new approaches were introduced including STimulated Emission Depletion (STED) microscopy. In case of STED microscopy resolution drops down to 60 nm by rapid bleaching of a donut around the central excited point by a STED laser. Additional benefit of STED microscopy represents use of standard fluorescent proteins. Independently, advanced fluorophores attached to antibodies are commercially available in these days as well. In this lecture we will present principles of the methods mentioned above and the properties of chromophores available for PALM/STORM. We will present several practical applications of PALM/STORM including mitochondrially targeted photoconvertible protein KikumeGR1 and outer mitochondrial membrane targeted protein Fis-EOS. Moreover, we will introduce the STED microscopy available in our laboratory and will present practical aspects to optimize the image acquisition.
Download slides: biophysics2018_cagalinec_optical_microscopy_01.pdf (2.7 MB)
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