2020 Winter School Les Houches
General introduction to fluorescence
Introduction to fluorescent proteins (FP)
Theoretical concepts on fluorescent protein properties: folding, structure and dynamics, chromophore maturation, protonation states, oligomerization tendency, phototransformations mechanisms, photostability.
Biological function, ecological niche, diversity and evolutionary tree of FPs
Biophysical characterization of FPs in practice. FP expression and purification, optical steady state and ultrafast spectroscopy, X-ray crystallography, NMR, molecular dynamics simulations. Genetic encoding, codon optimization, fusion proteins, functional assays
Novel Fluorescent proteins
FP engineering, directed evolution, rational engineering
FP photophysics at the single molecule level, photon budget, blinking, nano-environmental effects, nonlinear light-induced FP-based sensors
Introduction to chemical dyes
Photophysics and photo-transformations, anti-fade and switching buffers, environmental influences
Advanced fluorophore photophysics: metallic environment, plasmonic resonance, nanoantennas
How to label my target in practice: labeling of proteins, DNA, lipids, cellular compartments, membranes. Halo/Snap/Clip tags, nanobodies, click chemistry. Fluorogenic probes, spontaneously blinking probes
Labeling with Q-dots and nanoparticles, lipids and sugar labeling
Labeling with expanded genetic code
Resonance energy transfer with various fluorophores and luminophores
FPs and chemical dyes: pro’s and cont’s
Objectives of the school :
Fluorescence microscopy techniques play an essential role in biology and medicine. A very rapidly developing field concerns nanoscale imaging (“nanoscopy”), which today ties together different fields of biology such as structural and cell biology. Similarly, “single molecule” imaging and spectroscopy techniques are in rapid development and play a central role in life science research. All fluorescent microscopy methods are based on labelling samples with appropriate markers: fluorescent proteins, organic fluorophores or nanoparticles. All these type of markers are characterized by very complex photophysical behaviors, leading to blinking, chromatic convertion, photoreactivity, etc… However, nanoscopy and single molecule spectroscopy or imaging, specifically rely on the ability to understand and control these properties.
The school we offer is motivated by the fact that more and more laboratories around the world are committed to implementing advanced microscopy techniques, especially super-resolution, with an increasing number of dedicated platforms. However, there is a lack of overall knowledge about the selection of fluorescent markers, their properties and mechanisms, and the type of artifacts they can create. The school’s objective, through interdisciplinary teaching between physics, chemistry and biology, is to overcome this gap by transferring knowledge to the participants’ laboratories. Our school also aims to make participants aware of new developments in the field in order to prepare them to become major players in the breakthroughs that may result from them.
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March 29 - April 3 2020
France Les Houches is a village located in Chamonix valley, in the French Alps.