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