Single-molecule and super-resolution microscopies of complex organizations for biological applications
Résumé
The optical microscopy of single molecules has recently been beneficial for many applications, in particular in biology as acknowledged by the Nobel Prize of Chemistry in 2014. It allows a sub-wavelength localization of isolated molecules and subtle probing of their spatio-temporal nano-environments on living cells. It also allows designing innovative strategies to obtain super-resolved optical images i.e. with resolution below the diffraction limit.
In a first part, after introducing “standard” single molecule detection strategies currently used in biology, I will present novel developments in the field. In particular, for many single-molecule microscopy applications, more photostable nanoprobes than fluorescent ones are desirable. For this aim, we developed several years ago far-field photothermal methods based on absorption instead of luminescence. Such approaches do not suffer from the inherent photophysical limitations of luminescent objects and allows the ultra-sensitive detection of tiny absorbing individual nano-objects such as gold nanoparticles down to 5 nm in cells or carbon nanotubes. I will present our current efforts to reduce the size of the functional nano-objects and thus obtain single-molecule photothermal nanoprobes for biomolecules found in confined cellular environment (adhesion sites, synapses etc...)
The second part of my presentation will be devoted to the presentation of super-resolution microscopy methods. It is indeed crucial to study a large ensemble of molecules on a single cell while keeping the sub-wavelength localization provided by single molecule microscopy. In order to study the dynamical properties of endogenous membrane proteins found at high densities on living cells we developed a new single molecule super-resolution technique, named uPAINT. Interestingly, uPAINT does not require the use of photo-activable dyes allowing easy multi-color super-resolution imaging and single molecule tracking. Different applications of uPAINT will be presented, in particularly the first demonstration of super-resolution imaging of functional receptors in interaction. This last result was obtained combining super-resolution microscopy and single molecule FRET.