Abstract

The volumetric architecture of organelles and molecules inside cells can only be elucidated with microscopes featuring sufficiently high spatial resolution in 3D. However, current super resolution microscopy methods suffer from severe limitations when applied to live cell imaging such as insufficient resolving power along the optical axis, long recording times and/or photobleaching. Our lab developed MoNaLISA and 3D pRESOLFT techniques capable of 3D isotropic 50 nm imaging in living cells. The parallelization of the illumination module enables rapid (1-2 Hz) acquisition of large field of view (~40-100x40-100 µm2), which allow full-length cellular imaging without compromising speed and spatial resolution. It is achieved with the creation of a new interference pattern featuring an array of 3D-confined and equally spaced intensity minima. This pattern is used to transiently switch photochromic fluorescent probes such as reversible switchable fluorescent protein in dark states allowing a targeted 3D confinement of the fluorescence emission. The 3D organization of several cellular structures can be visualized, even dynamically, which open new possibilities for 3D in situ biological studies of proteins and organelles in living cells. We are currently using our methods to study organelle such as exosomes, ER and mitochondria proteins trafficking as well as synaptic plasticity in firing neurons.