Seminar by Emil Boye Kromann

LIVE-CELL IMAGING AT THE NANOSCALE

Emil B. Kromann

Biomedical Engineering Group, Dept. of Electrical Engineering, Technical University of Denmark

Bewersdorf Laboratory, Dept. of Cell Biology, Yale School of Medicine

Super-resolution fluorescence microscopy techniques have allowed us to observe the nanoscale organization of sub-cellular elements like specific lipid- or protein-species. Being conceptual derivatives of conventional fluorescence microscopes, the super-resolution techniques also allow observation of nanoscale dynamics. However, this application has been very limited due to constraints imposed by photobleaching, imaging speed, system reliability and available fluorescent markers.

     In my talk, I address these constraints by presenting a super-resolution STED microscope specifically geared for capturing nanoscale dynamics in living cells. The microscope incorporates a spatial light modulator for laser focus optimization and automatic alignment, enabling three-dimensional resolution improvement and making the system reliable for routine applications. Photobleaching is reduced by fast scanning (16 kHz) with a resonant mirror, controlled light exposure (RESCue STED), and the introduction of a novel photostable dye-pair (ATTO590 and SiR) suitable for intracellular labeling. Together, these advancements enable continuous (>90 frames) multi-plane imaging of intracellular dynamics, with nanoscale resolution (<50 nm) and acquisition of one two-color image frame (10.24 × 10.24 µm FOV) per second. 

The presented microscope was constructed in the Bewersdorf Lab at Yale University.

See: Bottanelli, Kromann et al., Nature Communications, 2016.