Dr. Emil B. Kromann, The Bewersdorf Laboratory,
Dept. of Cell Biology, Yale School of Medicine, CT, USA.
Dept. of Biomedical Engineering, Yale Graduate School of Arts and Sciences, CT, USA

Live-Cell Imaging at the Nanoscale

Super-resolution imaging techniques have enabled researchers to take a closer look at sub-cellular structures and dynamics. I will briefly review two of these imaging techniques: Fluorescence PhotoActivation Localization Microscopy (FPALM aka. PALM/STORM), and Stimulated Emission Depletion (STED) microscopy. I will present recent advancements made in the Bewersdorf Lab, emphasizing my own work with live-cell STED microscopy:

Using stimulated emission depletion (STED) microscopy, we can image distinctly labeled structures with 20-30 nm resolution in fixed cells. Despite being built upon confocal microscopy (a powerful technique for observing live-cell dynamics) STED microscopy is rarely used to image living specimens. One of the most dominant reasons has been the lack of STED-compatible, photostable dye-pairs for intracellular labeling.

Figure 1: STED image (deconvolved) of mitochondria (green) and endoplasmic reticulum (magenta) in a living cell.

I will present STED instrumentation and a novel labeling strategy specifically geared towards two-color live-cell STED imaging of intracellular targets. In my instrument, I apply a spatial light modulator for beam shaping and aberration correction, FPGA based laser control, gated detection and a 16 kHz resonant scanning mirror, which reduces pixel dwell time and photobleaching. In combination with a novel dye pair my instrument enables continuous (up to 90 frames) two-color STED imaging of intracellular targets in living cells, with acquisition of one two-color STED image every two seconds (10 x 10 µm field of view, <50 nm resolution). See: Bottanelli, Kromann et al., Nature Communications, 2016.

Host: Associate professor Victoria Birkedal, iNANO & Dept. of Chemistry, Aarhus University