Two-color STED microscopy in living cells

Abstract

Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells. Using improved SNAPf and CLIPf technologies to label epidermal growth factor (EGF) and EGF receptor (EGFR), we report resolutions of 78 nm and 82 nm for 22 sequential two-color scans in living cells.

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

“…Tagging of two different proteins by SNAP and CLIP allows for simultaneous labeling of two different proteins in different colors (Gautier et al, 2008;Prendergast et al, 2011). More recently, variants of SNAP and CLIP named SNAPf and CLIPf have been developed that present faster reaction kinetics (Pellett et al, 2011;Sun et al, 2011). We evaluated SNAPf and CLIPf performance in vivo by sideby-side comparison with SNAP and CLIP, using the intracellular protein CENP-A as a labeling target (data not shown and Figure 7A).…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…Finally, the third aspect of imaging that needs to be addressed is resolution, where we find great efforts directed towards super-resolution techniques, including stimulated emission depletion (STED) [63][64][65][66], structured illumination microscopy (SIM) [67,68], photoactivated localization microscopy (PALM) [69,70], and stochastic optical reconstruction microscopy (STORM) [71][72][73][74]. These approaches offer sub-diffraction-limited resolution and have opened new ways of exploring the submicron world within the living cell.…”

“…The STED approach can be extended to a more general principle as shown in reversible saturable/switchable optically linear fluorescence transition (RESOLFT) microscopy [76], which can be performed with multiple kinds of switchable fluorescent molecules, slightly reducing the intensity needed for imaging. With STED a resolution of 70 nm has been reached in living cells [65,77], as can be seen in Figure 3E where a two-color STED microscope is used to image a living HEK293 cell over an interval of more than 4 minutes [65]. Drawbacks of targeted switching techniques such as STED and RESOLFT are that they often require high intensities, resulting in high photo-bleaching and photo-toxic effects.…”

Fluorescence nanoscopy. Methods and applications

“…Besides being permeable to the plasma membrane, tagging intracellular proteins with synthetic fluorophores requires genetically encoded labelling systems (see "Probes and labelling systems" section). Using SNAP-and CLIP-tag labelling technologies, dualcolour live-cell STED imaging was demonstrated at ≈80 nm resolution [29].…”

“…This is the basis of SIM: the projection of a known pattern (structured illumination, a) on an unknown high-frequency pattern, f h (the sample, b), results in a pattern of lower frequency, f l (dark stripes on c), from which the unknown pattern can be recovered: f h =f l +Δf, where Δf is the frequency offset between the dark stripes on c and the known illumination pattern on a CLIP fusion proteins can be labelled simultaneously and specifically with different molecular probes, dual-colour STED imaging of living cells is also possible [29].…”