Time-Resolved STED Microscopy

Introduced more than 30 years ago, stimulated emission depletion (STED) microscopy has raised to a standard and widely used method for imaging in the life sciences. Thanks to continuous technological progress, STED microscopy can now provide effective sub-diffraction spatial resolution, while preserving most of the useful aspects of fluorescence microscopy, such as optical sectioning, molecular specificity and sensitivity, and live-cell compatibility.

 

This seminar starts by presenting the basic principles of STED microscopy and the three significant conditions required for its implementation, specifically the spatial, spectral, and temporal conditions. Towards these conditions, the seminar introduces the initial limitations and problems of STED microscopy and how modern STED microscopy architectures have circumvented these limitations. In particular, the webinar shows how the high illumination intensity - required to achieve tens of nanometer spatial resolution - can be mitigated by time-resolved STED microscopy, namely by recording and analyzing the temporal dynamics of the fluorescence signal (i.e., the fluorescence lifetime). In this context, the seminar explains and compares the time-gated and, more recently, separation-of-photons-by-lifetime-tuning (SPLIT) STED microscopy implementations.

 

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