Steffen Dietzel

LMU, Germany

1992: Diploma as a Biologist
1993 – 96: PhD at the University of Heidelberg
1996 – 98: Postdoc at the University of Heidelberg
1998 – 00: Postdoc at the University of Illinois at Urbana-Champaign
2000-2007: Junior group leader at the Ludwig-Maximilians-Universität (LMU) München
2006: Habilitation in Cell Biology
2007 – 15: Head of light microscopy at the Walter-Brendel-Zentrum, LMU
Since 2015: Head of the Core Facility Bioimaging at the Biomedical Center of the LMU

Five color super resolution microscopy with FLIM STED

For multi-color STED (Stimulated emission depletion microscopy) with multiple depletion laser wavelengths, alignment of depletion lasers requires highest precision in the nanometer range. It is thus preferable to apply a single depletion wavelength for several fluorochromes. But this limits the number of color channels that can be used.

Using fluorescence lifetime imaging (FLIM) to separate labels was introduced to STED in 2011 by the group of Stefan Hell. However, FLIM equipment at that time was slow and difficult to operate compared to a confocal microscope and not many studies followed that path.
Technical progress and the development of phasor based analysis of FLIM data nowadays permit easy and fast fluorochrome separation in confocal and STED microscopy. Moreover phasor based separation requires much smaller photon numbers when compared to curve fitting when there is background present.

A labeling scheme will be presented that can be applied in life science environments, e.g. on cultured human cells, with common staining protocols, commercially available fluorochromes and a turn-key FLIM confocal or FLIM-STED microscope. The number of usable fluorochromes in STED or confocal microscopy can generally be doubled by phasor based fluorescence lifetime separation of two dyes with similar emission spectrum but different fluorescent lifetimes.

In a five color FLIM-STED approach we depleted with 775 nm two fluorochromes each in the near red (exc. 594 nm, em. 600-630) and far red channel (633/641-680), supplemented by a single fluorochrome even further redshifted (670/701-750). This allowed us to record five color STED samples with a single depletion laser. Current data suggest that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified.