PMID- 35982114
OWN - NLM
STAT- MEDLINE
DCOM- 20220822
LR  - 20221025
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 12
IP  - 1
DP  - 2022 Aug 18
TI  - Stimulated emission depletion microscopy with a single depletion laser using five 
      fluorochromes and fluorescence lifetime phasor separation.
PG  - 14027
LID - 10.1038/s41598-022-17825-5 [doi]
LID - 14027
AB  - Stimulated emission depletion (STED) microscopy achieves super-resolution by 
      exciting a diffraction-limited volume and then suppressing fluorescence in its 
      outer parts by depletion. Multiple depletion lasers may introduce misalignment 
      and bleaching. Hence, a single depletion wavelength is preferable for multi-color 
      analyses. However, this limits the number of usable spectral channels. Using 
      cultured cells, common staining protocols, and commercially available 
      fluorochromes and microscopes we exploit that the number of fluorochromes in STED 
      or confocal microscopy can be increased by phasor based fluorescence lifetime 
      separation of two dyes with similar emission spectra but different fluorescent 
      lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near 
      red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), 
      supplemented by a single further redshifted fluorochrome (670/701-750) were all 
      depleted with a single laser at 775 nm thus avoiding potential alignment issues. 
      Generally, this approach doubles the number of fully distinguishable colors in 
      laser scanning microscopy. We provide evidence that eight color FLIM-STED with a 
      single depletion laser would be possible if suitable fluorochromes were 
      identified and we confirm that a fluorochrome may have different lifetimes 
      depending on the molecules to which it is coupled.
CI  - (c) 2022. The Author(s).
FAU - Gonzalez Pisfil, Mariano
AU  - Gonzalez Pisfil M
AD  - Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, 
      Biomedical Center, Ludwig-Maximilians-Universitat Munchen, Grosshaderner Strasse 9, 
      82152, Planegg-Martinsried, Germany.
FAU - Nadelson, Iliya
AU  - Nadelson I
AD  - Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, 
      Biomedical Center, Ludwig-Maximilians-Universitat Munchen, Grosshaderner Strasse 9, 
      82152, Planegg-Martinsried, Germany.
FAU - Bergner, Brigitte
AU  - Bergner B
AD  - Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, 
      Biomedical Center, Ludwig-Maximilians-Universitat Munchen, Grosshaderner Strasse 9, 
      82152, Planegg-Martinsried, Germany.
FAU - Rottmeier, Sonja
AU  - Rottmeier S
AD  - Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, 
      Biomedical Center, Ludwig-Maximilians-Universitat Munchen, Grosshaderner Strasse 9, 
      82152, Planegg-Martinsried, Germany.
FAU - Thomae, Andreas W
AU  - Thomae AW
AD  - Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, 
      Biomedical Center, Ludwig-Maximilians-Universitat Munchen, Grosshaderner Strasse 9, 
      82152, Planegg-Martinsried, Germany.
FAU - Dietzel, Steffen
AU  - Dietzel S
AD  - Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, 
      Biomedical Center, Ludwig-Maximilians-Universitat Munchen, Grosshaderner Strasse 9, 
      82152, Planegg-Martinsried, Germany. dietzel@lmu.de.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20220818
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
RN  - 0 (Fluorescent Dyes)
SB  - IM
MH  - *Fluorescent Dyes
MH  - *Lasers
MH  - Microscopy, Confocal/methods
MH  - Microscopy, Fluorescence/methods
MH  - Staining and Labeling
PMC - PMC9388687
COIS- The authors declare no competing interests.
EDAT- 2022/08/19 06:00
MHDA- 2022/08/23 06:00
PMCR- 2022/08/18
CRDT- 2022/08/18 23:21
PHST- 2022/04/13 00:00 [received]
PHST- 2022/08/01 00:00 [accepted]
PHST- 2022/08/18 23:21 [entrez]
PHST- 2022/08/19 06:00 [pubmed]
PHST- 2022/08/23 06:00 [medline]
PHST- 2022/08/18 00:00 [pmc-release]
AID - 10.1038/s41598-022-17825-5 [pii]
AID - 17825 [pii]
AID - 10.1038/s41598-022-17825-5 [doi]
PST - epublish
SO  - Sci Rep. 2022 Aug 18;12(1):14027. doi: 10.1038/s41598-022-17825-5.