Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain.
The precision of single-molecule localization-based super-resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV-induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single-color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error-free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD-dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno-electron microscopy, yet with much improved label density in two independent channels.
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany). martin.lehmann@fu-berlin.de.|Freie Universität Berlin, Department of Biochemistry, Takustrasse 6, 14195 Berlin (Germany). martin.lehmann@fu-berlin.de.
Martin
M
Lehmann
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).
Benjamin
B
Gottschalk
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).
Dmytro
D
Puchkov
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).
Peter
P
Schmieder
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).
Sergej
S
Schwagerus
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).|Humboldt Universität zu Berlin, Department of Chemistry, Brook-Taylor-Strasse. 2, 12489 Berlin (Germany).
Christian P R
CP
Hackenberger
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).|Freie Universität Berlin, Department of Biochemistry, Takustrasse 6, 14195 Berlin (Germany).
Volker
V
Haucke
Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany). jan.schmoranzer@fu-berlin.de.|Freie Universität Berlin, Department of Biochemistry, Takustrasse 6, 14195 Berlin (Germany). jan.schmoranzer@fu-berlin.de.|Charité - Universitätsmedizin Berlin, Charité Campus Mitte, Virchowweg 6, 10117 Berlin (Germany). jan.schmoranzer@fu-berlin.de.
Jan
J
Schmoranzer
D000818:Animals; D001921:Brain / Q000166:cytology / Q000648:ultrastructure*; D003116:Color; D005456:Fluorescent Dyes / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D013572:Synaptic Vesicles / Q000648:ultrastructure*
D005456:Fluorescent Dyes
NMR spectroscopy; electron microscopy; fluorescent probes; immunochemistry; single-molecule studies