Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength.
Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Ilaria
I
Testa
Christian A
CA
Wurm
Rebecca
R
Medda
Ellen
E
Rothermel
Claas
C
von Middendorf
Jonas
J
Fölling
Stefan
S
Jakobs
Andreas
A
Schönle
Stefan W
SW
Hell
Christian
C
Eggeling
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D003116:Color; D005456:Fluorescent Dyes / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D048328:Potoroidae; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes