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.
Testa
Ilaria
I
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Wurm
Christian A
CA
Medda
Rebecca
R
Rothermel
Ellen
E
von Middendorf
Claas
C
Fölling
Jonas
J
Jakobs
Stefan
S
Schönle
Andreas
A
Hell
Stefan W
SW
Eggeling
Christian
C
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