Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength.

Journal article

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.

Authors: Testa, I; Wurm, C; Medda, R; Rothermel, E; von Middendorf, C

• Publication status: Published
• Journal: Biophysical journal
• Volume: 99
• Issue: 8
• Pages: 2686-2694
• Publication date: 2010-10-01
• DOI: 10.1016/j.bpj.2010.08.012
• EISSN: 1542-0086
• ISSN: 0006-3495
• Language: English
• Keywords: Microscopy, Fluorescence; Cell Line; Potoroidae; Spectrometry, Fluorescence; Fluorescent Dyes; Nanotechnology; Animals; Cell Survival; Color; Microscopy, Atomic Force