Wide-Field Fluorescence Lifetime Imaging of Single Molecules
| Autor: | Ingo Gregor, Jan C. Thiele, Joerg Enderlein, Sebastian Isbaner, Nazar Oleksiievets, Roman Tsukanov, Oleksii Nevskyi, André Weber |
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| Rok vydání: | 2020 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
010304 chemical physics business.industry Chemistry Resolution (electron density) Optical Imaging Signal-To-Noise Ratio 010402 general chemistry Frame rate 01 natural sciences 7. Clean energy Noise (electronics) Fluorescence spectroscopy Single Molecule Imaging 0104 chemical sciences Förster resonance energy transfer Orders of magnitude (time) 0103 physical sciences Microscopy Optoelectronics Physical and Theoretical Chemistry business |
| Zdroj: | The journal of physical chemistry. A. 124(17) |
| ISSN: | 1520-5215 |
| Popis: | Fluorescence lifetime imaging (FLIM) has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Forster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy (CLSM) and time-correlated single-photon counting (TCSPC) and the other based on wide-field microscopy and phase fluorometry. Although the first approach (CLSM-TCSPC) assures high sensitivity and allows one to detect single molecules, it is slow and has a small photon yield. The second allows, in principal, high frame rates (by 2-3 orders of magnitude faster than CLSM), but it suffers from low sensitivity, which precludes its application for single-molecule imaging. Here, we demonstrate that a novel wide-field TCSPC camera (LINCam25, Photonscore GmbH) can be successfully used for single-molecule FLIM, although its quantum yield of detection in the red spectral region is only ∼5%. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. We performed single-molecule FLIM of different red fluorophores, and we use the lifetime information for successfully distinguishing between different molecular species. Finally, we demonstrate single-molecule metal-induced energy transfer (MIET) imaging which is a first step for three-dimensional single-molecule localization microscopy (SMLM) with nanometer resolution. |
| Databáze: | OpenAIRE |
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