Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters.
In: The journal of physical chemistry. B, Jg. 128 (2024-01-11), Heft 1, S. 125-136
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Zugriff:
Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.
Titel: |
Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters.
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Autor/in / Beteiligte Person: | Sandberg, E ; Demirbay, B ; Kulkarni, A ; Liu, H ; Piguet, J ; Widengren, J |
Zeitschrift: | The journal of physical chemistry. B, Jg. 128 (2024-01-11), Heft 1, S. 125-136 |
Veröffentlichung: | Washington, D.C. : American Chemical Society, c1997-, 2024 |
Medientyp: | academicJournal |
ISSN: | 1520-5207 (electronic) |
DOI: | 10.1021/acs.jpcb.3c06905 |
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