Single-molecule fluorescence experiments have transformed our understanding of complex materials and biological systems. Whether single molecules are used to report on their nano-environment or provide for localization, understanding their blinking dynamics (i.e., stochastic fluctuations in emission intensity under continuous illumination) is paramount. We recently demonstrated another use for blinking dynamics called blink-based multiplexing (BBM), where individual emitters are classified using a single excitation laser based on blinking dynamics, rather than color. This study elucidates the structure-activity relationships governing BBM performance in a series of model rhodamine, BODIPY, and anthraquinone fluorophores that undergo different photo-physical and-chemical processes during blinking. Change point detection and multinomial logistic regression analyses show that BBM can leverage spectral fluctuations, electron and proton transfer kinetics, as well as photostability for molecular classification—even within the context of a shared blinking mechanism. In doing so, we demonstrate two- and three-color BBM with ≥ 93% accuracy using spectrally-overlapped fluorophores.
- Award ID(s):
- 1841419
- NSF-PAR ID:
- 10125906
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 144
- Issue:
- 10
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 3250 to 3259
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Purpose The aim of this study is to identify the potential cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease and to evaluate these markers on independent CSF samples using parallel reaction monitoring (PRM) assays.
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c9an00297a
