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Investigating Organic Fluorophores for Blink-Based Multiplexing
Seabury, Amelia
Seabury, Amelia
Abstract
To deepen our understanding of both biological and material systems, visualization of the complexity occurring on the nanometer scale is critical. Multicolor super-resolved fluorescence studies allow for a nuanced understanding of intricate structures within cells, nanomaterials, and polymers but are limited by the necessity of spectrally distinct probes or time intensive procedures. Recently, we demonstrated an alternative method termed blink-based multiplexing (BBM) wherein spectrally-overlapped emitters are classified based solely on their blinking dynamics (i.e., fluctuations in emission intensity under constant excitation caused by intrinsic photo-physical or -chemical transitions) instead of spectral color. This study parses the relationships between structure and blinking activity of rhodamine, BODIPY, and anthraquinone fluorophores to ultimately optimize BBM performance. Through change point detection and multinomial logistic regression analyses, we show that BBM can harness differences in spectral diffusion, charge transfer kinetics, and photo-stability to distinguish and accurately classify fluorophores. Informed by their photo-physical or -chemical properties, BBM can simultaneously differentiate groups of 2 or 3 spectrally-overlapped emitters using a single laser with ≥ 93% accuracy. Ongoing and future studies utilizing a new optical set-up (i.e., an electron multiplying charge-coupled device) and fluorophores more conducive to practical imaging applications are also outlined.
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Date
2024-05-01
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Chemistry