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Blinking characteristics of organic fluorophores for blink-based multiplexing.

ABSTRACT: 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.

SUBMITTER: Seabury AG

PROVIDER: S-EPMC10821931 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Blinking characteristics of organic fluorophores for blink-based multiplexing.

Seabury Amelia G AG Khodabocus Alisha J AJ Kogan Isabelle M IM Hoy Grayson R GR DeSalvo Grace A GA Wustholz Kristin L KL

Communications chemistry 20240127 1

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 ...[more]

PMID: 38280979

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Project description:IMPORTANCE Facial paralysis remains one of the most challenging conditions to effectively manage, often causing life-altering deficits in both function and appearance. Facial rehabilitation via pacing and robotic technology has great yet unmet potential. A critical first step toward reanimating symmetrical facial movement in cases of unilateral paralysis is the detection of healthy movement to use as a trigger for stimulated movement. OBJECTIVE To test a blink detection system that can be attached to standard eyeglasses and used as part of a closed-loop facial pacing system. DESIGN, SETTING, AND PARTICIPANTS Standard safety glasses were equipped with an infrared (IR) emitter-detector unit, oriented horizontally across the palpebral fissure, creating a monitored IR beam that became interrupted when the eyelids closed, and were tested in 24 healthy volunteers from a tertiary care facial nerve center community. MAIN OUTCOMES AND MEASURES Video-quantified blinking was compared with both IR sensor signal magnitude and rate of change in healthy participants with their gaze in repose, while they shifted their gaze from central to far-peripheral positions, and during the production of particular facial expressions. RESULTS Blink detection based on signal magnitude achieved 100% sensitivity in forward gaze but generated false detections on downward gaze. Calculations of peak rate of signal change (first derivative) typically distinguished blinks from gaze-related eyelid movements. During forward gaze, 87% of detected blink events were true positives, 11% were false positives, and 2% were false negatives. Of the 11% false positives, 6% were associated with partial eyelid closures. During gaze changes, false blink detection occurred 6% of the time during lateral eye movements, 10% of the time during upward movements, 47% of the time during downward movements, and 6% of the time for movements from an upward or downward gaze back to the primary gaze. Facial expressions disrupted sensor output if they caused substantial squinting or shifted the glasses. CONCLUSIONS AND RELEVANCE Our blink detection system provides a reliable, noninvasive indication of eyelid closure using an invisible light beam passing in front of the eye. Future versions will aim to mitigate detection errors by using multiple IR emitter-detector units mounted on glasses, and alternative frame designs may reduce shifting of the sensors relative to the eye during facial movements.

Dataset Information

Blinking characteristics of organic fluorophores for blink-based multiplexing.

Publications

Blinking characteristics of organic fluorophores for blink-based multiplexing.

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