Optically activated delayed fluorescence anisotropy: a new approach for macromolecular size determination
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Lu, Yi-Han
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Abstract
Time-resolved fluorescence anisotropy (FA) is a popular technique to investigate the dynamics of different emission depolarizing processes following polarized excitation. Although molecular rotational motion is the most frequently studied process, time-resolved FA is also useful for determining the kinetics of other events, such as conformational changes and intermolecular interactions of biomolecules in solution. FA is a popular tool for biomolecular interaction studies because of its rapid, sensitive, and non-destructive properties, however, the short (~ ns) fluorescence lifetime of common fluorescent dyes limits the application of FA measurement on larger macromolecules and complexes. To overcome this limitation, we introduce a new approach called optically activated delayed fluorescence anisotropy (OADFA) that utilizes sequential two-photon excitation to achieve triplet shelving and recovery of optical excitation. This new approach enables stretching of fluorescence anisotropy measurement times from nanoseconds to couple microseconds, allowing for the measurement of slow depolarization processes of large macromolecular complexes. In this study, we first present a quantitative rate model and Monte Carlo simulations to describe the depolarization process of OADFA at the molecular level. We also conducted experiments to demonstrate that the results of OADFA measurements matched the values measured by other approaches well. Our findings have great potential to enable future colloidal and biomacromolecular studies.
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Date
2023-04-27
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Dissertation