Imaging the rotational mobility of a fluorophore by frequency domain time-resolved fluorescence anisotropy

Gilad Yahav; Shweta Pawar; Yitzchak Weber; Bar Atuar; Hamootal Duadi; Dror Fixler

doi:10.1117/12.2648025

16 March 2023 Imaging the rotational mobility of a fluorophore by frequency domain time-resolved fluorescence anisotropy

Gilad Yahav, Shweta Pawar, Yitzchak Weber, Bar Atuar, Hamootal Duadi, Dror Fixler

Author Affiliations +

Proceedings Volume 12394, Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XX; 1239409 (2023) https://doi.org/10.1117/12.2648025
Event: SPIE BiOS, 2023, San Francisco, California, United States

Conference Poster

Abstract

Although single point time-resolved fluorescence anisotropy (FA) measurements are well established and routinely used for various applications in many laboratories, only a few reports described their extension into two-dimensional (2D) time-resolved FA imaging (TR-FAIM). The ability to perform TR-FAIM can offer cellular imaging based on the rotational correlation time (θ) that depends on the viscosity and dynamic properties of the tissues. We extended existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FAIM, which produces visual maps of θ. The proof of concept of the FD TR-FAIM was validated on 7 fluorescein solutions with increasing viscosities (achieved by increasing glycerol concentration between 0-80%). The studies were performed using images of θ as well as by characterizing the peak (mode) and the full width half maximum (FWHM) of its histograms (of normal probability distribution) and extracting the limiting FA (r₀). The θ of the 7 solutions was significantly increased from 0.15±0.05 to 11.25±1.87ns, whereas r₀ decreased from 0.40±0.01 to 0.30±0.06. The FD TR-FAIM provides wide-field imaging of the θ of the fluorophore, and hence offers a potential simultaneous interrogation with great sensitivity of diverse chemical and physical phenomena. In addition, as θ can vary according to the local microenvironment and across the sample under investigation, it can characterize different compartments of complex structures such as cells. Through the FD TR-FAIM a large variety of information can be probed from each sample and therefore it may become a reliable and powerful diagnostic tool for cellular imaging and biosensing.

Citation Download Citation

Gilad Yahav, Shweta Pawar, Yitzchak Weber, Bar Atuar, Hamootal Duadi, and Dror Fixler "Imaging the rotational mobility of a fluorophore by frequency domain time-resolved fluorescence anisotropy", Proc. SPIE 12394, Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XX, 1239409 (16 March 2023); https://doi.org/10.1117/12.2648025

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