Super Stable Fluorescein Isothiocyanate Isomer I Monolayer for Total Internal Reflection Fluorescence Microscopy

Langmuir : the ACS Journal of Surfaces and Colloids
Przemyslaw Zarski, Alan G Ryder

Abstract

Total internal reflection fluorescence microscopy (TIRFM) is an important method in surface science and for the analysis of surface-bound macromolecules. Here, we developed and explored the use of a novel fluorescein isothiocyanate isomer I (FITC)-adsorbed monolayer for alignment and validation of TIRFM measurements and configurations. Aqueous solutions of FITC exist as several different protolytic forms (dianionic, anionic, neutral, and cationic) with each form having different emission characteristics. However, the emission behavior of FITC adsorbed on hydrophilic, hydrophobic, and unmodified glass surfaces at different pH was unknown. TIRFM imaging and spectroscopy were used to study FITC and FITC-labeled bovine serum albumin (BSA-FITC) monolayers generated on three different glass surfaces. Monolayer emission intensity, spectra, and the photobleaching profiles were all dependent on pH and the surface properties of the glass. Very strangely, however, at pH 5.0 on hydrophobic surfaces, the FITC monolayers produced were both bright and apparently unbleachable over ∼20 min of imaging (60 s total exposure). During monolayer formation at pH 5.0, we saw clear evidence for concentration-based quenching, indicating high surface cove...Continue Reading

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Citations

Jul 24, 2021·Langmuir : the ACS Journal of Surfaces and Colloids·Gaoyan MuChristopher B Gorman

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