Single molecule orientational microscopy: an accurate measurement?

Muneesh Maheshwari; Kishalay Mahato; John T. Fourkas; J. Alexander Liddle; Henri J. Lezec; Dhruv Fomra

doi:10.1117/12.3001259

13 March 2024 Single molecule orientational microscopy: an accurate measurement?

Muneesh Maheshwari, Kishalay Mahato, John T. Fourkas, J. Alexander Liddle, Henri J. Lezec, Dhruv Fomra

Proceedings Volume PC12849, Single Molecule Spectroscopy and Superresolution Imaging XVII; PC1284903 (2024) https://doi.org/10.1117/12.3001259
Event: SPIE BiOS, 2024, San Francisco, California, United States

Abstract

The ability to measure fluorophore molecular orientation and mobility can provide valuable information on the local physical and chemical environment. Analysis of polarized images can determine the orientation and mobility of fluorophores with well-defined uncertainties and bias, as well as enabling the use of two to four times greater emitter density per image frame than PSF engineering methods do. This study presents extensive Monte Carlo simulation and experimental data to determine quantitatively the degree of coupling among orientations and orientational mobility, and the dependence of orientational uncertainty on photon count and background noise. These results may be used for the rational design of experimental protocols and conditions to yield the levels of precision and accuracy necessary to effectively explore a wide range of physical phenomena.

Conference Presentation

Citation Download Citation

Muneesh Maheshwari, Kishalay Mahato, John T. Fourkas, J. Alexander Liddle, Henri J. Lezec, and Dhruv Fomra "Single molecule orientational microscopy: an accurate measurement?", Proc. SPIE PC12849, Single Molecule Spectroscopy and Superresolution Imaging XVII, PC1284903 (13 March 2024); https://doi.org/10.1117/12.3001259

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