Photobleaching of randomly-rotating fluorescently-decorated particles

TL;DR

This study investigates how randomly rotating fluorescent particles undergo non-isotropic photobleaching, revealing fluctuations and brightness variations influenced by particle rotation and fluorophore number, with implications for understanding fluorescence dynamics.

Contribution

It introduces a detailed analysis of photobleaching dynamics in rotating particles, highlighting the role of rotational diffusion and fluorophore count in brightness fluctuations and non-monotonic behaviors.

Findings

Significant interparticle fluctuations at intermediate Dτ.

Transient non-monotonic brightness changes up to 20%.

Photobleach-fluctuations dominate over single-fluorophore variability with over 1000 fluorophores.

Abstract

Randomly rotating particles that have been isotropically labeled with rigidly linked fluorophores will undergo non-isotropic (patchy) photobleaching under illumination due to the dipole coupling of fluorophores with light. For a rotational diffusion rate $D$ of the particle and a photobleaching timescale $\tau$ of the fluorophores, the dynamics of this process are characterized by the dimensionless combination $D \tau$. We find significant interparticle fluctuations at intermediate $D \tau$. These fluctuations vanish at both large and small $D \tau$, or at small or large elapsed times $t$. Associated with these fluctuations between particles, we also observe transient non-monotonicities of the brightness of individual particles. These non-monotonicities can be as much 20\% of the original brightness. We show that these novel photobleach-fluctuations dominate over variability of single-fluorophore orientation when there are at least $10^3$ fluorophores on individual particles.