Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM‐FRAP

Abstract

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single‐plane image acquisition within a subcellular‐sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

Actin dynamics sustains spatial gradients of membrane tension in adherent cells

“…The ACF analysis showed that lipid mobility does not change between the adhesive and non-adhesive areas (Figure S5G-H). The diffusion values are consistent with point FCS measurements of the same lipid probe from the literature (⟨D cross control ⟩ = 2.2±2.7μm 2 /s; ⟨D ring control ⟩ = 1.9±2.6μm 2 /s) 84 . Under JLY treatment, we noticed a weak trend decreasing overall diffusion and transport (⟨D cross JLY ⟩ = 1.7±2.0μm 2 /s; ⟨D ring JLY ⟩ = 1.5±2.0μm 2 /s).…”

Development of Molecular Dynamics and Research Progress in the Study of Slag

“…Molecular simulation refers to the use of theoretical methods and computational techniques to simulate or simulate the microscopic behavior of molecular motion, and is widely used in computational chemistry, computational biology, and materials science [43,44]. The object of its research can be as small as a single chemical molecule or as large as a complex biological system or material system [45][46][47].…”

Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching

“…The following recovery of fluorescence intensity directly informs on the diffusion characteristics of fluorescent molecules spreading into the photobleached volume from the surroundings due to an established concentration gradient. FRAP has been widely utilized as a routine and reliable method to probe the mobility and chemical properties of fluorescently labeled biomolecules within cell organelles and membranes. − FRAP has also found applications in materials science for studying the diffusion of polymer chains and the mobility of dopants in glass materials. , …”