Simultaneous measurement of rotational and translational diffusion of anisotropic colloids with a new integrated setup for fluorescence recovery after photobleaching

Abstract

This paper describes an integrated setup for fluorescence recovery after photobleaching (FRAP) for determining translational and rotational Brownian diffusion simultaneously, ensuring that these two quantities are measured under exactly the same conditions and at the same time in dynamic experiments. The setup is based on translational-FRAP with a fringe pattern of light for both the bleaching and monitoring of fluorescently labeled particles, and rotational-FRAP, which uses the polarization of a short bleach light pulse to create a polarization anisotropy. The fringe pattern of the probe beam is modulated in conjunction with a synchronized lock-in amplifier giving a fast, sensitive, ensemble-averaged measurement compared to microscope-image based techniques. The experimental polarization geometry we used ensures that the fluorescence emission is collected without polarization bias. Therefore, only the orientation of the absorption dipole moment of the fixed dye in the particles is measured, which simplifies interpretation of the data. The polarization is modulated rapidly between two orthogonal polarization states, giving the polarization anisotropy in one, single measurement. The rotational and translational Brownian diffusion of anisotropic colloids is measured for ellipsoids of revolution. This experiment shows that in this case the rotational correlation function matches a three-exponential decay in accordance with theoretical predictions.

Self-limiting aggregation of phospholipid vesicles

“…We model the translational diffusion behaviour of the vesicle pairs as if they are prolate ellipsoids. For such objects, the diffusion coefficient is given by Kuipers et al 51 In DLS and FCS, the diffusion coefficient is experimentally determined, and using the Stokes-Einstein equation, an effective hydrodynamic radius, R h , is calculated. It can be shown (see the ESI †) that for pairs of monodisperse vesicles, R h is expected to be between 1.32R 0 and 1.15R 0 : the first value refers to the case that the vesicles have a very small contact area ({R 0 2 ), and the second value is for an extensive contact area causing the vesicle pair to be perfectly spherical.…”

Spherical probes for simultaneous measurement of rotational and translational diffusion in 3 dimensions

“…This has been ascribed to a lack of experimental approaches for capturing rotational motion 20 . Rotational dynamics can shed a unique light onto various dynamic phenomena that cannot be accessed only with translational degrees of freedom, such as motion in glassy and supercooled states (where decoupling between translational and rotational diffusion emerges) [21][22][23] ; particle adsorption and self-assembly at fluid interfaces 24,25 ; interfacial dynamics at solid-liquid interfaces 26 and biological interactions; such as viruses binding to membranes 27 Ensemble averaged rotational diffusion of colloids has been studied by techniques such as fluorescence recovery after photobleaching (FRAP) 28,29 , depolarized dynamic light scattering 30,31 and nuclear magnetic resonance (NMR) spectroscopy 32 . These bulk methods fall short in identifying local (dynamic) heterogeneities.…”

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

“…Underpinning tr-FAIM, fluorescence lifetime imaging microscopy (FLIM) [2,3,[28][29][30][31][32][33] maps the fluorescence lifetime in every pixel of an image and is a powerful technique for probing the local environment of a fluorophore as the measured lifetime is largely independent of fluorophore concentration, but can be sensitive to pH [34], refractive index [35][36][37][38], reactive quenching species [39] and viscosity [30,31,[40][41][42]. Both fluorescence anisotropy and FRAP have previously been used independently for a study of aggregation states of alpha-synuclein, a protein which plays a role in Parkinson's disease [43], and an arrangement for dynamic FRAP and rotational diffusion measurements for colloids has been presented [44]. FRAP and FLIM have also recently been used independently in a study of keratinocyte migration [45], influenza virus association with lipid rafts [46], and cyanobacteria [47].…”