Manipulating and probing the spatio-temporal dynamics of nanoparticles near surfaces

Abstract

In this report, we combine total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) with a single optical trap to simultaneously manipulate and measure the dynamics of individual molecules near the substrate-solution interface. As a proof of principle, polystyrene particles (84 nm in diameter) are used as a model system to test our approach in studying their diffusion properties near surfaces, which are treated with polyethylene glycol 8000, bovine serum albumin or sodium hydroxide. The evanescent field of 543 nm excitation propagates ~100 nm into the solution, and the fluorescence detection is spatially confined by a 25 or 50 m pinhole that is parfocal with the specimen plane. The optical trap is generated using a cw Ti:sapphire laser at 780 nm. Our results indicate that the particles' diffusion is influenced by surface interactions, which might have further implications on biomembrane studies. Furthermore, the observed translational diffusion of individual particles can be manipulated using an optical trap. By combining the single molecule sensitivity of TIR-FCS with a noninvasive manipulation method, such as optical trapping, we will be able to probe molecular dynamics in biomimetic systems and living cells.

Vesicle Diffusion Close to a Membrane: Intermembrane Interactions Measured with Fluorescence Correlation Spectroscopy

“…The TIR-FCS setup is described and characterized elsewhere (31,33). Briefly, a HeNe laser (0.4 mW, 543 nm, Meredith Instruments) was used for prism-based TIR.…”

A versatile multimode microscope to probe and manipulate nanoparticles and biomolecules

“…Because the entrance path for the trap on the microscope is parallel to the epi‐path (Fig. 2b), we can easily combine optical trapping with DIC, epi‐ and TIR illumination to simultaneously measure temporally resolved dynamics using FCS (Kyoung & Sheets, 2006) or SPT.…”

“…Note that the FCS experiments described in this paper were carried out at room temperature (∼25°C). We can also combine FCS with TIR excitation to follow the dynamics of fluorescent particles (Nile red sulphate polystyrene beads, 84‐nm Ø, Invitrogen, Carlsbad, CA, USA) that are diffusing close to the surface (Kyoung & Sheets, 2006). Diffusion measured with TIR‐FCS are fit following Hassler et al .…”