Single Molecule Tracking With Light Sheet-Based Microscopy In Vivo

Abstract

Autofluorescence, rapid photobleaching and high particle concentrations present restrictions to single molecule observation in biological systems by epi-illumination. Light sheet-based microscopy overcomes these limitations [1]. By combining the speed of parallel image acquisition and the optical sectioning produced by light sheet illumination, we created a powerful tool to study single molecule dynamics on a millisecond timescale [2].We illuminated the sample perpendicular to the detection axis with a thin light sheet (FWHM 2-3μm). In this manner a simple optical sectioning microscope is created, because only the focal plane of the detection optics is illuminated and no out-of-focus fluorescence is generated. The background fluorescence is strongly reduced and the signal-to-noise-ratio (SNR) greatly improved.We constructed a miniaturized glass specimen chamber, which can be illuminated from the side in a very flexible manner and directly be mounted on a commercial inverse microscope. The specimen is easily accessible for micromanipulation and can be observed via the 0.17mm thin glass bottom of the chamber using high NA objective lenses.With this setup it was straightforward to observe trajectories of single protein molecules in aqueous solution with a D = 90μm2/s, and also in the cellular interior. By analysis of the diffusion behavior of single fluorescent dextran molecules we determined the viscosity of living C. tentans salivary gland cell nuclei. Similarly, molecular dynamics in adherent cells can be observed with greatly improved contrast.With this new experimental setup we use the ideal imaging scheme for single molecule visualization and push the limit of sensitivity far beyond the potential of conventional epi-illumination.[1] Huisken, J. et al. (2004). Science 305(5686): 1007-9.[2] Ritter, J.G. et al. (2008). Opt Express 16(10): 7142-52.