High-contrast single-particle tracking by selective focal plane illumination microscopy.

Wide-field single molecule microscopy is a versatile tool for analyzing dynamics and molecular interactions in biological systems. In extended three-dimensional systems, however, the method suffers from intrinsic out-of-focus fluorescence. We constructed a high-resolution selective plane illumination microscope (SPIM) to efficiently solve this problem. The instrument is an optical sectioning microscope featuring the high speed and high sensitivity of a video microscope. We present theoretical calculations and quantitative measurements of the illumination light sheet thickness yielding 1.7 microm (FWHM) at 543 nm, 2.0 microm at 633 nm, and a FWHM of the axial point spread function of 1.13 microm. A direct comparison of selective plane and epi-illumination of model samples with intrinsic background fluorescence illustrated the clear advantage of SPIM for such samples. Single fluorescent quantum dots in aqueous solution are readily visualized and tracked proving the suitability of our setup for the study of fast and dynamic processes in spatially extended biological specimens.