Open AccessHigh-contrast single-particle tracking by selective focal plane illumination microscopy
Author(s) -
Jörg Ritter,
R. Veith,
Jan Peter Siebrasse,
Ulrich Kubitscheck
Publication year - 2008
Publication title -
optics express
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.16.007142
Subject(s) - optics , microscope , microscopy , point spread function , materials science , optical microscope , cardinal point , fluorescence microscope , full width at half maximum , resolution (logic) , tracking (education) , light sheet fluorescence microscopy , total internal reflection fluorescence microscope , biological imaging , fluorescence , image resolution , physics , scanning electron microscope , computer science , psychology , pedagogy , artificial intelligence
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.