Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes

Abstract

Fluorescence microscopy offers a minimally perturbative approach to probe biology in vivo. However, available techniques are limited both in sensitivity and temporal resolution for commonly used fluorescent proteins. Here we present a new imaging system with a diagnostic toolkit that caters for the detection and quantification of fluorescent proteins for use in fast functional imaging at the single-molecule level. It utilizes customized microscopy with a mode of illumination we call “slimfield” suitable for rapid (∼millisecond) temporal resolution on a range of common fluorescent proteins. Slimfield is cheap and simple, allowing excitation intensities ∼100 times greater than those of widefield imaging, permitting single-molecule detection at high speed. We demonstrate its application on several purified fluorescent proteins in standard use as genetically-encoded reporter molecules. Controlled in vitro experiments indicate single protein molecules over a field of view of ∼30 μm² area, large enough to encapsulate complete prokaryotic and small eukaryotic cells. Using a novel diagnostic toolkit we demonstrate automated detection and quantification of single molecules with maximum imaging rates for a 128 × 128 pixel array of ∼500 frames per second with a localization precision for these photophysically poor fluorophores to within 50 nm. We report for the first time the imaging of the dim enhanced cyan fluoresecent protein (ECFP) and CyPet at the single-molecule level. Applying modifications, we performed simultaneous dual-colour slimfield imaging for use in co-localization and FRET. We present preliminary in vivo imaging on bacterial cells and demonstrate ∼millisecond timescale functional imaging at the single-molecule level with negligible photodamage.