Tracking Single Quantum Dots in Live Cells with Minimal Paths

We present here a novel method for automatically detecting and tracking semi-conductor quantum dots (QDs) in sequences of fluorescence images. QDs are new nanometersized fluorescent probes with great prospects for ultrasensitive biological imaging. When specifically attached to a biomolecule, they can be observed and tracked in live cells at the single molecule level over unprecedented durations. Due to QD complex optical properties, such as fluorescence intermittency, the quantitative analysis of image stacks is however challenging and requires advanced algorithms. Our tracking approach, instead of a frame by frame analysis, is based on perceptual grouping in a spatio-temporal volume. By applying a detection process based on an image fluorescence model, we first obtain a set of unstructured points. Individual molecular trajectories are then considered as minimal paths in a Riemannian metric derived from the fluorescence image stack. These paths are computed with the Fast Marching method, and few parameters are required. We illustrate our approach by showing experimental results issued from the tracking of individual glycine receptors in the membrane of live neurons.

1. Introduction

Single molecule tracking (SMT) is a new approach to investigate the mobility of specific objects in heteregeneous systems such as biological media. In live cells, SMT has notably been used to study the membrane dynamics of proteins or lipids specifically attached to a probe [14], [18]. To truly access single-molecule properties and not alter the dynamics of the biomolecules, the ideal probe should give a bright optical signal and be as small as possible. Among the fluorescent labels used in recent years, QDs appear specially promising for their small size (5 to 15 nm), brightness and photostability [13]. Using these nanoprobes, individual glycine receptors have for instance been tracked in the membrane of live neurons for durations over 20 minutes [6].