Single particle tracking of acyl carrier protein (ACP)-tagged TrkA receptors in PC12nnr5 cells.

There is a wide interest in studying the membrane mobility of Nerve Growth Factor (NGF) tropomyosin receptor kinase A (TrkA) at the single molecule level, in order to elucidate its diverse signaling responses related to different receptor functions. Here we present an experimental strategy based on the acyl carrier protein (ACP) tag in order to study the dynamics of the high-affinity NGF receptor TrkA in the membrane of PC12nnr5 cells. We present a single-particle tracking (SPT) study using highly photostable semiconductor quantum dots (Qdots) conjugated to ACP-tagged TrkA receptors. We demonstrate that ACP-TrkA shows biochemical and biological properties identical to those of its unmodified counterpart and that single receptor molecules in living cells display distinct diffusive regimes and a highly heterogeneous dynamics.

Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.

Two-stimuli manipulation of a biological motor.

F1-ATPase is an enzyme acting as a rotary nano-motor. During catalysis subunits of this enzyme complex rotate relative to other parts of the enzyme. Here we demonstrate that the combination of two input stimuli causes stop of motor rotation. Application of either individual stimulus did not significantly influence motor motion. These findings may contribute to the development of logic gates using single biological motor molecules.

Scanning electron microscope charging effect model for chromium/quartz photolithography masks.

We propose a new method for fitting a model of specimen charging to scanning electron microscope (SEM) images. Charging effects cause errors when one attempts to infer the size or shape of a specimen from an image. The goal of our method is to enable image analysis algorithms for measurement, segmentation, and three-dimensional (3-D) reconstruction that would otherwise fail on images containing charging effects. Our model is applied to images of chromium/quartz photolithography masks and may also work in the more general case of isolated metal islands on a flat insulating substrate. Unlike methods based on Monte Carlo simulation, our simulation method does not handle more general topographies or specimens composed entirely of an insulator; it is a crude approximation to the physical charging process described in more detail in Cazaux (1986) and Melchinger and Hofmann (1985), but can be fit with quantitative accuracy to real SEM images. We only consider changes in intensity and do not model charging-induced distortion of image coordinates. Our approach has the advantage over existing methods of enabling fast prediction of charging effects so it may be more practical for image analysis applications.