Low-cost multimodal light sheet microscopy for optically cleared tissues and living specimens.

Light sheet microscopy techniques have expanded with designs to address many new applications. Due to rapid advancements in computing power, camera/detector technologies, and tissue clearing techniques, light sheet methods are becoming increasingly popular for biomedical imaging applications at the cellular and tissue levels. Light sheet imaging modalities couple rapid imaging rates, low-levels of phototoxicity, and excellent signal to noise ratios, contributing to their popularity for experimental biology.

Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS.

Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells.

Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking.

Independent synchronized control and visualization of interactions between living cells and organisms.

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints.

Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells.

Mapping molecular diffusion in the plasma membrane by Multiple-Target Tracing (MTT).

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT). Single-molecule videomicroscopy, offering millisecond and nanometric resolution, allows a detailed representation of membrane organization by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.