Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications.

The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ. However, its use is limited to restricted regions, and consequently few cells, and frequently no more than one or two parameters. Correlative microscopy, obtained by the fusion of different optical technologies, can consequently surpass this barrier by merging results from different spatial scales. We discuss here the use of an acquisition and analysis correlative microscopy pipeline to obtain high statistical sampling, high content, and maximum spatial resolution by combining widefield, confocal, and molecular localization microscopy.

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes.

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Age Mosaicism across Multiple Scales in Adult Tissues.

Most neurons are not replaced during an animal's lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using 15N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization.