Dysfunctional and leaky blood vessels resulting from disruption of the endothelial cell (EC) barrier accompanies numerous diseases. The EC barrier is established through endothelial cell tight and adherens junctions. However, the expression patterning and precise contribution of different junctional proteins to the EC barrier is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability and commonalities of EC barrier patterning. Across tissues, Claudin5 exhibited diminishing expression along the arteriovenous axis, correlating with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage patterning and response to the leakage agonist histamine. Loss of Claudin5 enhanced histamine-induced leakage in an organotypic and vessel type-specific manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss left junction ultrastructure unaffected but altered its composition, with concomitant loss of zonula occludens-1 and upregulation of VE-Cadherin expression. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds, providing insights to modify EC barrier stability in a targeted, organ-specific manner.

Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing. Keywords Two-photon excitation • Fluorescence lifetime imaging microscopy (FLIM) • Nicotinamide adenine dinucleotide hydrogen (NADH) • Flavin adenine dinucleotide (FAD)

Intra-vessel heterogeneity establishes enhanced sites of macromolecular leakage downstream of laminin a5 Graphical abstract Highlights d Intra-vessel heterogeneity produces sites of predetermined leakage d Predetermined sites of leakage are preserved between agonists and vascular beds d Low laminin a5 defines sites of predetermined leakage d Laminin a5 promotes junctional VE-cadherin and attenuates Y685 phosphorylation