Luminescence lifetime imaging of three-dimensional biological objects

Abstract

A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.

Interaction With the Extracellular Matrix Triggers Calcium Signaling in Trypanosoma cruzi Prior to Cell Invasion

“…The invasion of host mammalian cells by T. cruzi trypomastigotes is a complex process which requires distinct surface molecules, and the activation of multiple signaling pathways in the parasites, as well as in the host cells ( De Souza et al., 2010 ; Caradonna and Burleigh, 2011 ; Maeda et al., 2012 ; Giorgi and de Lederkremer, 2020 ; Nájera et al., 2021 ). Molecules from the parasite surface, such as members of the gp85/Transialidase glycoproteins coded by a multigenic family, bind to the ECM components, as a first step of the invasion process ( Alves and Colli, 2008 ; De Souza et al., 2010 ; Nde et al., 2012 ), which triggers relevant changes in phosphorylation, S-nitrosylation, and nitration levels of proteins in T. cruzi trypomastigotes, Y strain, which elicit a reprogramming of the parasite metabolism ( Mattos et al., 2019 ) or modification of the DNA binding profile of nitrated histones ( Magalhães et al., 2020 ).…”

“…Moreover, remodeling of the ECM by T. cruzi , with an increment in the synthesis of collagen IV, fibronectin, and laminin ( Cardenas et al., 2010 ; Coelho et al., 2018 ), was described in cardiac cells. Increase in thrombospondin-1 in the case of human colon epithelial cells ( Suman et al., 2018 ) was associated with fibrosis, which occurs in chronic Chagas disease patients ( Magalhães-Santos et al., 2004 ; Garzoni et al., 2008 ; Nájera et al., 2021 ). Notwithstanding, biochemical events triggered by ECM on trypomastigotes during their mutual interaction are less explored ( Nde et al., 2012 ).…”

NAD(P)H fluorescence lifetime imaging of live intestinal nematodes reveals metabolic crosstalk between parasite and host

“…In order to account for cellular and tissue functionality, live tissue or in vivo metabolic imaging, is required for a full understanding of these pathophysiological phenomena, besides the analysis of cellular dynamics and communication. This has previously been achieved by combining NAD(P)H FLIM with two-photon microscopy in mammals, especially in mice 29 . However, there is only a single study using the free-living nematode Caenorhabditis elegans by means of NAD(P)H-FLIM showing specific metabolic pathways in various tissue regions of the worm, associated with the respective cellular differentiation stage 30 .…”

NAD(P)H Fluorescence Lifetime Imaging of Live Intestinal Nematodes Reveals Metabolic Crosstalk Between Parasite and Host

“…In order to account for cellular and tissue functionality, in vivo metabolic imaging, is required for a full understanding of these pathophysiological phenomena, besides the analysis of cellular dynamics and communication. This has previously been achieved by combining NAD(P)H FLIM with two-photon microscopy in mammals, especially in mice 14 . However, there is only a single study using the free-living nematode Caenorhabditis elegans by means of NAD(P)H-FLIM showing speci c metabolic pathways in various tissue regions of the worm, associated with the respective cellular differentiation stage 15 .…”

“…The uorescence of the ubiquitous co-enzymes NADH and NADPH has been extensively used for FLIM to monitor metabolic activity 27 . The NAD(P)H metabolic activity can be correlated to speci c cellular phenotypes and functions, with application in health and different pathologies 14 . While many technologies to perform FLIM have been proposed, both in time and in frequency domain [30][31][32][33] , time-correlated single-photon counting proved to be most reliable and found broad application for deep-tissue imaging.…”