Imaging molecular dynamics in vivo – from cell biology to animal models

Abstract

Advances in fluorescence microscopy have enabled the study of membrane diffusion, cell adhesion and signal transduction at the molecular level in living cells grown in culture. By contrast, imaging in living organisms has primarily been restricted to the localization and dynamics of cells in tissues. Now, imaging of molecular dynamics is on the cusp of progressing from cell culture to living tissue. This transition has been driven by the understanding that the microenvironment critically determines many developmental and pathological processes. Here, we review recent progress in fluorescent protein imaging in vivo by drawing primarily on cancer-related studies in mice. We emphasize the need for techniques that can be easily combined with genetic models and complement fluorescent protein imaging by providing contextual information about the cellular environment. In this Commentary we will consider differences between in vitro and in vivo experimental design and argue for an approach to in vivo imaging that is built upon the use of intermediate systems, such as 3-D and explant culture models, which offer flexibility and control that is not always available in vivo. Collectively, these methods present a paradigm shift towards the molecular-level investigation of disease and therapy in animal models of disease.

Intravital FLIM-FRET Imaging Reveals Dasatinib-Induced Spatial Control of Src in Pancreatic Cancer

“…Detailed topologic analysis at cellular and subcellular resolution in live tumor tissue has enabled precise detection and single cell quantification of subtle changes in protein activity over time that cannot faithfully be achieved using standard fixed endpoint approaches applied to in vitro cultured models or ex vivo immunohistochemical analysis (2,44). Using a genetically engineered model of pancreatic cancer that recapitulates the human disease and a novel therapeutic with shown activity in pancreatic cancer, we have quantified the improvement in drug response associated with combination therapy and illustrate the application and advantages of FLIM-FRET in the drug target validation process.…”

“…Examining cancer behavior in an intact host setting allows us to understand, in a more physiologic context, the aberrant regulation of critical events that drive tumor progression. Intravital imaging is providing new insights on how cells behave in a more native microenvironment, thereby improving our understanding of disease progression (1,2). In combination with adaptations of in vitro molecular techniques to threedimensional (3D) model systems, intravital imaging is helping to bridge the gap in our understanding of key biologic events that govern cancer progression and therapeutic response in vivo (2)(3)(4).…”

Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

“…To design customized modulation of the actin cytoskeleton at the first step, researchers can analyze in vivo behavior of the actin cytoskeleton using techniques, such as in vivo imaging 161 and fabrication of an in vivo topographical replica. 159 At the second step, the standard rule, which is for the design of micro/nanotopographical features to achieve the customized modulation of the actin cytoskeleton, can be applied in the same way regardless of whether one is dealing with in vivo or in vitro cues.…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

“…This is essential for imaging thick samples, without the need for a confocal pinhole, thereby reducing the effect of sample aberration on the image quality relative to confocal microscopy. The relative advantages of different microscope platforms are discussed in detail elsewhere (Follain et al, 2017;Pantazis and Supatto, 2014;Timpson et al, 2011).…”