Two-Photon Intravital Microscopy Animal Preparation Protocol to Study Cellular Dynamics in Pathogenesis

Abstract

Two-photon intravital microscopy (2P-IVM) is an advanced imaging platform that allows the visualization of dynamic processes at subcellular resolution in vivo. Dynamic processes like cell migration, cell proliferation, cell-cell interactions, and cell signaling have an interactive character and occur in complex environments. Hence, it is of pivotal importance to study these processes in living animals, using for example 2P-IVM. 2P-IVM can be performed on a variety of tissues, from the skin of the animal to internal organs, and a variety of methods can be utilized to perform 2P-IVM on these tissues. Here, we discuss the protocols and considerations for four of those 2P-IVM methods, namely tissue explant imaging, skin imaging, surgical exposure imaging, and multi-day window imaging. We carefully compare and explain in depth how to set up each method. Lastly, in the notes section we mention some alternative solutions for the 2P-IVM methods described. In conclusion, this protocol can be used as a guide towards deciding which 2P-IVM method to use and to enable the setup of this method.

Let’s shine a light on fungal infections: A noninvasive imaging toolbox

“…Intravital microscopy (IVM) enables us to image pathogens in a complex biological environment, such as living tissues and organs [1]. Two-photon intravital imaging (2P-IVM) is used for fluorescence imaging of tissues at microscopic resolution [21] through obtaining access to sites of interest by implantation of an optical window. This technique is often used to study the migration of fungal pathogens across the BBB (Fig 1D) [22].…”

“…Recently, an IVM by FCFM procedure was optimized to repeatedly image A. fumigatus and C. gattii infections in mouse lungs in a minimally invasive way ( Fig 1E) [1]. Currently, IVM by FCFM is limited to visualize only two colors, whereas 2P-IVM can be applied with multiple exogenous fluorescent markers [21].…”

Biosensors for spatiotemporal detection of reactive oxygen species in cells and tissues

“…Custom-made devices help to stabilize the organ and prevent artifacts due to cardiac and respiratory movement. Alternatively, skin imaging and multiday window imaging are suitable for certain organs and animals (61). Endomicroscopy is mostly used for the gastrointestinal tract in humans and animals (58), but new endoscopic fibers of Ͻ0.5-mm thickness can be introduced into many organs including the brain (59).…”