Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Abstract

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton. Video LinkThe video component of this article can be found at

In Vivo Tissue-wide Synchronization of Mitochondrial Metabolic Oscillations

“…The SGs and the body of the animal were immobilized using custom-made holders as previously shown (Masedunskas et al, 2013a; Masedunskas et al, 2013b). The blood flow was assessed visually by using the eyepiece.…”

Multiscale modelling of saliva secretion

“…As has been known for a long time, the ducts form a tree-like branching structure [29], and the acinar cells are situated at the terminals of the tree branches. However, the acinar cells themselves are distributed around a lumen which is itself a branching structure [30]. Thus, each acinar cell secretes fluid into a small lumenal tube, and there is no common lumen into which all the acinar cells secrete.…”

Probing the Role of the Actin Cytoskeleton During Regulated Exocytosis by Intravital Microscopy

“…Set up the stage and position the animal with the glands exposed and immobilized as previously described for rats [11] and mice [16]. …”