intravital-microscopy-for-imaging-subcellular-structures-live-mice

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

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, ...

National Institutes of Health

Rutgers University 

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e., secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane, and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules.

Multiple roles for the actin cytoskeleton during regulated exocytosis.

Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs.

Oleg Milberg

Intracellular Membrane Trafficking Unit,
Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research,
Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research

National Institutes of Health

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