Chapter 11: Imaging fluorescent mice in vivo using confocal microscopy.
In vivo imaging is the most direct way to uncover the dynamic events that occur during neural development. This approach is especially challenging in developing mammals where technical hurdles related to optical resolution, animal movement, phototoxicity, and postoperative complications need to be addressed. In our work concerning the process of naturally occurring synapse elimination at developing neuromuscular junctions, these technical issues are critical because we need to resolve multiple and very fine single axons that converge on the same synaptic site. In previous studies, we used wide-field microscopy with either intensified or high quantum efficiency cameras. We now have begun to use laser scanning confocal microscopy which improves contrast and resolution but comes with its own challenges. In this chapter, we describe the approaches we have taken to permit both rapid time-lapse (minutes to hours) and long-term time-lapse (days to months) to visualize the synaptic alterations associated with the development and maturation of the neuromuscular system.
Turney
Stephen G
SG
Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.
Lichtman
Jeff W
JW
D000818:Animals; D001369:Axons / Q000502:physiology / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000032:analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009420:Nervous System / Q000254:growth & development* / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000254:growth & development / Q000648:ultrastructure; D038761:Photobleaching; D013569:Synapses / Q000502:physiology / Q000648:ultrastructure
D049452:Green Fluorescent Proteins