Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.
In vivo imaging has revolutionized our understanding of biological processes in brain physiology and pathology. However, breathing-induced movement artifacts have impeded the application of this powerful tool in studies of the living spinal cord. Here we describe in detail a method to image stably and repetitively, using two-photon microscopy, the living spinal tissue in mice with dense fluorescent cells or axons, without the need for animal intubation or image post-processing. This simplified technique can greatly expand the application of in vivo imaging to study spinal cord injury, regeneration, physiology and disease.
Davalos
Dimitrios
D
Department of Pharmacology, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. davalos@ucsd.edu
Lee
Jae K
JK
Smith
W Bryan
WB
Brinkman
Brendan
B
Ellisman
Mark H
MH
Zheng
Binhai
B
Akassoglou
Katerina
K
D000075:Acepromazine / Q000494:pharmacology; D000758:Anesthesia / Q000379:methods*; D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000502:physiology / Q000648:ultrastructure; D002196:Capillaries / Q000166:cytology / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007649:Ketamine / Q000494:pharmacology; D007796:Laminectomy / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic; D008833:Microcirculation / Q000166:cytology / Q000502:physiology; D017628:Microglia / Q000166:cytology / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods*; D019635:Neurosurgical Procedures / Q000379:methods; D012143:Respiratory Physiological Phenomena / Q000187:drug effects; D013116:Spinal Cord / Q000098:blood supply / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*; D013238:Stereotaxic Techniques / Q000295:instrumentation; D014520:Urethane / Q000494:pharmacology; D014991:Xylazine / Q000494:pharmacology
D000777:Anesthetics; D049452:Green Fluorescent Proteins; D014991:Xylazine; D014520:Urethane; D000075:Acepromazine; D007649:Ketamine