Abstract
Confocal imaging of fluorescent probes offers a powerful, non-invasive tool which enables data collection from vast population of cells at high spatial and temporal resolution. Spinning disk confocal microscopy parallelizes the imaging process permitting the study of dynamic events in populations of living cells on the millisecond time scale. Several spinning disk microscopy solutions are commercially available, however these are often poorly configurable and relatively expensive. This chapter describes a procedure to assemble a cost-effective homemade spinning disk system for fluorescence microscopy, which is highly flexible and easily configurable. We finally illustrate a reliable protocol to obtain high-quality Ca2+ and voltage imaging data from cochlear preparations.
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References
Mammano F (2013) ATP-dependent intercellular Ca2+ signaling in the developing cochlea: facts, fantasies and perspectives. Semin Cell Dev Biol 24:31–39
Marcotti W (2012) Functional assembly of mammalian cochlear hair cells. Exp Physiol 97:438–451
Pawley J (2006) Handbook of biological confocal microscopy, vol 236. Springer, New York, NY
Gräf R, Rietdorf J, Zimmermann T (2005) Live cell spinning disk microscopy. Adv Biochem Eng Biotechnol 95:57–75
Confocal Microscopy System (2005) http://www.biosciencetechnology.com/articles/2005/01/confocal-microscopy-system
Beltramello M, Piazza V, Bukauskas FF, Pozzan T, Mammano F (2005) Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness. Nat Cell Biol 7:63–69
Piazza V, Ciubotaru CD, Gale JE, Mammano F (2007) Purinergic signalling and intercellular Ca2+ wave propagation in the organ of Corti. Cell Calcium 41:77–86
Ortolano S, Di Pasquale G, Crispino G, Anselmi F, Mammano F, Chiorini JA (2008) Coordinated control of connexin 26 and connexin 30 at the regulatory and functional level in the inner ear. Proc Natl Acad Sci U S A 105:18776–18781
Anselmi F, Hernandez VH, Crispino G et al (2008) ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear. Proc Natl Acad Sci U S A 105:18770–18775
Rodriguez L, Simeonato E, Scimemi P et al (2012) Reduced phosphatidylinositol 4,5-bisphosphate synthesis impairs inner ear Ca2+ signaling and high-frequency hearing acquisition. Proc Natl Acad Sci U S A 109:14013–14018
Miller EW, Lin JY, Frady EP, Steinbach PA, Kristan WB, Tsien RY (2012) Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires. Proc Natl Acad Sci U S A 109:2114–2119
Mammano F, Canepari M, Capello G, Ijaduola RB, Cunei A, Ying L, Fratnik F, Colavita A (1999) An optical recording system based on a fast CCD sensor for biological imaging. Cell Calcium 25:115–123
Acknowledgments
This work was supported by Telethon Italy grants GGP13114 and GGP12269 to FM and MB, respectively.
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Ceriani, F., Ciubotaru, C.D., Bortolozzi, M., Mammano, F. (2016). Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology, vol 1427. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3615-1_13
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DOI: https://doi.org/10.1007/978-1-4939-3615-1_13
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