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Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

Published online by Cambridge University Press:  26 July 2012

Manuel Castellano-Muñoz ,
Anthony Wei Peng ,
Felipe T. Salles  and
Anthony J. Ricci
Manuel Castellano-Muñoz
Affiliation:
Department of Otolaryngology, Stanford University, 300 Pasteur Drive, Edwards R145, Stanford, CA 94305, USA
Anthony Wei Peng
Affiliation:
Department of Otolaryngology, Stanford University, 300 Pasteur Drive, Edwards R145, Stanford, CA 94305, USA
Felipe T. Salles
Affiliation:
Department of Otolaryngology, Stanford University, 300 Pasteur Drive, Edwards R145, Stanford, CA 94305, USA
Anthony J. Ricci*
Affiliation:
Department of Otolaryngology, Stanford University, 300 Pasteur Drive, Edwards R145, Stanford, CA 94305, USA Department of Molecular and Cellular Physiology, Stanford University, 300 Pasteur Drive, Edwards R145, Stanford, CA 94305, USA
*
Corresponding author. E-mail: [email protected]
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Abstract

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality.

Keywords

swept field confocal microscopyprairie technologyRedShirt imaginglive-cell imaging
Type
Special Section: Seventh Omaha Imaging Symposium
Information
Microscopy and Microanalysis , Volume 18 , Issue 4 , August 2012 , pp. 753 - 760
Copyright
Copyright © Microscopy Society of America 2012

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References

REFERENCES

Beurg, M., Fettiplace, R., Nam, J.H. & Ricci, A.J. (2009). Localization of inner hair cell mechanotransducer channels using high-speed calcium imaging. Nat Neurosci 12, 553–558.CrossRefGoogle ScholarPubMed
Gasparini, S., Losonczy, A., Chen, X., Johnston, D. & Magee, J.C. (2007). Associative pairing enhances action potential back-propagation in radial oblique branches of CA1 pyramidal neurons. J Physiol 580, 787–800.CrossRefGoogle ScholarPubMed
Gratton, E. & vandeVen, M.J. (2006). Chapter 5: Laser sources for confocal microscopy. In Handbook of Biological Confocal Microscopy, 3rd ed., Pawley, J.B. (Ed.), pp. 80–125. New York: Springer.CrossRefGoogle Scholar
Inoue, S. (2006). Chapter 1: Foundations of confocal scanned imaging in light microscopy. In Handbook of Biological Confocal Microscopy, 3rd ed., Pawley, J.B. (Ed.), pp. 1–16. New York: Springer.Google Scholar
Minsky, M. (1988). Memoir on inventing the confocal microscope. Scanning 10, 128–138.CrossRefGoogle Scholar
Pawley, J.B. (2006). Fundamental limits in confocal microscopy. In Handbook of Biological Confocal Microscopy, 3rd ed., Pawley, J.B. (Ed.), pp. 20–41. New York: Springer.CrossRefGoogle Scholar
Ricci, A.J., Gray-Keller, M. & Fettiplace, R. (2000). Tonotopic variations of calcium signaling in turtle auditory hair cells. J Physiol (Lond) 524(Pt 2), 423–436.CrossRefGoogle ScholarPubMed
Santana, L.F., Navedo, M.F., Amberg, G.C., Nieves-Cintron, M., Votaw, V.S. & Ufret-Vincenty, C.A. (2008). Calcium sparklets in arterial smooth muscle. Clin Exp Pharmacol Physiol 35, 1121–1126.CrossRefGoogle ScholarPubMed
Schnee, M.E., Santos-Sacchi, J., Castellano-Munoz, M., Kong, J.H. & Ricci, A.J. (2011). Calcium-dependent synaptic vesicle trafficking underlies indefatigable release at the hair cell afferent fiber synapse. Neuron 70, 326–338.CrossRefGoogle ScholarPubMed
Toomre, D. & Pawley, J.B. (2006). Chapter 10: Disc scanning confocal microscopy. In Handbook of Biological Confocal Microscopy, 3rd ed., Pawley, J.B. (Ed.), pp. 221–237. New York: Springer CrossRefGoogle Scholar
Tucker, T. & Fettiplace, R. (1995). Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells. Neuron 15, 1323–1335.CrossRefGoogle ScholarPubMed