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Wide-Field and Two-Photon Imaging of Brain Activity with Voltage and Calcium-Sensitive Dyes

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Dynamic Brain Imaging

Part of the book series: METHODS IN MOLECULAR BIOLOGY™ ((MIMB,volume 489))

Abstract

This chapter presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes. Because experimental measurements are limited by low sensitivity, the chapter then discusses the methodological aspects that are critical for optimal signal-to-noise ratio. Two of the examples use wide-field (1-photon) imaging and the third uses two-photon scanning microscopy. These methods have relatively high temporal resolution ranging from 10 to 10,000 Hz.

The three examples are the following: (1) Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. These experiments are directed at understanding how individual neurons convert the complex input synaptic activity into the output spike train. (2) Recently developed methods for staining many individual cells in the mammalian brain with calcium-sensitive dyes together with two-photon microscopy made it possible to follow the spike activity of many neurons simultaneously while in vivo preparations are responding to stimulation. (3) Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb glomeruli. There, the population signals can be used as a measure of the input from the nose to the bulb.

Three kinds of noise in measuring light intensity are discussed: (1) Shot noise from the random emission of photons from the preparation. (2) Extraneous (technical) noise from external sources. (3) Noise that occurs in the absence of light, the dark noise. In addition, we briefly discuss the light sources, the optics, and the detectors and cameras.

The commonly used organic voltage and ion sensitive dyes stain all of the cell types in the preparation indiscriminately. A major effort is underway to find methods for staining individual cell types in the brain selectively. Most of these efforts center around fluorescent protein activity sensors because transgenic methods can be used to express them in individual cell types.

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Acknowledgments

The authors are indebted to their collaborators Vicencio Davila, Amiram Grinvald, Kohtaro Kamino, Les Loew, Bill Ross, Brian Salzberg, Dejan Vucinic, Alan Waggoner, Matt Wachowiak, and Jian-young Wu for numerous discussions about optical methods. The experiments carried out in our laboratories were supported by NIH grants DC05259 and NS42739, Deutsche Forschungsgemeinschaft (SFB 391 and SFB 596) and the Bundesministerium für Bildung und Forschung (NGFN-2).

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Authors and Affiliations

  1. Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, CT

    Ryota Homma, Bradley J. Baker, Lei Jin, Lawrence B. Cohen, Chun X. Bleau & Dejan Zecevic

  2. Institute of Neuroscience, Technical University Munich, Munich, Germany

    Olga Garaschuk & Arthur Konnerth

  3. RedShirtImaging, LLC, Decatur, GA

    Lawrence B. Cohen & Chun X. Bleau

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  1. Ryota Homma
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  2. Bradley J. Baker
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  3. Lei Jin
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  4. Olga Garaschuk
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  5. Arthur Konnerth
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  6. Lawrence B. Cohen
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  7. Chun X. Bleau
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  8. Marco Canepari
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  9. Maja Djurisic
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  10. Dejan Zecevic
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Editors and Affiliations

  1. Yale University, New Haven, CT, USA

    Fahmeed Hyder

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Homma, R. et al. (2009). Wide-Field and Two-Photon Imaging of Brain Activity with Voltage and Calcium-Sensitive Dyes. In: Hyder, F. (eds) Dynamic Brain Imaging. METHODS IN MOLECULAR BIOLOGY™, vol 489. Humana Press. https://doi.org/10.1007/978-1-59745-543-5_3

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  • DOI: https://doi.org/10.1007/978-1-59745-543-5_3

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-74-9

  • Online ISBN: 978-1-59745-543-5

  • eBook Packages: Springer Protocols

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