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Alignment and calibration of a focal neurotransmitter uncaging system

Nature Protocols volume 1, pages 828–832 (2006)Cite this article

Abstract

Photolysis of caged compounds is a powerful tool for studying subcellular physiological functions. Here we describe protocols for the alignment and calibration of a focal uncaging system. We also report procedures for convenient quantitative calibration of uncaging. Using these methods, we can achieve submicron lateral resolution of photolysis and probe biological function in spines, the smallest signaling compartments of neurons. Initially, the entire alignment procedure takes 4–6 h to perform; periodic fine-tuning of the system takes 1–2 h.

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Figure 1: Design and alignment of a system for focal uncaging.
Figure 2: Two methods for the fine adjustment of the size and position of the uncaging spot.
Figure 3: Estimating the uncaging efficiency of the system.

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Acknowledgements

We thank G.M. Wittenberg and B. Kuhn for advice and discussion. This work was supported by the Rita Allen Foundation, the Whitehall Foundation, and National Institutes of Health grant NS045193. S.S.-H.W. is a W.M. Keck Foundation Distinguished Young Scholar. D.V.S. was supported by a Burroughs-Wellcome Interfaces of Science fellowship.

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Authors and Affiliations

  1. Department of Physics, Princeton University, Princeton, 08544, New Jersey, USA

    Dmitry V Sarkisov

  2. Program in Neuroscience, Princeton University, Princeton, 08544, New Jersey, USA

    Dmitry V Sarkisov & Samuel S-H Wang

  3. Department of Molecular Biology, Princeton University, Princeton, 08544, New Jersey, USA

    Samuel S-H Wang

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  1. Dmitry V Sarkisov
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Correspondence to Samuel S-H Wang.

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Sarkisov, D., Wang, SH. Alignment and calibration of a focal neurotransmitter uncaging system. Nat Protoc 1, 828–832 (2006). https://doi.org/10.1038/nprot.2006.124

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