High‐Speed Multineuron Calcium Imaging Using Nipkow‐Type Confocal Microscopy

Naoya Takahashi1, Shigeyuki Oba2, Naoto Yukinawa2, Sakiko Ujita1, Mika Mizunuma1, Norio Matsuki1, Shin Ishii2, Yuji Ikegaya1

1 Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 2 Laboratory for Integrated Systems Biology, Graduate School of Informatics, Kyoto University, Kyoto, Japan
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.14
DOI:  10.1002/0471142301.ns0214s57
Online Posting Date:  October, 2011
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Abstract

Conventional confocal and two‐photon microscopy scan the field of view sequentially with single‐point laser illumination. This raster‐scanning method constrains video speeds to tens of frames per second, which are too slow to capture the temporal patterns of fast electrical events initiated by neurons. Nipkow‐type spinning‐disk confocal microscopy resolves this problem by the use of multiple laser beams. We describe experimental procedures for functional multineuron calcium imaging (fMCI) based on Nipkow‐disk confocal microscopy, which enables us to monitor the activities of hundreds of neurons en masse at a cellular resolution at up to 2000 fps. Curr. Protoc. Neurosci. 57:2.14.1‐2.14.10. © 2011 by John Wiley & Sons, Inc.

Keywords: imaging; microscopy; calcium; neuron; spike

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Loading Neurons with Calcium Indicator (Immersion‐Loading) and Imaging
  • Alternate Protocol 1: Spot Loading of Brain Tissue with Dyes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Loading Neurons with Calcium Indicator (Immersion‐Loading) and Imaging

  Materials
  • Oregon Green 488 BAPTA‐1 (OGB‐1) acetoxymethyl ester (AM) (Invitrogen)
  • Dimethyl sulfoxide (DMSO)
  • Pluronic F‐127 (Invitrogen)
  • Extracellular artificial cerebrospinal fluid (aCSF; see recipe), oxygenated with carbogen gas (95% O 2/5% CO 2)
  • Organotypic brain slices on early DiV or acute slices from young animals
  • Carbogen gas (95% O 2/5% CO 2)
  • 1.5‐ml microtubes
  • 35‐mm dishes
  • 37°C incubator
  • Submerged recording chamber (1.5 ml volume)
  • Temperature controller
  • Upright microscope and high‐aperture (>0.8) water‐immersion objective lens (e.g., Nikon or Zeiss)
  • High‐speed CCD camera: e.g., iXon EM+ DU897 (512 × 512 pixels) for <200 frames per second (fps) or iXon EM+ DU860 (128 × 128 pixels) for 100–2000 fps (Andor)
  • Nipkow‐type spinning‐disk confocal unit, e.g., CSU‐X1 (Yokogawa Electric)
  • 488‐nm laser diode
  • ImageJ software

Alternate Protocol 1: Spot Loading of Brain Tissue with Dyes

  • Brain slices, such as late‐DiV organotypic slices and acute slices from adult animals
  • Oregon Green 488 BAPTA‐1 (OGB‐1) acetoxymethyl ester (AM) (Invitrogen)
  • Pluronic F‐127
  • Extracellular artificial cerebrospinal fluid (aCSF; see recipe), oxygenated with carbogen gas (95% O 2/5% CO 2)
  • 45‐µm‐pore‐diameter filter
  • Patch pipet (2–5 MΩ)
  • Micromanipulator
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Figures

Literature Cited

Literature Cited
   Ishikawa, D., Takahashi, N., Sasaki, T., Usami, A., Matsuki, N., and Ikegaya, Y. 2010. Fluorescent pipettes for optically targeted patch‐clamp recordings. Neural Netw. 23: 669–672.
   Sasaki, T., Matsuki, N., and Ikegaya, Y. 2007. Metastability of active CA3 networks. J. Neurosci. 27: 517–528.
   Sasaki, T., Takahashi, N., Matsuki, N., and Ikegaya, Y., 2008. Fast and accurate detection of action potentials from somatic calcium fluctuations. J. Neurophysiol. 100: 1668–1676.
   Sasaki, T., Minamisawa, G., Takahashi, N., Matsuki, N., and Ikegaya, Y. 2009. Reverse optical trawling for synaptic connections in situ. J. Neurophysiol. 102: 636–643.
   Smetters, D., Majewska, A., and Yuste, R. 1999. Detecting action potentials in neuronal populations with calcium imaging. Methods 18: 215–21.
   Takahara, Y., Matsuki, N., and Ikegaya, Y. 2011. Nipkow confocal imaging from deep brain tissues. Journal of Integrative Neuroscience 10: 121–129.
   Takahashi, N., Sasaki, T., Usami, A., Matsuki, N., and Ikegaya, Y. 2007. Watching neuronal circuit dynamics through functional multineuron calcium imaging (fMCI). Neurosci. Res. 58: 219–225.
   Takahashi, N., Sasaki, T., Matsumoto, W., Matsuki, N., and Ikegaya, Y. 2010. Circuit topology for synchronizing neurons in spontaneously active networks. Proc. Natl. Acad. Sci. U.S.A. 107: 10244–10249.
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Supplementary Materials