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, null, null, 2 Laboratory for Integrated Systems Biology, Graduate School of Informatics, Kyoto University, Kyoto, Japan, null, null
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

  •   FigureFigure 2.14.1 Overview of Nipkow‐type spinning‐disk confocal microscopy and experimental procedures. (A) Photograph of a Nipkow‐type spinning‐disk confocal microscope. The high‐speed EM‐CCD camera and the Yokogawa CSU‐X1 confocal unit are mounted on a Nikon FN1 upright microscope. (B) Schematic illustration of the Nipkow‐type confocal system. A laser source passes through a microlens‐array disk and a rotating pinhole‐array disk and splits into multiple beams before reaching the specimen. The microlens‐array disk is used to minimize light loss by concentrating each laser beam on the corresponding pinhole. (C) Schematic illustration for the in vitro loading of neuronal populations with calcium indicators. The immersion‐loading method (left) is suitable for slice cultures and acute slices prepared from juvenile animals. The spot‐loading method (right) is more versatile.
  •   FigureFigure 2.14.2 Post‐hoc data processing of fMCI movies. (A) Example fMCI from CA3 neurons in hippocampal slice cultures. OGB‐1AM was applied using the spot‐loading method. The original movie (left, 100 fps) was image‐processed post‐hoc with a custom‐made denoising program and separated into signals (middle) and periodic noise artifact (right); see Supplementary Movie 1. (B) Representative raw fluorescence trace (left), denoised signal trace (middle), and noise artifact (right) of the neuron indicated by the red ellipse in A. (C) Spatial locations of all neurons identified in A. (D) Calcium‐signal traces for the four neurons labeled in C. Reconstructed spike times are indicated at the bottom of each trace. To access the supplemental movie (NS0214_denoise example.mp4), see the supplemental file at http://www.currentprotocols.com/protocol/ns0214.
  •   FigureFigure 2.14.3 Post‐hoc processing of calcium‐imaging data. (A) S/N ratios of action‐potential‐induced calcium transients decrease with the image‐acquisition rate (20 to 2000 fps). Preparations were illuminated at a laser power of 2 mW. Neurons were fired with a brief current injection in the current‐clamp mode (top right). Representative fluorescent signals observed at 100 and 2000 fps are shown in the traces on the right. The S/N ratio was defined as the ratio of the calcium‐transient amplitude to the standard deviation (SD) of its baseline fluctuation. Data are reported as the means ± SDs of four neurons. (B) S/N ratios increase with laser power (0.5 to 16 mW). Movies were taken at 200 fps. Neurons were fired with a short current injection with a patch‐clamp electrode (top right). Representative fluorescence signals observed at 0.5 and 4 mW are shown in the right traces. Data are reported as the means ± SDs of four neurons. (C) Baseline fluorescence intensity bleaches as a function of time under continuous laser illumination. Left traces represent the average photobleaching functions at various laser intensities ranging from 0.5 to 16 mW (four slices each). The right plot shows the relationship between the laser power and the photobleaching decay constant. Data are reported as the means ± SDs of four neurons. Abbreviations: S/N, signal‐to‐noise.

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