Real‐Time Chemiluminescence Imaging Using Nano‐Lantern Probes

Yoshiyuki Arai1, Takeharu Nagai1

1 Osaka University, Osaka
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140168
Online Posting Date:  December, 2014
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Chemiluminescence imaging can be performed without excitation light sources at various spatial levels ranging from a single cell to the whole body. Thus far, chemiluminescence imaging has been primarily performed with long exposure times because of weak signals, resulting in low temporal resolution. Recently, the brightest‐known chemiluminescent proteins—Nano‐lantern and Nano‐lantern‐based functional indicators—have been developed. Nano‐lantern probes break the limitation of temporal resolution and enable chemiluminescence imaging of living samples such as cells, plants, and small animals at video rates. This unit describes one protocol for observation of a freely moving unshaved mouse transplanted with Nano‐lantern‐expressing tumor cells, and another for compatible use of optogenetic tools and a Nano‐lantern calcium indicator. Both protocols utilize the synchronization of illumination and camera acquisition sessions, thereby enabling real‐time chemiluminescence imaging. © 2014 by John Wiley & Sons, Inc.

Keywords: Nano‐lantern; Nano‐lantern (Ca2+); optogenetics; channelrhodopsin2 (ChR2); dead time; CCD camera

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Table of Contents

  • Introduction
  • Basic Protocol 1: Nano‐Lantern for Video‐Rate Luminescence Imaging of Tumor Cells in Freely Moving Unshaved Mice
  • Basic Protocol 2: Optogenetic Activation During Dead Time of Detector for Compatible Chemiluminscence Imaging in Real Time
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Nano‐Lantern for Video‐Rate Luminescence Imaging of Tumor Cells in Freely Moving Unshaved Mice

  • Murine colon adenocarcinoma colon26 cells (RIKEN BRC)
  • Supplemented RPMI‐1640 (see recipe)
  • Nano‐lantern/pcDNA3 (Plasmid 51970, Addgene)
  • Lipofectamine 2000 (Life Technologies)
  • 0.25% trypsin/EDTA with phenol red (Life Technologies)
  • Hank's solution “Nissui” 2 (Nissui, cat. no. 05906)
  • 5‐week‐old male BALB/c mice
  • Coelenterazine‐h (Promega, cat. no. S2011)
  • 100% ethanol
  • 1× phosphate‐buffered saline (PBS)
  • 37°C humidified incubator with 5% CO 2
  • Fluorescence microscope (e.g., Nikon TS100 bright‐field microscope with fluorescence observation unit)
  • 10‐cm culture dishes
  • Burker‐Turk hemacytometer (Erma, cat. no. 2422)
  • Disposable 1‐ml, 30‐G Lo‐dose syringes (BD, cat. no. 326638)
  • Metal angle box (aluminum or steel, ∼30 × 30 × 30 cm, e.g., made in a DIY shop)
  • Shade curtain sufficient to block light (e.g., for dark room; ∼130 × 130 cm)
  • C‐mount lens (Fujifilm, cat. no. HF12.5SA‐1)
  • EMCCD camera (Evolve512, Roper Scientific) with input/output ports
  • Personal computer (PC) and software for camera control
  • Cardboard box (∼60 × 60 × 60 cm)
  • Stainless steel box (∼10 × 10 × 10 cm) with inside painted matte black
  • Light source for bright‐field illumination with optical fiber (e.g., LED; SPECTRA‐X light engine, Lumencor)
  • Pole stand (∼40 cm high) and clamp
  • Oscilloscope
  • Two 50‐Ω BNC cables
  • BNC branch connector (T3285, Thorlabs)
  • Multifunction generator (WF1973, NF Corporation)
  • Image analysis software (e.g., ImageJ, Metamorph)

Basic Protocol 2: Optogenetic Activation During Dead Time of Detector for Compatible Chemiluminscence Imaging in Real Time

  • Pregnant Wistar rats at embryonic day 18
  • Dissection buffer (see recipe), freshly prepared
  • 0.25% trypsin/EDTA with phenol red (Life Technologies)
  • Plating medium (see recipe)
  • Maintenance medium (see recipe)
  • 20× DNase I (see recipe)
  • pcDNA3.1/hChR2‐mCherry (Plasmid 20938, Addgene)
  • Nano‐lantern(Ca2+_600)/pcDNA3 (Plasmid 51982, Addgene)
  • Coelenterazine‐h (see recipe) in 25‐μg aliquots
  • 15‐ml Falcon tube
  • Burker‐Turk hemacytometer (e.g., Erma, cat. no. 2422)
  • 35‐mm PEI‐coated glass‐bottom dishes (see recipe)
  • Electron multiplying charge coupled device (EMCCD) camera (Evolve512, Roper Scientific)
  • Inverted fluorescence microscope (TE2000E, Nikon) on vibration‐free table, with high−numerical aperture objective lens (60×, N.A. 1.30, Nikon)
  • Filter sets for Venus and mCherry fluorescence signals
  • Dichroic mirrors for optogenetic stimulation (FF458‐Di02‐25 × 36, Semrock) that can reflect blue light (438 nm) and allow light in the wavelength range of 467 to 950 nm to pass through
  • Fluorescence tube
  • LED base light source with fluorescence microscopy adaptor (SPECTRA‐X light engine, Lumencor)
  • Light power meter (PM100D and S120C, Thorlabs)
  • Multifunction generator with external trigger input (WF1973, NF Corporation)
  • Two 50‐Ω BNC (or SMA) cables (∼0.5 to 1 m length)
  • Two BNC branch connectors (T3285, Thorlabs)
  • Oscilloscope
  • Personal computer (PC) and imaging software for camera control (Metamorph, Molecular Device)
  • Black curtain to cover entire microscopy system (Meiritz Seiki)
  • Additional reagents and equipment for calcium phosphate precipitation (Mizuno et al., )
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Literature Cited

Literature Cited
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