Practical Methods for Molecular In Vivo Optical Imaging

Hannah Chen1, Stephen H. Thorne1

1 Departments of Surgery and Immunology, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.24
DOI:  10.1002/0471142956.cy1224s59
Online Posting Date:  January, 2012
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Abstract

Traditional approaches for translating observations of molecular events into the context of a living organism have suffered from the requirements for either sacrificing animals at multiple time points prior to labor‐intensive analyses of multiple tissues, or have relied on subjective observations or measurements of the animals over time. Recently, a plethora of dedicated animal‐imaging modalities and the release of modified clinical imaging devices dedicated for animal imaging have allowed for the design of quantitative real‐time experiments incorporating fewer animals and providing whole‐animal analyses. Of these modalities, optical imaging (bioluminescence and fluorescence) has emerged as a powerful research tool, allowing investigators with limited whole‐animal imaging expertise to rapidly and inexpensively translate models produced in cellular assays into the context of a living animal. Here we will outline the steps necessary for translation of models established in culture systems into rodents. Curr. Protoc. Cytom. 59:12.24.1‐12.24.11. © 2012 by John Wiley & Sons, Inc.

Keywords: bioluminescence; fluorescence; whole animal; molecular imaging; non‐invasive reporter

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bioluminescence Imaging of Adoptively Transferred Eukaryotic Cells
  • Support Protocol 1: Lentiviral Labeling of Cell Lines
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Bioluminescence Imaging of Adoptively Transferred Eukaryotic Cells

  Materials
  • Cells expressing luciferase ( protocol 2)
  • Cells of interest (e.g., mouse tumor cell line)
  • Mice, typically obtained form commercial vendors (e.g., Jackson Laboratories or Charles River)
  • D‐luciferin, 30 mg/ml in PBS, filter sterilized
  • 5 mg/ml coelenterazine in methanol (Millipore Chemicon)
  • Anesthetic (ideally inhaled, such as isoflurane, but may be injected, such as avertin)
  • 70% ethanol
  • Whole‐animal bioluminescence imaging system (e.g., IVIS, Caliper Life Sciences) with heated (37°C) stage and appropriate image analysis software (e.g., Living Image, Caliper Life Sciences)
  • Black paper
  • Software for image analysis (e.g., Living Image, Caliper Life Sciences)
NOTE: Although there are no restrictions of age, gender, weight, or breed of mice, larger mice and black mice tend to absorb more light and so display reduced sensitivity.

Support Protocol 1: Lentiviral Labeling of Cell Lines

  Materials
  • 293T cell lines
  • DMEM containing 10% FBS, 1% penicillin‐streptomycin, and 1% glutamine
  • Lipofectamine 2000 (Invitrogen)
  • OPTI‐MEM medium
  • Insert DNA plasmid (e.g., from an Endo‐free midi or maxi‐prep kit)
  • Packaging DNA plasmids
  • Polybrene
  • Phosphate‐buffered saline (PBS)
  • Lentiviral expression kits (third generation, four plasmid packaging systems are recommended, can be cloned to express luciferase)
  • 10‐cm plates
  • 32°C incubator
  • 15‐ml tubes
  • Centrifuge
  • 6‐well plates
  • 37°C shaking incubator
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Figures

Videos

Literature Cited

Literature Cited
   Contag, C.H. and Bachmann, M.H. 2002. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed. Eng. 4:235‐260.
   Shaner, N.C., Steinbach, P.A., and Tsien, R.Y. 2005. A guide to choosing fluorescent proteins. Nat. Methods 2:905‐909.
   Thorne, S.H. and Contag, C.H. 2005. Using in vivo bioluminescence imaging to shed light on cancer biology. Proc. IEEE 93:750‐762.
   Villanueva, F.S. 2008. Molecular imaging of cardiovascular disease using ultrasound. J. Nucl. Cardiol. 15:576‐586.
   Zhao, H., Doyle, T.C., Coquoz, O., Kalish, F., Rice, B.W., and Contag, C.H. 2005. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J. Biomed. Opt. 10:41210.
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