Whole‐Body Imaging of Infection Using Fluorescence

Ying Kong1, Ali R. Akin2, Kevin P. Francis2, Ning Zhang2, Tamara L. Troy2, Hexin Xie3, Jianghong Rao3, Suat L. G. Cirillo1, Jeffrey D. Cirillo1

1 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, Texas, 2 Caliper Life Sciences, Alameda, California, 3 Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, Stanford University, Stanford, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2C.3
DOI:  10.1002/9780471729259.mc02c03s21
Online Posting Date:  May, 2011
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Abstract

Optical imaging is emerging as a powerful tool to study physiological, neurological, oncological, cell biological, molecular, developmental, immunological, and infectious processes. This unit describes the use of fluorescent reporters for biological organisms, components, or events. We describe the application of fluorescence imaging to examination of infectious processes, in particular subcutaneous and pulmonary bacterial infections, but the same approaches are applicable to nearly any infectious route. The strategies described use mycobacterial infections as an example, but nearly identical systems can be used for Pseudomonas, Legionella, Salmonella, Escherichia, Borrelia, and Staphylococus, suggesting that the approaches are generally applicable to nearly any infectious agent. Two strategies for fluorescence imaging are described: the first method uses reporter enzyme fluorescence (REF), and the second uses fluorescent proteins for fluorescence imaging. Methods are described in detail to facilitate successful application of these emerging technologies to nearly any experimental system. Curr. Protoc. Microbiol. 21:2C.3.1‐2C.3.21. © 2011 by John Wiley & Sons, Inc.

Keywords: bioluminescence; fluorescence; mice; in vivo imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Reporter Enzyme Fluorescence for Imaging Bacterial Infections
  • Basic Protocol 2: Imaging M. smegmatis Lung Infection with Fluorescent Proteins in Live Mice
  • Basic Protocol 3: Imaging M. bovis BCG Intratracheal Lung Infection with Fluorescent Proteins in Live Mice
  • Support Protocol 1: Preparation of Mycobacterial Cultures for Inoculation
  • Support Protocol 2: Subcutaneous and Intratracheal Infection of Mice
  • Support Protocol 3: Isoflurane Gas Anesthesia
  • Support Protocol 4: 3D Fluorescent Imaging via FLIT Acquisition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Reporter Enzyme Fluorescence for Imaging Bacterial Infections

  Materials
  • 5‐ to 7‐week‐old female Balb/c mice (The Jackson Laboratory) housed in polycarbonate microisolator cages in a temperature‐, humidity‐, and light‐controlled environment
  • Mycobacterium tuberculosis H37Rv strain grown in M‐OADC‐TW broth (see recipe) or on M‐OADC agar (see recipe) or Middlebrook 7H11 selective agar (BD Difco)
  • M. bovis BCG strain Pasteur grown in M‐OADC‐TW broth (see recipe) or on M‐OADC agar (see recipe) or 7H11 selective agar (BD Difco)
  • 5 µM CNIR5 [substrate for the β‐lactamase, having a Cy5.5 fluorochrome and a quencher (QSY21) (Kong et al., )] in PBS (see appendix 2A for PBS; prepare from 1.8 mM CNIR5 stock in DMSO; protect from light); CNIR5 was provided by Dr. Rao's lab ( )
  • IVIS imaging system (Caliper Life Sciences; http://www.caliperls.com/)
  • Living Image software (Caliper Life Sciences; http://www.caliperls.com/)
  • Additional reagents and equipment for preparation of mycobacterial cultures ( protocol 4) and inoculation of mice with mycobacteria ( protocol 5), isoflurane anesthesia of mice ( protocol 6), and FLIT reconstruction ( protocol 7)

Basic Protocol 2: Imaging M. smegmatis Lung Infection with Fluorescent Proteins in Live Mice

  Materials
  • Sterile LB+ADC+TW growth medium (see recipe)
  • LB+ADC agar plate (see recipe) containing M. smegmatis strain having a plasmid pFJS8 (Wagner et al., ), in which the gfp gene has been replaced with the mCherry gene by inserting it between HindIII and KpnI sites under the control of the L5 promoter (M. smegmatis expressing mCherry is available from the Cirillo Lab: )
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Female nu/nu mice at 6 to 8 weeks of age
  • Antimicrobial (e.g., iodine)
  • Alcohol swabs
  • 1000‐ml Erlenmeyer flask
  • 14‐ml culture tubes (VWR, cat. No. 60818‐725)
  • Disposable sterile inoculation loop
  • 37°C incubator containing platform shaker or roller‐tube apparatus
  • Spectrophotometer
  • Gas anesthesia manifold apparatus (see protocol 6)
  • Insulin syringe with 29‐G needle (Becton Dickinson, cat. no. 309301)
  • IVIS imaging system (Caliper Life Sciences; http://www.caliperls.com/)
  • Living Image software (Caliper Life Sciences; http://www.caliperls.com/)
  • Additional reagents and equipment for isoflurane anesthesia of mice ( protocol 6)

Basic Protocol 3: Imaging M. bovis BCG Intratracheal Lung Infection with Fluorescent Proteins in Live Mice

  Materials
  • M. bovis BCG strain carrying a plasmid expressing tdTomato (Kong et al., ; available from from the Cirillo Lab, )
  • 5‐ to 7‐week‐old female Balb/c mice (The Jackson Laboratory) housed in polycarbonate microisolator cages in a temperature‐, humidity‐, and light‐controlled environment
  • IVIS imaging system (Caliper Life Sciences)
  • Additional reagents and equipment for preparing bacteria (see protocol 2), intratracheal infection of mice ( protocol 5), IVIS image analysis and spectral unmixing ( protocol 1), and 3D (FLIT) analysis ( protocol 7)

Support Protocol 1: Preparation of Mycobacterial Cultures for Inoculation

  Materials
  • Mycobacterial frozen stocks, prepared by growth at 37°C until an OD 600 = 0.5 to 1 is obtained, and stored in aliquots in half medium/half sterile glycerol at −80°C until use
  • M‐OADC‐TW broth (see recipe)
  • LB‐ADC‐TW growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Refrigerated centrifuge

Support Protocol 2: Subcutaneous and Intratracheal Infection of Mice

  Materials
  • Mouse
  • Bacterial inoculum ( protocol 4) and negative control (BCG strain carrying the backbone alone)
  • M‐OADC agar plates (see recipe)
  • 10 mg/ml ketamine/1 mg/ml xylazine in distilled H 2O for intratracheal infection (store at 4°C)
  • Pet Pocket Pro Trimmer (Wahl Clipper Corporation) to trim mouse hair off for subcutaneous infection
  • 1‐ml syringes with 26‐G needles
  • Horizontal surface, e.g., dissecting pan
  • Marker (optional)
  • Tape
  • Forceps
  • Otoscope with intubation speculum
  • 22‐G catheter for intratracheal infection
  • Guide wire: mouse endotracheal tube introducer (Hallowell EMC, http://www.hallowell.com/)
  • Additional reagents and equipment for isoflurane anesthesia of the mouse ( protocol 6) and preparation of cultures for inoculation ( protocol 4)

Support Protocol 3: Isoflurane Gas Anesthesia

  Materials
  • Isoflurane, Abbott Laboratories, 250 ml (Burns Veterinary Supply; https://www.accessbutler.com/)
  • 10% bleach or similar approved commercial disinfectant
  • XGI‐8 gas anesthesia system (Caliper Life Sciences; http://www.caliperls.com/)
  • Nose cone apparatus manifold (Caliper Life Sciences; http://www.caliperls.com/))
  • Activated charcoal evacuation filters, Omnicon (Burns Veterinary Supply; https://www.accessbutler.com/)

Support Protocol 4: 3D Fluorescent Imaging via FLIT Acquisition

  Materials
  • IVIS imaging system (Caliper Life Sciences; http://www.caliperls.com/)
  • Living Image software (Caliper Life Sciences; http://www.caliperls.com/)
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Figures

Literature Cited

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