Three‐Dimensional Second‐Harmonic Generation Imaging of Fibrillar Collagen in Biological Tissues

Jiansong Xie1, John Ferbas1, Gloria Juan1

1 Department of Medical Sciences, Amgen, Thousand Oaks, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.33
DOI:  10.1002/0471142956.cy0633s61
Online Posting Date:  July, 2012
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Abstract

Multiphoton‐induced second‐harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three‐dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues. Some technical considerations are also addressed. Curr. Protoc. Cytom. 61:6.33.1‐6.33.11. © 2012 by John Wiley & Sons, Inc.

Keywords: multiphoton microscopy; second‐harmonic generation; laser scanning microscopy; collagen; extracellular matrix; articular cartilage

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Setting Up an Imaging System for Three‐Dimensional SHG Microscopy
  • Basic Protocol 2: Three‐Dimensional SHG Imaging of Fibrillar Collagen in Biological Tissues on Inverted Microscope
  • Basic Protocol 3: SHG Signal Quantitation by Spectral Fingerprinting
  • Support Protocol 1: SHG Imaging of Fibrillar Collagen in Ex Vivo Cultured Tissue
  • Support Protocol 2: SHG Imaging of Fibrillar Collagen in Tissue Cryosection
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Setting Up an Imaging System for Three‐Dimensional SHG Microscopy

  Materials
  • Inverted or upright microscope (e.g., ZEISS, Leica, Nikon, Olympus) including:
    • Transmitted‐light illuminator
    • Fluorescence illuminator
    • Suitable objective lenses
    • Detector unit including beam splitters, BP filters, PMT, and spectral separator (e.g., ZEISS META detector, optional)
    • Motorized microscope stage with computer‐driven z‐position control
  • Multiphoton laser (e.g., Coherent Chameleon two‐photon laser)
  • Visible lasers for confocal imaging (e.g., Argon, green HeNe, red HeNe, optional)
  • Computer hardware and software for image acquisition, analysis and export
  • Vibration isolation table (e.g., KINETIC SYSTEMS)

Basic Protocol 2: Three‐Dimensional SHG Imaging of Fibrillar Collagen in Biological Tissues on Inverted Microscope

  Materials
  • Biological tissue samples of interest
  • Phosphate‐buffered saline (PBS) or other physiological saline solution
  • Glass‐bottom dish (plastic petri dish with high optical quality glass coverslip attached to the bottom)
  • Additional reagents and equipment for SHG imaging ( protocol 1)

Basic Protocol 3: SHG Signal Quantitation by Spectral Fingerprinting

  Materials
  • Excised biological tissues of interest
  • Storage medium of choice (e.g., 50/50 DMEM/F12 with 10% FBS)
  • Incubation buffer (e.g., PBS or other physiological saline solution)
  • Treatment agents (e.g., molecule/compound to test)
  • Multi‐well tissue culture plate
  • 37°C tissue culture incubator
  • Glass‐bottom dish
  • Additional reagents and equipment for SHG imaging ( protocol 1)

Support Protocol 1: SHG Imaging of Fibrillar Collagen in Ex Vivo Cultured Tissue

  Materials
  • Embedding tissue
  • Phosphate‐buffered saline (PBS) or other physiological saline solution
  • OCT (Optimal cutting temperature) compound
  • Liquid nitrogen
  • Mounting solution (e.g., 75% glycerol in PBS)
  • Nail polish
  • Paper towels
  • Embedding mold
  • Standard microtome
  • Glass slide suitable for cryosections
  • −80°C freezer
  • Glass coverslips
  • Additional reagents and equipment for SHG imaging ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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