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

  •   FigureFigure 6.33.1 Schematic illustration of a typical acquisition light path for multiphoton‐induced SHG imaging. (A) Photomultiplier‐based. SHG signal is induced using an 800‐nm line of the multiphoton laser as excitation wavelength. A short‐pass (SP) 650‐nm filter is used as the main dichroic beam splitter (MBS) and mirrors are used as secondary dichroic beam splitters (DBS). A band‐pass (BP) 390‐ to 465‐nm IR‐blocking filter serves as emission filter. (B) A spectral separator‐based 800‐nm laser line is used as excitation wavelength and the spectral range of 370 to 700 nm is used for SHG detection.
  •   FigureFigure 6.33.2 Three‐dimensional projection of SHG images from fresh human cartilage explants from normal donor. Fresh human cartilage explants were obtained from Articular Engineering, LLC, and placed in a glass‐bottom dish for imaging. SHG signal was induced using an 800‐nm laser line as excitation wavelength. BP 390‐ to 465‐nm IR‐blocking filter served as emission filter. Z‐stack SHG imaging was performed at 0.5‐µm intervals. The Z‐stack thickness is 30 µm. Three‐dimensional projection of SHG images was generated using Imaris software and snapshot series were shown (+180° horizontal rotation). Note that the empty space with no SHG signal indicates the absence of collagen fibrils, therefore representing the lacunae regions.
  •   FigureFigure 6.33.3 Characterization of fresh human cartilage explants from normal and OA+ donors by SHG and multiphoton microscopy. (A) Multiphoton and SHG images of human cartilage explants. Fresh human cartilage explants were obtained from Articular Engineering, LLC, and placed in a glass‐bottom dish for imaging. An 800‐nm laser line was used as excitation wavelength. Emitted light was detected by the spectral separator. The spectral range of 383 to 415 nm was used for SHG detection. The two‐photon excited fluorescence (TPF) image is also shown for comparison (emission: 469 to 522 nm). Bar: 20 µm. (B) Spectral fingerprinting of fresh human cartilage explants. To confirm that the emission setting used in panel A is appropriate, an 800‐nm laser line was used as excitation wavelength and spectral stacks were ascertained at emission wavelengths of 373 to 629 nm (in 10‐nm increments) using the spectral separator. The graph represents peak SHG intensity of collagenous structure in the spectral stack image. The crosses (ROIs 1 to 6) depict the SHG signal of collagen emitting at about 400 nm. (C) Comparison of SHG intensity in human cartilage explants. The peak SHG intensity values for each condition in panel B were calculated before comparison using Student's t‐test. n = 6 and *, significant at p ≤ 0.05. Error bars represent SD (standard deviation).

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Literature Cited

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