Biological Second and Third Harmonic Generation Microscopy

Peter Friedl1, Katarina Wolf1, Gregory Harms1, Ulrich H. von Andrian2

1 University of Würzburg, Würzburg, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.15
DOI:  10.1002/0471143030.cb0415s34
Online Posting Date:  March, 2007
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Abstract

Multiphoton microscopy has become a standard method for noninvasive imaging of thick specimens with subcellular resolution. Higher harmonic generation microscopy (HHGM), based on nonlinear multiphoton excitation, is a contrast mechanism for the structural and molecular imaging of native samples in cell culture and in fixed and live tissues, for both, three‐dimensional and four‐dimensional reconstructions. HHGM comprises second and third harmonic generation (SHG, THG) of ordered molecules, can be obtained without exogenous labels, and provides detailed real‐time optical reconstruction of fibrillar collagen, myosin, microtubules, and membrane potential, as well as cell depolarization. This unit presents the principles of SHG and THG and the basic setup of a HHGM system, and summarizes current applications in cell biology. Multimodal multiphoton microscopy using HHGM together with two‐photon excited fluorescence will develop into a key approach to real‐time imaging of cell dynamics in the context of live tissues.

Keywords: microscopy; second and third harmonic generation microscopy; living cells; higher harmonic generation microscopy

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Designing A Microscope System for HHGM
  • Basic Protocol 2: Detection of Fibrillar Collagen in Connective Tissue ex vivo
  • Basic Protocol 3: Detection of SHG in Mouse Tissues by Intravital Microscopy
  • Basic Protocol 4: Simultaneous Detection of Cells and Collagen Fibers In Vitro and In Vivo
  • Support Protocol 1: Cytoplasmic Staining of Live Cells
  • Support Protocol 2: Establishment of 3‐D Collagen Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Designing A Microscope System for HHGM

  Materials
  • Upright or inverted microscope and optics suitable for epifluorescence detection including:
    • Epi‐illuminator (bright field)
    • High numeric aperture objective (NA > 0.7)
    • Detector unit using photomultipliers, beam splitters and band‐pass filters
    • Sample stage including computer‐driven z‐position control (alternatively xyz‐position control)
    • Heating system to maintain 37°C (optional)
  • Multiphoton laser including:
    • Diode‐pump laser coupled to titanium‐sapphire laser (Table 4.15.2)
    • Incoupling of the laser beam into the scan head via mirrors (beam path)
  • Laser scanning unit including computer‐controlled operation system for scanning conditions, signal acquisition, primary image reconstruction and storage (Table 4.15.3)
  • Software for 3‐D and 4‐D image reconstruction (Table 4.15.4)
  • Vibration isolation table designed for stable mounting of optical instruments
  • Powermeter to measure laser light intensities along the beam path, including infra‐objective level

Basic Protocol 2: Detection of Fibrillar Collagen in Connective Tissue ex vivo

  Materials
  • Freshly excised piece of tissue or organ
  • Medium of choice (e.g., RPMI, DMEM, or PBS; appendix 2A)
  • Additional reagents and equipment for designing a microscope system for HHGM ( protocol 1)

Basic Protocol 3: Detection of SHG in Mouse Tissues by Intravital Microscopy

  Materials
  • Mouse sample
  • Additional reagents and equipment for preparing mouse samples for intravital microscopy (unit 4.11), acquiring a z‐stack ( protocol 2) and, designing a microscope system for HHGM ( protocol 1)

Basic Protocol 4: Simultaneous Detection of Cells and Collagen Fibers In Vitro and In Vivo

  Materials
  • Cells expressing GFP or labeled with fluorescent dye ( protocol 5)
  • Collagen lattice containing fluorescent cells ( protocol 6)
  • Additional reagents and equipment for designing a microscope system for HHGM ( protocol 1)

Support Protocol 1: Cytoplasmic Staining of Live Cells

  Materials
  • Cultured cells in culture flask or petri dish
  • Calcein‐AM (Molecular Probes)
  • DMSO (Invitrogen)
  • Dulbecco's modified Eagle's medium (DMEM) or other medium (e.g., PBS; appendix 2A); may contain serum and antibiotics

Support Protocol 2: Establishment of 3‐D Collagen Cultures

  Materials
  • Adherent cells
  • 2 mM EDTA solution in PBS, pH 7.4
  • Dulbecco's modified Eagle's medium (DMEM) or other medium; may contain serum and antibiotics ( appendix 2A)
  • 10× concentrated minimal essential Eagle's medium (MEM; Flow Laboratories) or other 10× concentrated medium
  • 7.5% (w/v) sodium bicarbonate solution (Life Technologies)
  • 3.0 mg/ml collagen stock solution, acidified (e.g., bovine dermal collagen; Vitrogen; Nutacon BV or rat tail collagen; Becton Dickinson)
  • Migration chamber (see recipe)
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Figures

Videos

Literature Cited

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