UNIT 4.15 Biological Second and Third Harmonic Generation Microscopy

  1. Peter Friedl1,
  2. Katarina Wolf1,
  3. Gregory Harms1,
  4. Ulrich H. von Andrian2

Published Online: 1 MAR 2007

DOI: 10.1002/0471143030.cb0415s34

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Friedl, P., Wolf, K., Harms, G. and von Andrian, U. H. 2007. Biological Second and Third Harmonic Generation Microscopy. Current Protocols in Cell Biology. 34:4.15:4.15.1–4.15.21.

Author Information

  1. 1

    University of Würzburg, Würzburg, Germany

  2. 2

    Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAR 2007
  2. Published Print: MAR 2007


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.


  • microscopy;
  • second and third harmonic generation microscopy;
  • living cells;
  • higher harmonic generation microscopy