Biological second and third harmonic generation microscopy.
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.
Friedl
Peter
P
University of Würzburg, Würzburg, Germany.
Wolf
Katarina
K
von Andrian
Ulrich H
UH
Harms
Gregory
G
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured / Q000648:ultrastructure; D003094:Collagen / Q000648:ultrastructure; D003238:Connective Tissue / Q000648:ultrastructure; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000032:analysis; D007202:Indicators and Reagents; D007834:Lasers; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D003094:Collagen