30 March 2005 Second harmonic generation imaging microscopy of cellular structure and function
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Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, with many advantages over existing one- or two-photon fluorescence techniques. A non-linear phenomenon employing mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry. Many important structural proteins such as collagen and cellulose show intrinsic SHG, thus providing access to sub-resolution information on symmetry. However, we are particularly interested here in "resonance-enhanced" SHG from styryl dyes. In general SHG is a combination of a true second-order process and a third-order process dependent on a static electric field, such that SHG from membrane-bound dyes depends on a cell's trans-membrane potential. With simultaneous patch-clamping and non-linear imaging of cells, we have found that SHG is a sensitive probe of trans-membrane potential with sensitivities that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging. With the sensitivity of SHG to local electric fields from other sources such as the membrane dipole potential as well as the quadratic dependence of SHG on concentration, we have found that SHG imaging of styryl dyes is also a powerful technique for the investigation of lipid phases and rafts and for the visualization of the dynamics of membrane-vesicle fusion following fertilization of an ovum.
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Andrew C. Millard, Andrew C. Millard, Lei Jin, Lei Jin, Leslie M. Loew, Leslie M. Loew, "Second harmonic generation imaging microscopy of cellular structure and function", Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); doi: 10.1117/12.590919; https://doi.org/10.1117/12.590919

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