Physical Chemistry Chemical Physics vol:12 issue:41 pages:13484-13498
Nonlinear optical imaging has revolutionized microscopy for the life sciences. Second harmonic generation (SHG), the younger sibling of two-photon excited fluorescence (2PF), is a technique that can produce high resolution images from deep inside biological tissues. Second harmonic light is generated by the coherent scattering of an ensemble of aligned chromophores in a focused, pulsed laser beam. SHG is only generated at the focal spot, reducing the background signal, and requires ordered chromophores, so is highly structure-specific. In contrast to two-photon fluorescence, the physical process that creates the signal does not require the formation of excited states, allowing elimination of harmful photochemistry. While the SHG of native proteins and biopolymers is well known, the use of exogenous dyes can provide SHG contrast from areas without a sufficiently high intrinsic quadratic hyperpolarizability, β. Dyes for SHG primarily target lipid bilayers; a trait that, combined with sensitivity to transmembrane potential, allows monitoring of action potentials in a variety of excitable cells, most importantly mammalian neurons. This article summarizes the principles of SHG imaging and explores approaches for maximizing the SHG signal from a biological specimen. We survey methods of optimizing the optical set-up, enhancing the β of the dye and achieving biological compatibility. In conclusion, we examine novel applications of SHG imaging and highlight promising directions for the development of the field.