Application of Second Harmonic Imaging Microscopy in Biological Studies

Abstract

The fact that some crystalline substances can generate the second harmonic (SH) – light of twice the frequency – under intense irradiation was discovered soon after lasers were available. Later, it was realized that some biological polymers were also able to generate the SH, offering a label‐free way of imaging cell components in the microscope. To obtain sufficiently intense light without damaging the sample, pulsed lasers are needed, typically delivering 100 fs pulses at ∼10 ns intervals. The signal is only generated at the focus of the beam so the image is inherently focal‐plane selective. Molecules that generate the SH must have no center of symmetry, and the signal is greatly enhanced if they are crystalline. Collagen has been the most studied biopolymer, and its structure and arrangement is a wide range of normal and diseased tissues has been investigated. The proteins myosin and tubulin have also been much studied. The polysaccharides starch and cellulose also give an SH signal, but to date they have received less attention. As second harmonic generation (SHG) is an electric field effect, changes in the field around a molecule will affect the signal, and this has been used to devise fast‐responding reporter dyes to measure membrane potential at high resolution.