Image Formation in Coherent Nonlinear Optical Microscopy

Coherent nonlinear optical microscopy has allowed real-time visualization of objects and biological processes on small length scales, where molecule-specific imaging can be achieved without the use of labels. Given the coherent nature of the nonlinear processes involved (which includes, for example, coherent anti-Stokes Raman scattering, second and third harmonic generation), the images produced are complex and not always straightforward to interpret, especially when object sizes are much smaller than the focal volume. Images are subject to, for example, interference from multiple scatterers, interference due to competing nonlinear processes, and distortion to due inhomegeneities. This can lead to bright spots that appear in the wrong place or are 10x brighter than they should be, unexpected shadows, false dark and bright signals, spectral shifts that depend on the spatial position of objects along the laser axis, and signals that depend on the precise shapes and relative positions of sub-wavelength objects within the focal volume (rather than just the total number of molecules present), to name a few. I will discuss the origin of these strage effects, and review our work and others in this area over the last several years.