The combination of femtosecond lasers and the high numerical aperture optics found in mi­croscopy makes it possible to create high intensities (100 GW/cm2) with extremely modest energies (~ 100 pJ). This high intensity results in inducing a dynamic, nonlinear polarization in virtually any media located within the focal volume of the microscope objective. This nonlinear, time-varying polarization response acts as a driving force in the wave equation that can result in new source terms. These new sources can be used to create image con­trast. Because they scale nonlinearly with the excitation intensity they are strictly confined to the focal volume (no out-of-focus contributions), and in essence are naturally confo — cal. The net result — nonlinear microscopy — is a high-resolution (sub-micrometer lateral resolution, micrometer axial resolution), three-dimensional imaging modality capable of effectively probing material structure and function. While these intensities may seem ex­treme, the combination of modest energy (44) and infrared wavelengths actually results in a relatively benign excitation source. In comparison to continuous wave excitation at UV or near-UV wavelengths, delicate systems are minimally perturbed under femtosecond laser excitation.

The recently developed nonlinear microscopy has been applied to imaging biologi­cal systems, such as nonlinear signals of second (SHG) and third harmonic generation (THG) (45), coherent anti-stokes Raman spectroscopy (CARS) (46, 47). These tech­niques combine spectroscopy (chemical) and microscopy (spatial) approaches, which have particular potential in characterizing plant cell wall structures and their bioconversion processes.