ABSTRACT
Harmonic microscopy is based upon a non-linear effect that takes place within certain materials and has a totally different physical origin to fluorescence. In order to observe harmonic generation in microscopy, the molecules need to be aligned such that the interference process is constructive rather than destructive. This chapter examines the physical basis for harmonic generation before considering how excellent contrast mechanism can be utilized for microscopy and the optimal instrumentation for practical imaging in this manner. It presents methods by which a user can determine if some of the signal being detected in a multiphoton microscope is from harmonic generation rather than non-linearly excited fluorescence. In optical harmonic generation, light enters the sample and the output is detected at exactly half, or a third, of the input wavelength. There is one crucial aspect to the light production and driving of the molecular dipoles that is vital in determining if any second harmonic light will be detected.
