This chapter deals with some of the most fascinating topics in fluorescence
microscopy and optics, in general: non-linear optics. All the optical phenomena
explained in the previous chapters, with some particular exceptions, have dealt with
linear optics; this means that a single photon is involved in the optical process during
and after being absorbed. However, it is possible to have a multiphoton optical
response, one that depends on more than one photon interacting with the system “at the
same time”. This leads to a whole new range of optical phenomena; many of them
unobservable at the relatively low light intensities with characteristic of linear optics.
Some of the non-linear optical phenomena provide very interesting and useful
applications in fluorescence microscopy. In this chapter, we will introduce and explain
the main non-linear optical processes employed in fluorescence microscopy, enumerate
their advantages and drawbacks, and highlight their importance in the life sciences.
Keywords: Biphotonic microscopy, Coherent anti-Stokes Raman scattering
(CARS), Excited state absorption, Harmonic microscopy, Lanthanide
upconversion, Multiphoton microscopy, Non-linear optics, Photon upconversion
(UC), Raman microscopy, Second harmonic generation, Stimulated Raman
scattering (SRS), Third harmonic generation, Triplet-triplet annihilation
upconversion, Triplet state, Virtual state.
