Photobleaching resistance of stimulated parametric emission in microscopy

Photobleaching of organic fluorescent labels is a ubiquitous problem in fluorescence microscopy, limiting imaging capabilities and presenting hurdles to quantitative biophysical measurements. We report here that a nonlinear optical signal from some organic fluorophores persists in the presence of photobleaching. Specifically, a four-wave mixing process that is enhanced by a two-photon absorption resonance in the target fluorophore, termed stimulated parametric emission (SPE), is essentially unaffected by the photobleaching of the fluorophore, for rhodamine 6G and other commercial green and red fluorophores. The stability of the SPE signal, and the ability to image weakly or nonfluorescent chromophores, should make this nonlinear microscopy useful for quantitative biophysical measurements.