This proposal addresses the development of multiphoton photoacoustic microscopy via resonant and non-resonant two-photon absorption. The weak attenuation of acoustic emission in tissue holds the promise of improving the maximum imaging depth of nonlinear microscopy by a factor of 4. An imaging depth of ~2 mm would allow subcellular imaging completely through nearly all areas of the human epithelium where the majority of cancer begins. 2 mm also reaches a significant portion of the mouse anatomy which is used extensively in drug testing and as the model for numerous human diseases. Absorption based nonlinear microscopy increases the pool of potential target molecular species. Essentially all biomolecules are capable of absorbing light. If they exist in high enough concentrations they could be imaged with the developed technology. Using endogenous molecular contrast simplifies the application of nonlinear microscopy to higher organisms including humans because it obviates the need for the delivery of molecular probes to the target molecule or morphology. These techniques potentially open up new avenues of research by enabling the high-resolution 3-D visualization of concentrations/dynamics of biomolecular species (e.g. hemoglobin and cytochrome c) which are essentially invisible to nonlinear microscopy at imaging depths several times deeper than the current state of the art. The imaging techniques do not require exogenous molecular tags for molecular contrast so they are readily applicable to higher organisms including humans.
