Long wavelength three-photon microscopy is increasingly being used for deep in-vivo bio-imaging, however there are still areas where quantitation is needed. Two of these areas include a standardized method for judging binary image quality, and three-photon excitation cross-section measurements of standard fluorophores over a large spectral range (i.e., reference standards). This the- sis attempts to bridge this gap by providing a statistical framework for judg- ing the quality of a binary image, measurements of the three-photon action cross-section of fluorescein (thus providing a reference standard), and a method for improving the in situ temporal characterization of a pulsed laser for three- photon cross-section measurements.The statistical framework for quantifying image quality is based on detection theory and provides a metric to quantify the quality of an image when binary detection (i.e., distinguishing an object from background) is of interest. Our treatment does not require acquired or reference images, and thus allows for a theoretical comparison of different imaging modalities and systems. The measurements of the three-photon action cross-section of fluorescein (dissolved in water, pH ∼11.5) are presented in the excitation wavelength range from 1154 to 1500 nm in ∼50 nm steps. Cube-law power dependance is con- firmed at the measurement wavelengths, and the three-photon excitation spectrum is found to differ from both the one- and two-photon excitation spectra. The three-photon action cross-section at 1154 nm is more than an order of magnitude larger than those at 1450 and 1500 nm (approximately three times the wavelength of the one-photon excitation peak), which possibly indicates the presence of resonance enhancement. In situ temporal characterization of a pulsed laser for three-photon cross- section measurements is improved by showing theoretically that the third order coherence at zero delay can be obtained by measuring the second and third order autocorrelation traces of a pulsed laser. The theory enables the measurement of a fluorophore’s three-photon cross-section without prior knowledge of the temporal profile of the excitation pulse by using the same fluorescent medium for both measurement of the third order coherence at zero delay as well as the cross-section. Such an in situ measurement needs no assumptions about the pulse shape nor group delay dispersion of the optical system. This is demonstrated experimentally by measuring the three-photon action cross-section of Alexa Fluor 350, and showing that the measured values remain approximately constant despite varied amounts of chirp on the excitation pulses.