The Cornell Dr. Bio 2-Photon Excitation Spectra Database is an invaluable online resource for researchers engaged in advanced microscopy, specifically focusing on two-photon excitation techniques. This database provides comprehensive data on the two-photon excitation spectra of various fluorophores and fluorescent proteins, which are critical for optimizing imaging parameters. This resource is particularly beneficial for scientists utilizing two-photon microscopy, a technique known for its advantages such as reduced out-of-focus photobleaching, minimized autofluorescence, and enhanced tissue penetration depth compared to traditional one-photon excitation methods. The database aids in the informed selection of appropriate fluorophores and their corresponding optimal excitation wavelengths. The database typically includes crucial photophysical parameters such as two-photon absorption (2PA) cross-sections and two-photon excitation action cross-sections (brightness) for a wide range of molecules. These values are essential for designing experiments that require high-resolution, deep-tissue imaging and for achieving precise control over light-matter interactions in biological samples. By serving as a central reference for the two-photon properties of fluorescent probes, this database supports the design of cutting-edge experiments for imaging living systems, studying dynamic cellular processes, and developing new bioimaging applications with improved signal-to-noise ratios and reduced photodamage.