In some cases, it may be necessary to visualize multiple ex/em wavelengths simultaneously, which cannot be done with basic fluorescence microscopy. Here multi-photon microscopy becomes advantageous, performed with very high powered, pulsed lasers that serve to package photons. Many times, red light is preferred over blue light as an excitation source because the longer wavelength penetrates deeper into tissues better than shorter wavelengths, and the absorption of high-energy blue light is more likely to damage cells. Either two-photon or three-photon microscopy are the most common variations of the technique.

Three-photon microscopy is similar in principle to two-photon, except three photons must interact and converge with the fluorophore simultaneously. The quantum mechanics of fluorescence absorption means that only, roughly, a 10-fold greater photon density is needed for three photon excitation than is required for two-photon absorption. Three-photon microscopy has extended fluorescent imaging into the realm of deep UV, where 720 nm light can be used to excite a fluorophore that normally absorbs at 240 nm.

Two-photon excitation occurs when two photons are simultaneously absorbed in a single quantized event. First, excitation is produced by a single pulsed laser focused through the microscope. As the laser beam focuses, photons become more crowded and the chance of simultaneous interaction of two photons on a single fluorophore increases. Excitation relies on simultaneous absorption, and the resultant emission is dependent on the square of the excitation intensity. Though two-photon microscopy is extremely expensive, this technique is superior for live cell imaging, particularly in thick, multicellular preparations like developing embryos or brain slices. Additionally, no background is present in produced images, this technique negates the need for pinholes or other light sources, and two-photon microscopy is extremely well-suited for 3D imaging.