ABSTRACT
Light microscopy is in the middle of a revolution, and one of the greatest challenges for the aspiring microscopist is to appreciate what is available in terms of technology, software, and dyes or labeling techniques. Most fluorescence microscopy selects input and output wavelengths using filter cubes, but there are important limitations to this approach. Because each filter has a fixed central wavelength and passband, several filters must be used to image multiple fluorophores of different colors, and the filters are often mechanically interchanged by a rotating turret mechanism. The probability of the two-photon process occurring is proportional to the square the intensity of the excitation, so only those fluorophores right at the focal point are excited efficiently. The laser is the only needed additional equipment and may be fitted to an ordinary confocal microscope. Pulsed lasers are costly, so only a few individual investigators have two-photon instruments, but most universities have two-photon capabilities in imaging facilities.
