A Start on Parts for Multiphoton Microscopy

Daniel C. McCarthy, Senior News Editor

Ten years ago, in a Cornell University laboratory in Ithaca, N.Y., researchers unveiled the first multiphoton microscope for biological imaging. The biomedical research community quickly realized the virtue of the technology, which demonstrated that two photons are better than one for maintaining cell or tissue viability during imaging.

The multiphoton microscope borrowed from confocal laser scanning devices in its application of laser light to excite fluorescence in a sample, but two significant differences distinguished it from confocal techniques. First, multiphoton fluorescence is excited only at the focal spot under the microscope where the photon flux density is high enough for the nonlinear process to occur. This eliminates the need for the confocal detector pinhole and prevents other sections of the sample from being photobleached before they can be imaged.

The other distinction is that multiphoton technology uses two or more infrared photons to do the work of confocal's single visible or ultraviolet photon. Besides infrared's ability to penetrate deeper into tissue, the longer wavelengths do less damage to cells and tissue than visible and UV wavelengths outside of the focal plane, and thus extend the length of time a living sample can be imaged. This has made multiphoton microscopy a popular method for in vivo research because it does not arrest cell processes as quickly as confocal techniques.