The Center for Biomedical Imaging Technology has engineered a two-photon laser scanning microscope, which is available for use. Dr. Paul Campagnola has adapted the center's BioRad MRC600 for two-photon excitation of fluorescence while still maintaining the MRC600's one-photon CLSM.

Two-photon excitation of fluorescence is based on the principle that two photons of longer wavelength light are simultaneously absorbed by a fluorochrome which would normally be excited by a single photon, with a shorter wavelength. The nonlinear optical absorption property, of two-photon excitation, limits the fluorochrome excitation to the point of focus. This is graphically demonstrated in the image above. The images below, of pollen granules which were obtained on our system, clearly show the difference between one and two-photon excitation. The two-photon image was generated using 704nm excitation light while the one-photon image was generated using 488nm.

Limiting the excitation light to the point of focus rather than exposing the entire sample greatly reduces total photobleaching and photodamage. This is one major advantage to using two-photon excitation.

Two-photon microscopy has several other advantages over confocal microscopy; optical sections can be obtained deeper within a specimen, a detection pinhole is no longer necessary, thus not limiting the number of photons being detected. (However, a pinhole may still be used to slightly improve the resolution of two-photon excitation.) In addition, the use of UV fluorophores is no longer limited to UV corrected objectives. Two-photon excitation utilizes visible wavelengths to excite UV fluorescent stains and indicators, therefore the objectives do not have to pass UV light.

The image above shows a solution of fluorescein being excited by 704nm laser light, supplied from the Mira 900-F femtosecond Modelocked Titanium-sapphire laser. Note the arrow pointing to the fluorescence limited to the point of focus.

Individuals who are interested in using CBIT's two-photon microscope are encouraged to call the center at 860-679-1452 or send email to CBIT