Multiphoton excitation microscopy has several limitations. For a given fluorophore the resolution is slightly lower than with a confocal microscope. The insertion of a confocal pinhole in front of the detector can eliminate this difference, but there is a large loss of signal with the use of a pinhole.
Multiphoton excitation microscopes only work in the fluorescence mode. It is possible to collect the backscattered reflected light with a confocal pinhole in front of the detector, which provides simultaneous imaging of the specimen in both multiphoton excitation and confocal microscopy modes. The advantage of multimode imaging, with its increase of information about the object, is often very important.
The depth of imaging limitation is also very important. Depending on the nature of the specimen, nonlinear multiphoton microscopes can achieve a penetration depth that is 2 to 3 times that of a confocal microscope. The value of depth penetration is highly dependent on the concentration of absorbing and scattering molecules in the tissue under microscopic observation.
This problem has two components. The first limitation is the free working distance of the microscope objective. That is a fixed limitation of any type of microscopy and is based on the distance that the microscope objective can focus into the specimen. In an ideal, semitransparent specimen, that is the focal distance when the tip of the microscope objective just makes contact with the specimen. When a cover glass is used, this distance is reduced by the thickness of the cover glass.
The second process that limits the depth within the specimen is the amount of light scatter and absorption. Within the focal volume, multiphoton excitation processes cause the chromophore to attain higher-energy electronic states. The electronic transition from the first singlet state to the ground state is accompanied by the production of a photon with the energy of the transition. Photons that are either scattered out of the microscope objective or absorbed within the sample never are detected. If the specimen has a scattering coefficient and absorption coefficient that is constant within the depth of the specimen, then increasing depth of the specimen will scatter and absorb increasing amounts of the fluorescence; therefore, the signal will decrease.
Photobleaching of fluorescent probes is another problem. In the multiphoton excitation microscope the zone of photobleaching is constrained to the focal volume; nevertheless, the problem still exists. Perhaps the design of new fluorescent probes could mitigate this problem.