Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging

Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal Volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.