Multiphoton Excitation Provides Optical Sections from Deeper within Scattering Specimens than Confocal Imaging

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