The limit on imaging-depth in two-photon microscopy depends on parameters of the imaging system such as available power, wavelength, numerical aperture, and fluorescencecollection field-of-view and on properties of the sample such as scattering and absorption cross-sections and fluorophore distribution. These dependencies are discussed and strategies for optimizing the imaging depth are presented.
It is shown that two-photon fluorescence images can be obtained almost throughout the entire grey-matter layers of the mouse neocortex by using optically amplified femtosecond pulses. The maximum imaging depth is not limited by the available excitation power but instead by the generation of out-of focus fluorescence.
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