Image filtering for two-photon deep imaging of lymphonodes

Non-linear excitation microscopy is considered an ideal spectroscopic method for imaging thick tissues in vivo due to the reduced scattering of infrared radiation. Although imaging has been reported on brain neocortex at 600–800 μm of depth, much less uniform tissues, such as lymphonodes, are characterized by highly anisotropic light scattering that limits the penetration length. We show that the most severe limitation for deep imaging of lymphonodes appears to be the tissue scattering and the diffuse fluorescence emission of labeled cell (lymphocytes) in layers above the focusing plane. We report a study of the penetration depth of the infrared radiation in a model system and in ex vivo lymphonodes and discuss the possibility to apply Fourier filtering to the images in order to improve the observation depth.