Ultra-deep imaging of turbid samples by enhanced photon harvesting
Published Web Locationhttps://doi.org/10.1117/12.2002101
We constructed an advanced detection system for two-photon fluorescence microscopy that allows us to image in biological tissue and tissue phantoms up to the depth of a few mm with micron resolution. The innovation lies in the detection system which is much more sensitive to low level fluorescence signals than the fluorescence detection configuration used in conventional two-photon fluorescence microscopes. A wide area photocathode photomultiplier tube (PMT) was used to detect fluorescence photons directly from a wide (1 inch diameter) area of the turbid sample, as opposed to the photon collection by the microscope objective which can only collect light from a relatively small area of the sample. The optical path between the sample and the photocathode is refractive index matched to curtail losses at the boundaries due to reflections. The system has been successfully employed in the imaging of tissue phantoms simulating brain optical properties and in biological tissues, such as murine small intestine, colon, tumors, and other samples. The system has in-depth fluorescence lifetime imaging (FLIM) capabilities and is also highly suitable for SHG signal detection, such as collagen fibers and muscles, due to the intrinsically forward-directed propagation of SHG photons. © 2013 SPIE.