Application of the Diver Detection Method to Multiphoton Microscopy and Flim

Abstract

In two-photon microscopy of turbid media, such as biological tissues, achievable imaging depth depends on ability of detection system to detect weak emission signals induced in deep tissue layers. In the conventional epi-detection method photons are collected from a relatively narrow sample area and acceptance angle, so most of emitted photons remain undetected. The DIVER (Deep Imaging Via Emission Recovery) method utilizes wide photocathode area detector and matching refractive index in the optical path from the sample surface to photocathode; that allows efficient collection of photons from wide area of a turbid sample at almost any angle. Using this method, the imaging depth was increased by factor of 4-6 compared to conventional epi-detection. In addition, the DIVER method offers superior sensitivity in SHG imaging and allows detection of short wavelength emission, up to 320nm. This, in turn, allows THG and three-photon imaging using conventional Ti:Sa laser excitation wavelengths. Coupling with the FLIMBox enables Fluorescence Lifetime Imaging Microscopy (FLIM) in depth and acquiring 3D-FLIM images of tissue samples. Using combination of SHG and FLIM techniques we were able to separate various types of collagens (I to V) in tissue samples. These techniques also allowed us to detect kidney fibrosis at early stages in UUO mouse model. THG imaging is particularly useful for lipid droplets imaging and was used in imaging of adipose tissues and mouse livers in diabetes studies. The special UV-detection capability of the DIVER was also applied to study three-photon excitation FCS of unlabeled protein oligomers.