3D Tissue Reconstruction by using Deep Tissue Fluorescence Imaging System and FLIM

Abstract

We have demonstrated a new detection method used in two-photon fluorescence microscope called the DIVER (Deep Imaging via Enhanced-Photon Recovery) with enhanced capabilities for deep tissue imaging. The microscope uses a large area photo-detector to collect scattered emission photons directly from the wide area of a specimen. This detection scheme allows for increasing the imaging depth in tissues by about 6 folds due to a unique detector design and its increased photons collection efficiency compared to conventional methods. The DIVER system is also capable of performing Fluorescence Lifetime Imaging (FLIM), a very powerful tool to segregate various features in cells and tissues. The combination of deep tissue imaging and fluorescence lifetime provides contrasted images based on physiological parameters at depths that are not achievable by conventional microscopes. The DIVER potentially can be useful for imaging of various tissue samples, in particular for human skin cancer diagnostics, providing 3D cellular resolved images. We present the principle of operation of the microscope and show images obtained in unstained tissues in the heart, liver, kidney, lungs and other organs of mice. As an example of SHG in biological tissue, we have imaged the medial thigh muscle of a mouse obtained from the upper region of the leg. The muscle, roughly 3-4 mm thick, multiple z-stack images were acquired at field of view ranging from 400-900 μm, we can see the organization of the sarcomeres as well as the striations present in each sarcomere. We also have taken z-stack of the mouse heart where upon 3D reconstruction we can differentiate the different layers of the heart muscle as well as the ventricle.This work is supported in part by NIH grants P50 GM076516 and P41 GM103540 and the Keck grant KF 50242.