Using adaptive optics for deep in-vivo multiphoton FLIM

Abstract

Multiphoton microscopy (MPM) is a high resolution (sub-μm) 3D optical imaging technique that has seen widespread use for microscopy at moderate depth within biological tissue (~1 mm). MPM combined with Fluorescence lifetime imaging (FLIM) and Fluorescent resonant energy transfer (FRET) provides the ability to image protein-protein interactions. When applied in-vivo at depth, it will be a key component to identifying and evaluating drug interaction in tumours. Unfortunately as one images more deeply into biological tissue, depth is restricted due to the specimen induced aberrations, which result in deterioration in both the image quality and resolution. Adaptive optics (AO), a technique first developed for astronomy, has been shown to be successful in overcoming problems associated with imaging in depth in confocal, multiphoton, CARS and SHG microscopy. The principle relies on shaping the wavefront with a wavefront modulator to compensate for the distortions introduced by the biological tissue sample. The success of such a technique relies on being able to correctly determine the mirror shape required In this paper we will discuss the development a dedicated MPM FLIM-FRET microscope incorporating an AO for use in-vivo applications. Using a deformable membrane mirror as a wavefront modulator, a strategy for implementation will be discussed.