Fluorescence Lifetime Imaging in Ophthalmology

Abstract

Beginning disease in biological systems is often accompanied or preceded by changes in the metabolism of the tissue. Changes in the metabolism induce changes in the fluorescence decay functions of endogenous fluorophores. FLIM of the fundus of the eye is therefore a promising technique of detection early stages of eye diseases. Ophthalmic FLIM faces the problem that the excitation power is limited to very low levels, the transmission wavelength range of the ocular media is limited, the fluorescence intensities are low, and the decay functions of the fluorophores in the fundus are multi-exponential with extremely fast decay components. The task is further complicated by strong fluorescence of the lens of the eye and by the fact that the eye is constantly moving. We will show that these problems can be solved by a combination of multi-dimensional TCSPC with confocal scanning. This chapter gives a survey of the fluorophores found in the fundus, their fluorescence-decay properties, and the options to excite them at wavelengths that pass the ocular medium. It discusses the technical requirements to an ophthalmic FLIM system and describes the implementation of TCSPC FLIM in an ophthalmic scanning system for clinical use. The results obtained with the system allow fluorescence decay components to be associated to lateral and longitudinal anatomical structures of the eye, and to pathological alterations in the fundus. Statistical evaluation of the fluorescence decay parameters improves the identification of eye diseases and even detects phospho τ 181 protein in the eyes of Alzheimer patients.

Two-Photon Imaging for Non-Invasive Corneal Examination

“…This is a time-consuming procedure and requires a priori knowledge such as the number of fluorescence lifetime components. It also requires a high number of photons for accurate fit of the data [47][48][49]. A minimum of 1000 photons per pixel is required for an accurate fit of a bi-exponential decay curve [49].…”