Time-resolved fluorescence imaging in biology and medicine

Abstract

Fluorescence lifetime imaging is a rather new and effective tool that can be used to study complex biological samples, either at microscopic or macroscopic levels. The map of the fluorescence lifetime allows one to discriminate amongst different fluorophores and to achieve valuable insights into the behaviour of emitting molecules, leading to information like local pH, oxygen concentration in cells, etc. Moreover, the distribution in space of any fluorescent marker achievable with this technique can be exploited for diagnostic purposes in medicine. After a brief introduction on the motivations for applying fluorescence lifetime imaging in biology and medicine, the basic principles of this technique will be addressed. Then, the two possible implementations of fluorescence lifetime imaging (i.e. the frequency domain and the time domain methods) will be presented. For this purpose, special attention will be devoted to practical aspects of image acquisition and processing, especially for what concerns the time domain method. Then, the analysis of the state-of-the-art systems will include a brief discussion on new concepts that have recently been introduced in this research field. Finally, two interesting applications of fluorescence lifetime imaging will be presented. The former refers to skin tumour detection and has been successfully applied in a preliminary clinical trial, the latter regards DNA chips reading and has been tested only at laboratory level, yet it has produced promising results for its future implementation in commercial systems.

A flexible wide‐field FLIM endoscope utilising blue excitation light for label‐free contrast of tissue

“…The frequency domain system developed by Mizeret et al could operate at up to video rate but with a field of view (FOV) of 32 2 or 64 2 pixels, while the first time-gated systems provided images of >256 2 pixels but required data acquisition times of the order of minutes. With improved fluorescence decay sampling strategies and optimised optical setups, full-field time-gated FLIM endoscopy of biological tissue with mode-locked Ti:Sapphire laser-based excitation has been demonstrated at 7.2 Hz in a rigid endoscope [21] and 5.5 Hz in a flexible endoscope [22].…”

“…The measured fluorescence decay profiles, S(t), can be modelled as the convolution of the instrument response function, IRF(t), and the sample's fluorescence response to a delta function exci-tation pulse F(t), i.e. S(t) ¼ IRF(t) Á F(t) [1,2]. Our FLIM detection scheme samples the fluorescence decay profiles in each pixel of the FOV and these measurements are then fitted to a model of the sample's fluorescence decay profile convolved with the IRF, which can be measured experimentally using a short lifetime dye or a scattered excitation pulse.…”

“…Fluorescence lifetime imaging (FLIM) [1,2] of tissue autofluorescence has been shown to provide labelfree contrast between different types and states of tissue that may help differentiate between normal and diseased human tissue, e.g. [3][4][5][6][7][8].…”

Time-domain fluorescence lifetime imaging applied to biological tissue

“…This paper aims to describe our approach to timedomain FLIM and to review our recent work, as presented at the ESP 2003 conference [3] in Vienna, with particular emphasis on its application to biological tissue. It is not intended as a general discussion of FLIM -for this we could suggest the papers by Cubeddu et al [4], Wouters et al [5] and Tadrous [6]. For a discussion of FLIM applied to FRET, we suggest the paper by Bastiaens and Squire [7].…”

Phosphorescence decay time measurements using intensity correlation spectroscopy

“…In particular, the measurements in tumor under photodynamic diagnosis and therapy is one of the very interesting applications. (Glanzmann et al, 1999;Cubeddu et al, 2002) The simplicity is a great advantage when the use of the system is extended to clinical situations.…”