Multi-dimensional fluorescence imaging

Abstract

Fluorescence offers many opportunities for optical molecular imaging and can provide information beyond simply the localisation of fluorescent labels. At Imperial we are developing technology to analyse and image fluorescence radiation with respect to wavelength, polarisation and, particularly, fluorescence lifetime, in order to maximise the information content. This talk will review our recent progress applying fluorescence lifetime imaging (FLIM) and multidimensional fluorescence imaging (MDFI) to tissue imaging and in vitro cell microscopy. Applying FLIM to autofluorescence of biological tissue can provide label-free contrast for non-invasive diagnostic imaging, as we have demonstrated in various tissues including atherosclerotic plaques, cartilage, pancreas and cervical tissue. FLIM and MDFI are also applicable to image intracellular structure and function for cell biology and drug discovery: hyperspectral imaging and FLIM can provide (quantitative) information concerning the local fluorophore environment and facilitate robust fluorescence resonant energy transfer (FRET) experiments while information concerning structure and rotational mobility may be obtained by applying polarisation resolution. Our most recent work includes high-speed and optically-sectioned FLIM for automated imaging and live cell studies, hyperspectral FLIM for acquiring excitation-emission –lifetime matrices to distinguish different fluorophores and microenvironments and imaging of rotational correlation time, particularly applied to microfluidic devices. Excitation sources are a particular challenge for confocal microscopy and other FLIM modalities including endoscopy, owing to the complexity and limited spectral coverage of available technology. Increasingly we are exploiting ultrafast fibre lasers and continuously tunable ultrafast sources based on continuum generation in photonic crystal fibres for wide-field and confocal FLIM applications.