Advanced time-correlated single photon counting technique for spectroscopy and imaging of biological systems

Abstract

ABSTRACT We present a muhi-dimensional TCSPC technique that simultaneously records the photon distribution over the time in thefluorescence decay, the wavelength, and the coordinates of a two-dimensional scan or the time since the start of the experi-ment. We demonstrate the application ol the technique to diffuse optical tomography, single-point autofluorescence meas- urenients of skin, and imilti—spectra autofluorescence lifetime imaging of tissue. Keywords: TCSPC, DOT, Fluorescence, Autofluorescence, FLIM, FRET, Microscopy 1. INTRODUCTION Optical techniques in biomedical applications have the advantage of being non-destrnctive and minimum invasive. Moreo-ver, optical techniques deliver spectroscopic information that is directly related to the composition or the metabolic state ofthe tissue. Depending on the thickness ofthe tissue two different optical techniques are used.Tissue thicker than the mean free path length between scattering events is investigated by diffuse optical tomography (DOT)techniques. Typical DOT applications are optical mammography, static and dynamic brain imaging, and non-invasive in-vestigations of drug effects in small animals. DOT is based on illuminating the tissue at a large number of source positionsand detecting the diffusely reflected or transmitted light or the fluorescence at a large number of detector positions. Despiteof its relatively poor spatial resolution, DOT has the benefit that the measured absorption coefficients are related to thebiochemical constitution of the tissue, such as haemoglohin concentration and blood oxygenation.The effects of scattering. absorption, and fluorescence can better he distinguished if pulsed or modulated illumination andtime-resolved detection are used [31. Moreover, the depth of scattering and absorption changes in the tissue can be derivedfrom time-resolved data [5].Single cells, small organisms, or superficial tissue layers are usually investigated by fluorescence imaging techniques. Oftenmulti-spectral imagmg is used to distinguish between different flu.orophores. Fluorescence imaging of biological specimenscan be considerably improved by including the fluorescence lifetime in the recordii'ig ProceSs. The fluorescence lifetime of afluorophore is virtually independent of the concentration hut depends on environment parameters such as pFl. ion concen-trations, and oxygen saturation [4]. A particularly powerful tool of fluorescence imaging is fluorescence resonance energytransfer (FRET). Different proteins are labelled with different fluorophores which are acting as a donor and an acceptor forFRET. The fluorescence decay functions can be used to obtain both the fraction of interacting proteins and the distancebetween the donor and the acceptor [ I }.