Introduction to Fluorescence Spectroscopy with Applications to Biological Systems

Abstract

Optical technologies can provide novel opportunities and tools for the life sciences. In turn, biological applications continue to stimulate novel optical technologies. The purpose of this article is to present several applications to biological systems of the techniques of fluorescence spectroscopy. This presentation is divided into seven parts. In the first part we review the fundamental principles of fluorescence spectroscopy, starting with the consideration of fluorophores and of the characteristics of fluorescence spectroscopy. The techniques that produce absorption spectra, excitation spectra, fluorescence under continuous excitation and response to pulsed excitation are also examined. In the second part we present the technique of Fluorescence Anisotropy together with the information it can provide. In the third part we present the technique of Fluorescence Resonance Energy Transfer (FRET) and its applications to distance-distribution analysis in biological systems. In the fourth part we deal with the excitation of fluorescent systems by multiple photons and present its advantage in some instances over the one-photon excitation. In the fifth part we present the technique of fluorescence correlation spectroscopy and we show how it may provide information regarding concentration and diffusion of fluorescent molecules. The sixth part deals with single molecule detection whose application may avoid ensemble averaging. The final part deals with the tools that may increase the quantum efficiency and radiative emission rate by placing emitting fluorophores close to metals. We offer some conclusions at the end.