Monitoring biological relevant reactions on the single molecule level by the use of fluorescent probes has become one of the most promising approaches for understanding a variety of phenomena in living organisms. By applying techniques of fluorescence spectroscopy to labelled molecules a manifold of different parameters becomes accessible i.e. molecular dynamics, energy transfer, DNA fingerprinting, etc... can be monitored at the molecular level. However, many of these optical methods rely on oversimplified assumptions, for example a three-dimensional Gaussian observation volume, perfect overlap volume for different wavelength, etc. which are not valid approximations under many common measurement conditions. As a result, these measurements will contain significant, systematic artifacts, which limit their performance and information content. Based on Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Spectroscopy we will present representative examples including a thorough signal analysis with a strong emphasis on the underlying optical principles and limitations. An outlook to biochip applications, parallel FCS and parallel Lifetime measurements will be given with cross links to optical concepts and technologies used in industrial inspection.

Date Published: 30 May 2003
PDF: 9 pages
Proc. SPIE 5144, Optical Measurement Systems for Industrial Inspection III, (30 May 2003); doi: 10.1117/12.508363

Published in SPIE Proceedings Vol. 5144:
Optical Measurement Systems for Industrial Inspection III
Wolfgang Osten; Malgorzata Kujawinska; Katherine Creath, Editor(s)