Quantitative confocal spectroscopy — rectifying the limitations of single molecule detection

Quantitative confocal spectroscopy (QCS) is a new implementation of single molecule detection that allows highly quantitative and accurate analysis of single fluorescent molecules. It rectifies the limitations of standard confocal spectroscopy by significantly enhancing mass detection efficiency and observation volume uniformity while preserving high signal to noise ratio and single molecule sensitivity. This is accomplished by incorporation of a cylindrical lens and a novel microfabricated confocal aperture to perform a 1-D expansion of the observation volume such that it spans the entire cross-section of a typical microchannel. First, QCS is theoretically compared to standard SMD using a combination of optical modeling and Monte Carlo simulation. Then the performance of QCS is experimentally validated against standard SMD using PicoGreen stained pBR322 DNA.