Cylindrical illumination confocal spectroscopy: rectifying the limitations of confocal single molecule spectroscopy through one-dimensional beam shaping.
Cylindrical illumination confocal spectroscopy (CICS) is a new implementation of single molecule detection that can be generically incorporated into any microfluidic system and allows highly quantitative and accurate analysis of single fluorescent molecules. Through theoretical modeling of confocal optics and Monte Carlo simulations, one-dimensional beam shaping is used to create a highly uniform sheet-like observation volume that enables the detection of digital fluorescence bursts while retaining single fluorophore sensitivity. First, we theoretically show that when used to detect single molecules in a microchannel, CICS can be optimized to obtain near 100% mass detection efficiency, <10% relative SD in burst heights, and a high signal/noise ratio. As a result, CICS is far less sensitive to thresholding artifacts than traditional single molecule detection and significantly more accurate at determining both burst rate and burst parameters. CICS is then experimentally implemented, optically characterized, and integrated into separate two microfluidic devices for the analysis of fluorescently stained plasmid DNA and single Cy5 labeled oligonucleotides. CICS rectifies the limitations of traditional confocal spectroscopy-based single molecule detection without the significant operational complications of competing technologies.
Liu
Kelvin J
KJ
Biomedical Engineering Department, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Wang
Tza-Huei
TH
D016477:Artifacts; D002232:Carbocyanines / Q000032:analysis; D004247:DNA / Q000032:analysis; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D005453:Fluorescence; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009010:Monte Carlo Method; D055095:Optics and Photonics; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*
D002232:Carbocyanines; D004129:Dimethylpolysiloxanes; C085321:cyanine dye 5; C013830:baysilon; D004247:DNA