Confocal data acquisition for digital quantification using amplified single molecule detection

We have recently presented a method that enables single molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target specific padlock probe ligation, followed by rolling circle amplification (RCA) resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible by counting the number of generated RCPs using standard epi-fluorescence or confocal fluorescence microscopes. We have characterized the performance of the epi-fluorescence and the confocal readout formats. Both formats exhibit a linear response of the number of counted objects as a function of starting circles, and the dynamic range is three orders of magnitude employing epi-fluorescence readout and four when using confocal. In the epi-fluorescence format flow rate has to be below 1 μ1/min and flow variations are likely to be the limiting factor for precision. If the flow rate is above 3 μ1/min the precision of the confocal readout format is limited only by Poisson counting statistics, due to the accurate volume definition of the confocal optics. The limit of detection in the confocal format was reduced by a factor of three by increasing the data acquisition rate by a factor of ten.