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Single molecule detection schemes promise the ability to reach the ultimate limit of detection: one molecule. In this project we use Total Internal Reflection Fluorescence Microscopy (TIRFM) to detect individual biomolecules for quantification. TIRFM has the advantage of delivering high contrast images and has become a standard method for the optical detection of single molecules. Here TIRFM is ... read morepresented as a method to determine the concentration of molecules in solution by correlating digital counts of single fluorescent molecules immobilized on a TIRF probed surface. The luminescent reporters used in this project are quantum dots (QDs), semiconductor nanocrystals known for their remarkable brightness and stability. Despite the numerous outstanding photophysical properties of QDs, observations of single QDs also display a pronounced intermittent fluorescent behavior, posing a challenge for detecting single QDs. This thesis demonstrates a reliable method of detection taking advantage of these fluctuations of the signal. The quantitative methodology developed here for digital quantification of single QDs enables detection at the sub-femtomolar level, and can be applied to biological applications for quantification of low levels of DNA.read less
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