FRET-type remote excitation tip-enhanced fluorescence microscopy for super-resolution DNA mapping

DNA, deoxyribonucleic acid, is a nucleic acid with a double helical strand containing the genetic instructions for the development and function of all living organisms. Since 1953, when Watson and Crick first reported the molecular structure of DNA, extensive research has been put into decoding genetic information and understanding how it works. The most commonly used method is by DNA sequencing in which the DNA is first cut into small pieces (200 – 600 base-pair (bp)), targeted, amplified, and the sequences are determined by incorporating specific fluorescent labeling. While this is a powerful method, it is very expensive, time-consuming and not a perfect technique. For some applications, it does not require the entire sequence but it is sufficient to know the location and copies of certain recognition sequences as this will give long rage sequence information. Such information can be used for the identification and classification of organism but also to find mutations in cells that are the result of evolutionary changes. The method to realize this is called “DNA mapping”. The current resolution barrier of DNA mapping is 20 ~ 40 nm corresponding to 60 ~ 120 bp. This project will develop a novel FRET-type remote-excitation tip-enhanced fluorescence (RE-TEF) microscopy to break the current resolution barrier of 20 nm, avoid the risk of irreversible quenching effect of probe molecules and demonstrate it in a high-density optical DNA mapping.