Super resolution microscopy for biological applications

The use of quantum dots in single molecule microscopy is becoming a common approach due to their high quantum yield, broad absorption spectrum and narrow emission spectrum. In addition, the development of biocompatible quantum dots with site-specific conjugation properties has made quantum dots available to many biophysics and nanotechnology researchers.Chapter 1 describes the technique of DNA molecular combing and presents a novel approach for the silanization of the glass coverslips, which are used in many DNA molecular combing experiments. Two novel techniques for site-specifically labeling genomic DNA with quantum dots are also presented. The development of a multidisciplinary approach (DNA combing, FISH, quantum dot labeling, and protein crosslinking) for the high throughput and high-resolution mapping of DNA binding proteins on combed genomic DNA is also discussed.Chapter 2 describes ultra-high resolution distance measurements on quantum dots connected by short dsDNA's. First, a method for the attachment and purification of a ssDNA to a single quantum dot is presented. Next, two ssDNA/quantum dot molecules are conjugated through DNA hybridization. Finally, using a scanning stage confocal microscope, nanometer resolution distance measurements between two dsDNA linked quantum dots (quantum dot dimer) are carried out. The resolution measured is the highest achieved to date.Quantum dot blinking is an intrinsic property of quantum dots in which there are periods of dark states (no fluorescence) during excitation. The most widely accepted theory is that the quantum dot electron becomes trapped in an excited state and remains in that state for up to several seconds. Chapter 3 demonstrates the effectiveness of embedding quantum dots in a DTT doped polymer film to almost completely eliminate quantum dot blinking. Both water-soluble and inorganic quantum dots can be successfully embedded in these DTT doped films if the appropriate polymers are used. This technique has applications in super resolution microscopy, particle tracking, immuno-cyto/histo-chemistry, as well as for the fabrication of quantum dot containing devices requiring a reliable and stable photon source (including a single photon source) or stable charge characteristics. Intimate contact between the quantum dot and the surrounding matrix is maintained in this method. Direct applications to both super-resolution microscopy and immunochemistry are presented in this chapter.