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Advanced DNA imaging: improving spatial resolution and contrast through photoswitching

Final Report Summary - IMAGINDNA (Advanced DNA imaging: improving spatial resolution and contrast through photoswitching)

The general aim of this proposal is to improve the methodology for advanced imaging of DNA using different and complementary approaches, and to use the improved methods in the investigation of chromosomes and other complex structures at the nanoscale. To this end, a novel microscope in which is possible to correlate atomic force microscopy (AFM) and super-resolution fluorescence imaging has been implemented. A protocol for sample preparation that is compatible with both techniques has been developed and applied to proof-of-principle experiments with fluorescently labeled DNA. This novel methodology can be used to benchmark novel super-resolution imaging methods, as well as to obtain complementary information about the structure and properties of (bio)materials. The latter has been exemplified on a hybrid nanomaterial that consists of protein fibrils functionalized with organic fluorophores and quantum dots. Our experiments allow combining information about the topography and number of filaments that compose a fibril, as well as the emissive properties and nanoscale spatial distribution of the attached fluorophores. This technique offers great potential for the characterization of multifunctionalized hybrid materials, a key challenge in nanoscience.
Progress in fluorescent labeling of DNA has also been achieved by understanding and exploiting the properties of CyDNA, a biopolymer consisting in DNA labeled in high density with cyanine dyes. The fluorescence quenching mechanisms have been elucidated, and the knowledge has allowed redesigning CyDNA to improve its properties. CyDNA has also been re-engineered into a reversible fluorescence photoswitchable biopolymer, and constitutes a new class of photoactive DNA-based nanomaterial that is of great interest for advanced microscopy applications. We have exploited fluorescence photoswitching in CyDNA to perform proof-of-principle super-resolution and optical lock-in detection (OLID) imaging.
The grant has greatly contributed to the consolidation of the fellow and her group at the host institution, and to the establishment of new national and international collaborations.