Rational design of multi-chromophoric labels: Universal methods to improve the brightness of single molecules and fluorophores in the solid state

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

Over the last century fluorescence-based assays revolutionized our understanding of nature by revealing complex microstructures of materials and organisms.The never-ending curiosity and the development of new techniques puts a lot of pressure on the fluorophores which are the main signal reporters. This thesis investigates the current limitations of common organic fluorescent labels and seeks novel methods for further improvement of their photophysical properties, in order to push the current resolution and sensitivity limits in fluorescence spectroscopy and microscopy even further. By focusing on rational design of the fluorophores and engineering of their photophysical properties, two independent methods to increase the brightness of the fluorophores in solid state as well as single molecule level are suggested here. First, experimental results backing up theoretical predictions confirm that the brightness of the intrinsically dim long fluorescence lifetime fluorophores can be increased significantly by the covalent binding of an energy donor. Together with an acceptor it acts as an efficient energy transfer pair. As a proof-of-concept experiment, a dyad consisting of a perylene antenna and a triangulenium emitter with close to 100% energy transfer, retained long fluorescence lifetime (~17 ns), high quantum yield (75%) and an absorption coefficient up to 5 times higher in comparison to the free trianguleniumis introduced. These dyads with significantly improved brightness can now be detected at the single molecule level and easily discriminated from bright auto-fluorescence by time-gated and other fluorescence lifetime-based detection schemes. Second part in the experimental section deals with the centuries-long problem of fluorescence quenching in dense solid state, where the universal method based on anion-binding and structure-directing macrocycle is introduced as a way to isolate cationic fluorophores and reinstate their photophysical properties. By incorporating common, commercially available fluorophores, like rhodamines, cyanines, oxazines or trianguleniums, into Small Molecule Ionic Isolation Lattices (SMILES) provided by an anion receptor (cyanostar), electronic isolation between the dyes is shown to been sured while maintaining dense and predictable structure. In practice, SMILES crystals are shown to have the highest known brightness per volume and solve the issue of concentration quenching for cationic fluorophores. Furthermore,the applications of cyanostar are shown not to be limited by the solid state and can also be exploited in the solvents with varying or complex polarities. The last part of the thesis briefly explores the potential of long fluorescence lifetime triangulenium dyes as superior probes for time-gated continuous wave stimulated emission depletion imaging. Even though the results reported here reveal that the potential of DAOTA and BDATA trianguleniums is compromised by the excited state absorption spanning throughout their entire emission spectrum, experimental work led to another, unrelated discovery. Unexpectedly, live HeLa cells are shown to exhibit weak, but detectable intrinsic anti-Stokes emission originating in/on mitochondria and peaking around 590 nm when excited with intense green-red excitation sources. This discovery not only provides an additional modality for mitochondria visualization, but is also a precautionary statement, show in gevidence of autofluorescence in the range that was so far considered to be completely background-free.
OriginalsprogEngelsk
ForlagDepartment of Chemistry, Faculty of Science, University of Copenhagen
Antal sider296
StatusUdgivet - 2020

ID: 251186628