@phdthesis{Khayenko2023,
  author    = {Khayenko, Vladimir},
  title     = {Functional peptide-based probes for the visualization of inhibitory synapses},
  doi       = {10.25972/OPUS-32043},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320438},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Short functional peptidic probes can maximize the potential of high-end microscopy techniques and multiplex imaging assays and provide new insights into normal and aberrant molecular, cellular and tissue function. Particularly, the visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques and relies either on genetic manipulation or on antibodies that underperform in tissue immunofluorescence. Starting from an endogenous activity-related ligand of gephyrin, a universal marker of the inhibitory post-synapse, I developed a short peptidic multivalent binder with exceptional affinity and selectivity to gephyrin. By tailoring fluorophores to the binder, I have obtained Sylite, a probe for the visualization of inhibitory synapses, with an outstanding signal-to-background ratio, that bests the "gold standard" gephyrin antibodies both in selectivity and in tissue immunofluorescence. In tissue Sylite benefits from simplified handling, provides robust synaptic labeling in record-short time and, unlike antibodies, is not affected by staining artefacts. In super-resolution microscopy Sylite precisely localizes the post-synapse and enables accurate pre- to post-synapse measurements. Combined with complimentary tracing techniques Sylite reveals inhibitory connectivity and profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the periaqueductal gray brain region. Lastly, upon probe optimization for live cell application and with the help of novel thiol-reactive cell penetrating peptide I have visualized inhibitory synapses in living neurons. Taken together, my work provided a versatile probe for conventional and super-resolution microscopy and a workflow for the development and application of similar compact functional synthetic probes.},
  subject      = {Fluoreszenzsonde},
  language  = {en}
}
@article{EndesfelderMalkuschFlottmannetal.2011,
  author    = {Endesfelder, Ulrike and Malkusch, Sebastian and Flottmann, Benjamin and Mondry, Justine and Liguzinski, Piotr and Verveer, Peter J. and Heilemann, Mike},
  title     = {Chemically Induced Photoswitching of Fluorescent Probes - A General Concept for Super-Resolution Microscopy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74896},
  year      = {2011},
  abstract  = {We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.},
  subject      = {Super-Resolution Microscopy},
  language  = {en}
}