Horrocks, Mathew

Vendrell Escobar, Marc

Mowat, Chris

Konieczna, Zuzanna

2025-03-13T16:32:55Z

2025-03-13T16:32:55Z

2025-03-13

Testing new function - does this work?? Fluorescence microscopy is a tool routinely utilised to address biological questions. Direct visualisation of features of interest, confirmation of protein identity or detecting the presence of a posttranslational modification enable researchers to study differences between healthy and pathological phenotypes. As the questions get more complex, more advanced imaging techniques are required to address the limit of resolution, probe specificity to target and fluorescent background. This thesis explores two advancements to the field of fluorescence microscopy: the development of a novel imaging platform and the introduction of a new imaging modality. The study covers the design and construction of the Full Spectrum Fluorescence Lifetime Imaging Microscope (FS-FLIM): a new imaging platform. The FS-FLIM maximises the information collected in a fluorescence imaging experiment. It is capable of 3-in-1 imaging, collecting fluorescence intensity and lifetime data at 512 wavelengths simultaneously. The instrument is compatible with a wide range of samples. The spectral region observed can be matched to the emitters present in the sample. Moreover, a wide range of lifetimes (from sub-nanosecond to tens of nanoseconds) can be recorded using the FS-FLIM. The performance of the constructed instrument is validated through solution lifetime measurements of several fluorescent dyes, and its application in environment sensing is described using a model system. Next, the thesis focuses on developing fluorescent peptides for imaging applications. Although peptides can be labelled with a range of fluorophores, this study mostly uses a selenium-derivatised nitrobenzoxadiazole dye - SeNBD, a recent exciting addition to the imaging toolbox. The fluorogenic properties of the new dye are characterised in this study, and the incorporation of the dye on-column using standard solid state peptide synthesis methods is described. The switch-on character, as well as the small size of the dye, are leveraged throughout the work, with short peptide sequences and internally-labelled peptide sequences developed as a new generation of peptide probes. The specificity of peptides enables their application as therapeutic agents for otherwise undruggable proteins. One such example is α-synuclein, a protein associated with Parkinson’s disease (PD). It is commonly thought that aggregation of α-synuclein and formation of cytotoxic inclusions is involved in PD progression. An α-helical bacteria-derived peptide, phenol soluble modulin α3 (PSMα), was previously shown to bind to small oligomeric α-synuclein species. Here, the peptide sequence was derivatised with several labels to explore its applications in imaging. Pilot experiments to detect α-synuclein species in vitro were conducted to confirm the binding to the protein-of-interest. A biotin-labelled peptide analogue was used to perform immunohistochemistry staining on patient tissue for in situ detection. Lastly, first attempts at fluorescence lifetime imaging of α-synuclein on the FS-FLIM instrument were made. To further illustrate the capabilities of SeNBD dye specifically, and its potential for super-resolution imaging, a range of sequences targeting the PDZ domain (a common structural motif of anchoring and signalling proteins) were then considered. The chosen sequences were only a few amino acids long, however they demonstrated retained transient binding to the protein-of-interest upon labelling with SeNBD. Moreover, an internally-labelled sequence was also synthesised and successfully used to target PDZ domains. The transient nature of the peptideprotein binding was utilised in a Point Accumulation in Nanoscale Topography (PAINT) experiment, where the on-off binding enabled precise localisation of the dye molecules and super-resolution image reconstruction. The PDZ contained in post-synaptic density proteins of a brain-derived sample was successfully imaged, and nanoclusters of the protein super-resolved, corroborating literature findings. Furthermore, an extension to the approach using a coiled-coil interacting peptide pair: 101A and 101B, was investigated as an alternative approach for proteiniv peptide interaction pairs for super-resolution microscopy. Whilst the delivery of the synthesised targeting sequence (101B) proved challenging, expressing 101B attached to the target protein in cellulo, and subsequent staining with fluorescent 101A sequence produced promising results and enabled direct visualisation and super-resolution imaging of TOM-20 and LAMP-1 in fixed HEK cells. Overall, the work presented herein showcased a range of fluorescent peptides that could be used across versatile fluorescence imaging platforms. The small novel dye, SeNBD, enabled super-resolution imaging using peptide sequences containing less than 10 amino acids. The small size of the peptides decreased the linkage error and ensured the fluorescent signal was localised at target of interest. The environment sensing properties of the dye were explored in preliminary experiments to observe α-synuclein on a purpose-built novel microscope, the FS-FLIM. It is hoped the techniques developed here could further progress research in the field of neurodegeneration and beyond.

https://hdl.handle.net/1842/43209

http://dx.doi.org/10.7488/era/5749

en

The University of Edinburgh

F. de Moliner, Z. Konieczna, L. Mendive-Tapia, R. S. Saleeb, K. Morris, J. A. Gonzalez-Vera, T. Kaizuka, S. G. N. Grant, M. H. Horrocks and M. Vendrell, Angew. Chem. Int. Ed., 2023, 62, DOI: 10.1002/anie. 202216231

Z. Gidden, C. Oi, E. J. Johnston, Z. Konieczna, H. Bhaskar, L. Mendive- Tapia, F. de Moliner, S. J. Rosser, S. G. J. Mochrie, M. Vendrell, M. H. Horrocks and L. Regan, Nano Letters, 2023, 23, 10633–10641

A. Baibek, Z. Konieczna, M. ¨U¸c¨unc¨u, Z. S. Alghamdi, R. Sharma, M. H. Horrocks and M. Bradley, ChemRxiv (pre-print), 2024

Fluorescence microscopy

FS-FLIM (Full Spectrum Fluorescence Lifetime Imaging Microscope)

SeNBD dye

Peptide probes

Super-resolution imaging

Design and synthesis of peptides for fluorescence imaging applications

The design and synthesis of peptides for fluorescence imaging applications

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy