Developing and characterising genetically encoded vibrational probes for next- generation bioimaging applications using electronic pre-resonant Raman spectroscopy

Aksakal, Ozan 2024. Developing and characterising genetically encoded vibrational probes for next- generation bioimaging applications using electronic pre-resonant Raman spectroscopy. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The advancement of molecular imaging techniques is paramount for revealing cellular and molecular processes in their native environments. This thesis explores the development of genetically encoded Raman-active probes, utilising far-red and near-infrared fluorescent proteins (FPs) and non-canonical amino acids (ncAAs) to enhance Raman spectroscopy applications. By integrating experimental and computational methodologies, this work addresses limitations of fluorescence microscopy, including photobleaching, cytotoxicity, and limited multiplexing capabilities, paving the way for next-generation imaging techniques. In Chapter 3, selected far-red and near-IR FPs were characterised and spectroscopically analysed to identify the most suitable candidates for Raman-active probes. Structural insights into mRhubarb720 were obtained through micro-focused crystallography, revealing key residues within its biliverdin (BV)-binding pocket. Chapter 4 examines Raman enhancement in near-IR FPs, focusing on chromophore properties and their compatibility with Raman techniques. The study evaluates the suitability of these proteins for electronic pre-resonant Raman scattering (epr-RS) in bacterial and mammalian systems. Chapter 5 explores the incorporation of para-cyano-phenylalanine in attempt to induce through-space Raman enhancement within the biologically silent region. Molecular dynamics (MD) simulations were employed to assess structural and dynamic effects of the ncAA incorporation, while quantum mechanics (QM) simulations predicted electronic and vibrational properties of BV in protein-bound and free states. These simulations provided insights into unexplored vibrational modes and informed future epr-RS studies. Finally, Chapter 6 evaluates the incorporation of ncAAs into mCherry chromophores, analysing changes in spectral and structural properties. MD simulations highlighted structural stability, while QM methods elucidated optical behaviour, emphasising how ncAA modifications influence chromophore functionality. This thesis highlights the cooperation between experimental and computational approaches in developing Raman-active probes, offering a robust framework for enhancing molecular imaging. The findings contribute to a deeper understanding of chromophore interactions within biological environments and demonstrate the potential of ncAAs and far- red/near-IR FPs to transform Raman-based imaging technologies.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	16 May 2025
Last Modified:	22 May 2025 10:21
URI:	https://orca.cardiff.ac.uk/id/eprint/178321

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