Fluorophore studies and instrumentation development for tumour margin estimation

  • Hazel Stewart

Student thesis: Doctoral Thesis


Surgery remains a key treatment option for tumour removal, where surgeons primarily rely on eye and touch to assess the boundary between healthy and cancerous tissue with no cellular information as guidance. There is therefore a need for a device or instrument that can be used by the surgeon in real-time during the surgical procedure that will ensure as many of the cancerous cells have been removed as possible. Fluorescence-based techniques have great potential for such an application, wherein this thesis explores various aspects related to the use of fluorescence techniques for estimating tumour margins.The intrinsic fluorophore NAD(P)H has been studied due to the significant interest it has gained for its use in tumour margin estimation, where the influence of environmental factors on the properties of NAD(P)H in various solvents were initially investigated. An increase in the fluorescence lifetime of NADH was observed in water and ethylene glycol upon oxygen removal, where the average lifetimes increased from 0.40 ns to 0.45 ns and 0.76 ns to 0.94 ns respectively. In addition to this, fluorescence anisotropy measurements revealed two rotational correlation times of 0.43 ± 0.24 ns and 5.49 ± 0.04 ns for NADH in ethylene glycol. These times correspond to particle sizes of ~ 0.6 nm and 1.4 nm which have been attributed to the folded and unfolded conformations of NADH free in solution respectively, where such methods have not previously been reported for resolving the folded and unfolded conformations of NADH.NAD(P)H was also studied in the cellular environment, where a decrease in every lifetime component of NAD(P)H in cancerous prostate cells incubated in high glucose media compared to those in low glucose media has been observed, where decreases of 0.52 ns, 2.1 ns and 0.10 ns were found for τ1, τ2, (bound NAD(P)H) and τ3 (unbound NAD(P)H) respectively. In contrast to this, no change in lifetime was observed in healthy prostate cells incubated in both high and low glucose media. It is believed that the observed decrease in lifetime components in the cancer cells is due to the preferential use of the glycolysis pathway in these cells, which has been previously linked to a decrease in fluorescence lifetime of certain cancerous cell lines.;Additionally, a low-cost liquid light guide-based fluorescence lifetime system incorporating a translational stage has been designed for phantom margin assessments, where the margins were created using silica sol-gels. Measurements of phantom margins with Rhodamine 6G and NADH in the doped sol-gel region demonstrated that the sol-gel only region could be clearly identified 1 mm after the margin position based on the fluorescence decays obtained. Measurements were also performed using FDA-approved Indocyanine Green in the doped sol-gel region of the phantom margin, however only scatter from the sol-gel could be detected 7 mm before the margin. A comparison between light guide and single-photon avalanche diode (SPAD) array fluorescence lifetime imaging microscopy (FLIM) measurements demonstrated improved spatial resolution provided by the FLIM system for assessing the margin position with short-lived fluorophores such as ICG, where the sol-gel only region of the ICG-doped sample could be clearly identified 1 mm after the margin using this system.
Date of Award28 Feb 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorDavid Birch (Supervisor)

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