Thesis
Advancing widefield nanodiamond biological sensing with CMOS detection
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2026
- Thesis identifier
- T17680
- Person Identifier (Local)
- 202061926
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The prospect of using fluorescent nanodiamonds for biological imaging guided the work in this thesis. Fluorescent nanodiamonds have a small size (5 nm - 140 nm), which makes them an optimum choice for targeting cellular structures within an organism. Diamond is also biocompatible with no photobleaching effects, which allows for longterm imaging without toxic effects to the sample or concerns over signal loss. My PhD aimed to determine the feasibility of using a diffraction-limited, widefield microscope with CMOS camera detection for optically detected magnetic resonance spectroscopy of nitrogen-vacancy ensembles in fluorescent nanodiamonds in biological samples. First, a widefield microscope capable of such sensing was designed, built and characterised. The optical design involved widefield epifluorescence and prism-based total internal reflection fluorescence microscopy; both of these imaging modalities were characterised for their noise properties. The total internal reflection fluorescence mode and the evanescent field propagation depth was measured for total internal reflection. Both modes were used to image focal adhesions in HeLa cells to evaluate the excitation benefits of total internal reflection fluorescence over widefield epifluorescence. After optical characterisation, the optically detected magnetic resonance schemes were applied and investigated. To fully explore the novel challenges of widefield nitrogen vacancy spectroscopy, the achievable image contrast enhancements from super-resolution radial fluctuations were compared with those from contrast-limited adaptive histogram equalisation to improve the selection of regions of interest. After investigating the overall imaging performance, we characterised the optically detected magnetic resonance produced by each imaging modality, demonstrating the magnetic sensing capabilities for dynamic measurements with suitable resolution for quantitative outputs. Measurement can also be performed using temperature-sensing protocols, which this work attempts with limited success. We attribute the lack of success to hardware limitations in the temperature control system we employed. In particular, we identified an interaction between the temperature sensor used in our feedback control system and the microwaves required for optically detected magnetic resonance-based sensing. To characterise noise, which can also affect measurement stability within the system, Allan Variance was used to assess fluorescence stability in each imaging modality for frame integration time, camera gain, and laser power, using fluorescent microspheres. Using these fluorescent microspheres, the fluorescent stability of nanodiamond over time can also be compared to evaluate its photostability, and the noise characterisation using Allan Variance can improve continuous-wave optically detected magnetic resonance measurements by informing imaging criteria selection. The Allan Variance shows promise as a means to improve continuous-wave optically detected magnetic resonance measurements in high-noise datasets by indicating the number of repeats required to improve the accuracy when fitting the data to a suitable model function, as demonstrated in the widefield epifluorescence modality. However, no improvement in imaging parameter selection is observed using Allan Variance in the total internal reflection fluorescence modality. Finally, biological samples are used to validate the microscope’s application for biological sensing using the nitrogen vacancy centre defect. Initially, Caenorhabditis elegans are used to demonstrate that in situ measurement is possible using our microscope system. C. elegans has the additional advantage that it has been used previously for nitrogen vacancy centre sensing with some success. Next, Tetrahymena pyriformis demonstrates the ability of live-cell nitrogen vacancy centre sensing from within an organism. Allan Variance is used to determine whether additional noise arises from the inclusion of biological material in fluorescence imaging, which could lead to less accurate spectral measurements, thereby demonstrating the novelty of Allan Variance as a tool in biological fluorescence imaging. The final biological experiment is an investigation into the feasibility of using fluorescent nanodiamond to evaluate the thermal response in photosynthesis during light and dark reaction time which was done using Pond Weed and Spirogyra varians, it is possible to induce a blue shift in the spectra indicating a measurable thermal event but the thermal response is not characterised due to the interaction of the microwaves with the thermistor.
- Advisor / supervisor
- McConnell, Gail
- Patton, Brian R.
- Resource Type
- DOI
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PDF of thesis T17680 | 2026-03-26 | Public | Download |