Algorithms for the Correction of Photobleaching
University of Oxford
Preface
Ways to read
This thesis may be read on the web at https://rorynolan.github.io/phdthesis/. If you are reading this on the web now but would like a PDF version, click on the download symbol at the top left of the page (to the right of the A) and select PDF.
Apportion of credit
For clarity, I include the following reliable rules of thumb:
- Molecular biology was done by Maro Iliopoulou and Luis Alvarez.
- Imaging was done by Maro Iliopoulou, Luis Alvarez and Sergi Padilla-Parra.
- The idea that correction for bleaching was the crucial step for FFS analysis was formulated by Luis Alvarez, Sergi Padilla-Parra and I.
- I formulated the solutions for how to correctly correct for bleaching, i.e. the automatic parameter choice and the Robin Hood algorithm.
- I wrote all of the software and maintain all of it.
- All FFS analysis was performed using my software. The software was used to analyse data by Maro Iliopoulou, Luis Alvarez, Sergi Padilla Parra and I.
- Structural modelling was done by Thomas Bowden and Yasunori Watanabe.
- On all papers where I am the first listed author, I wrote the paper, taking suggested amendments and augmentation from other listed authors. The NSMB paper (Iliopoulou et al. 2018) was written by Sergi; I also made significant contributions to the writing of that manuscript but my main role in that project was in data analysis.
Publications
I hereby list my publications. These can all be downloaded from https://github.com/rorynolan/phdthesis/tree/master/papers, where the naming convention is JournalYEAR.pdf.
Other
- D. M. Jones, L. A. Alvarez, R. Nolan, et al. “Dynamin-2 stabilizes the HIV-1 fusion pore with a low oligomeric state”. In: Cell reports 18.2 (2017).
- G. M. Jakobsdottir, M. Iliopoulou, R. Nolan, et al. “On the whereabouts of HIV-1 cellular entry and its fusion ports”. In: Trends in molecular medicine (2017).
- Q. F. Wills, E. Mellado-Gomez, R. Nolan, et al. “The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq”. In: BMC genomics 18.1 (2017).
Acronyms used in this thesis
| Acronym | Term |
|---|---|
| ACF | AutoCorrelation Function |
| ALEX | Alternating Laser EXcitation |
| APD | Avalanche Photo-Diode |
| BMC | BioMed Central |
| CCD | Charge-Coupled Device |
| ccN&B | cross-correlated Number and Brightness |
| CCR5 | C-Chemokyne Receptor 5 |
| CD4 | Cluster of Differentiation 4 |
| CMOS | Complementary Metal–Oxide Semiconductor |
| CXCR4 | C-X-C motif Chemokyne Receptor 4 |
| DNA | Deoxyribinucleic Acid |
| dSTORM | direct STochastic Optical Reconstruction Microscopy |
| EM | Electron Microscopy |
| EMCCD | Electron-Multiplied Charge-Coupled Device |
| FCS | Fluorescence Correlation Spectroscopy |
| FFS | Fluorescence Fluctuation Spectroscopy |
| FIJI | FIJI Is Just ImageJ |
| FKBP | FK Binding Protein |
| FRET | Forster Resonance Energy Transfer |
| GaAsP | Gallium Arsenide Phosphide |
| GFP | Green Fluorescent Protein |
| HIV | Human Immunodeficiency Virus |
| HOMO | Highest Occupied Molecular Orbital |
| HXB2 | Subtype B2 HIV-1 isolate |
| HyD | Hybrid Detector |
| I/O | Input/Output |
| JR-FL | JR (patient name) Frontal Lobe |
| LOMO | Lowest Occupied Molecular Orbital |
| MSD | Mean Squared Displacement |
| mTFP | monomeric Turquoise Fluorescent Protein |
| N&B | Number and Brightness |
| NSM | Non-Stationary Mean |
| NSMB | Nature Structural and Molecular Biology |
| NSV | Non-Stationary Variance |
| PCF | Pair Correlation Function |
| PDB | Protein DataBase |
| Portable Document Format | |
| PhD | Philosophiae Doctor |
| PMT | Photon Multiplier Tube |
| QE | Quantum Efficiency |
| RNA | Ribonucleic Acid |
| sCMOS | scientific Complementary Metal–Oxide Semiconductor |
| SP | Scanning Platform |
| TIFF | Tagged Image File Format |
| TIR | Total Internal Reflection |
| TIRF | Total Internal Reflection Fluorescence |
| TZM-bl | Stable cell line expressing CD4 and CCR5 |
Mathematical symbols used in this thesis
| Symbol | Meaning |
|---|---|
| \(\theta\) | angle of incident light ray |
| \(B\) | apparent brightness of entities |
| \(N\) | apparent number of entities |
| \(G\) | autocorrelation |
| \(\varphi\) | concentration |
| \(\text{corr}\) | correlation |
| \(\theta_c\) | critical angle |
| \(\text{crosscor}\) | cross-correlation |
| \(B_\text{cc}\) | cross-corrlated brightness |
| \(\sigma^2_\text{cc}\) | cross-variance |
| \(\tilde{x}\) | deviation of \(x\) from the expected value \(\mu\) of \(X\) |
| \(D\) | diffusion constant |
| \(J\) | diffusive flux |
| \(\epsilon\) | entity brightness |
| \(E\) | expectation operator |
| \(\tau\) | exponential smoothing parameter |
| \(j\) | frame number |
| \(S_0\) | highest occupied molecular orbital |
| \(I\) | intensity |
| \(l\) | length parameter of boxcar |
| \(T_1\) | lowest energy triplet state |
| \(S_1\) | lowest unoccupied molecular orbital |
| \(\text{msd}\) | mean squared displacement |
| \(\mu\) | mean; expected value |
| \(n\) | moment number; number of entities; number of replicates |
| \(\mathbb{N}\) | natural numbers (excluding zero) |
| \(\mathbb{N}_0\) | natural numbers (including zero) |
| \(d\) | number of dimenions |
| \(\alpha\) | number of swaps |
| \(p\) | particle position; pixel position |
| \(x\) | position in space; horizontal position; instance of random variable \(X\) |
| \(k\) | positive real number |
| \(X\) | random variable |
| \(\mathbb{R}\) | real numbers |
| \(n_2\) | refractive index of cover slip |
| \(n_1\) | refractive index of sample on cover slip |
| \(\tau_D\) | residence time of particle (in confocal volume) |
| \(i\) | slice number |
| \(\text{exp}\) | the exponential function |
| \(t\) | time |
| \(\text{Var}\) | variance function |
| \(\sigma^2\) | variance in intensity |
| \(y\) | vertical position |
Acknowledgements
Thanks to my parents Kate and Dave who were good enough to house me rent-free for 18 years. They encouraged me academically after it became clear that I was not the next Kevin Kilbane. Thanks to my wife Naomi who puts up with me now that my parents have had enough of me. Thanks to my brother Brendan who taught me (perhaps wrongly) that maths is cool.
Thanks to my supervisor Sergi Padilla-Parra who had many good ideas of what to study, for his intelligent input and generosity with time. Moreover, thanks to him for emphasising that it’s possible to work too hard. It’s largely thanks to his attitude that my time in Oxford was so enjoyable.
Thanks to all of my lab mates: Dan, Keith, Xenia, Raquel, Luis, Margarita, Marina, Maro, Marina, Maria, Chad and Irene; it was interesting and fun to spend time with you all. I’ll look back upon my time in the lab fondly, I hope you can say the same.
Thanks to my friends Kathrin and Zammy from my PhD programme; we shared the journey to becoming doctors and come out of the other end with lifelong friendships as well as new titles.
Thanks to my college friends Bas, Jessica, Francis, Sarah, Andy, Frankie, Jonny, Suze, Walid, Sophia, Martin, Max, Maj, Nirmalie, Luiza, LD and many more. It was a privilege to have so many good friends at once.
There are plenty more people I should thank, but I’m tired so I’m going to stop now.
References
Iliopoulou, Maro, Rory Nolan, Luis Alvarez, Yasunori Watanabe, Charles A. Coomer, G. Maria Jakobsdottir, Thomas A. Bowden, and Sergi Padilla-Parra. 2018. “A dynamic three step mechanism drives the HIV-1 prefusion reaction.” Nat. Struct. Mol. Biol. 25 (9). doi:10.1038/s41594-018-0113-x.