The diffraction limit restricts conventional light microscopes to approximately 250 nm laterally and 500 nm axially, these limits being first proposed by Abbe in 1873. Despite this, optical microscopes have found many applications in biological research and single cells that are 10 - 100 um in size. Furthermore by coupling the non-invasive nature of a light microscope with highly sensitive fluorescent probes, fluorescence microscopy has also become a standard imaging technique. Recent advances in fluorescence microscopy now provide a number of methods to circumvent the Abbe diffraction limit, with many techniques becoming prevalent over the last 10 years including direct Stochastic Optical Reconstruction Microscopy (dSTORM). A dSTORM system has been constructed and calibrated using a commercially available inverted florescence microscope and total internal reflection florescence (TIRF) imaging. dSTORM relies on the ability to switch sparse subsets of fluorophores and temporally separate them. Provided the spatial separation is sufficient between any member of a subset, the average error with which the emission can be localized is much less than size of the emission profile itself. The underlying mechanism for this switching is detailed based on the principle of photoinduced electron transfer (PET). The switching characteristics of the common florescent dye Alexa Fluor 568 are investigated and shown to be controlled by a number of factors including the excitation intensity and concentration of the primary thiol cysteamine beta-MEA. A number of parameters are defined, including the dye switching rate, for a given set of physical parameters. U2OS cells are labelled for the microtubule protein Tubulin using immunofluorescent labelling strategies. A direct comparison is made between diffraction limited TIRF images and dSTORM reconstructed images, with an average width for microtubules determined to (58.2 ± 8.1) nm. Further measurements are made by labelling the Rab5 effector Early Endosome Antigen 1 (EEA1). From this the aspect ratio for early endosomes is determined to be 1.68 ± 0.7 with an average radius of (45.8 ± 18.8) nm. The point spatial distribution of EEA1 is investigated by using the linearised form of Ripley's K-function H(r) and the null hypothesis of complete spatial randomness tested. EEA1 is shown to cluster at radius of 58.7 nm on individual endosomes, thought to be due to the well defined binding domains present on early endosomes for EEA1. Further evidence suggests that clustering is also exhibited at another maximum of approximately 500 nm when looking at an ensemble of EEA1 and early endosomes.
|Date of Award||1 Aug 2015|
- The University of Manchester
|Supervisor||Thomas Waigh (Supervisor), Mark Dickinson (Supervisor) & Viki Allan (Supervisor)|
- fluorescence microscopy
Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures
Sanders, J. (Author). 1 Aug 2015
Student thesis: Phd