Work Undertaken During CoMPLEX MRes
Summer Project - In vitro Microtubule Tip Tracking: A quantitative study of the accuracy and precision.
Abstract from report.
A new method for the simulation of TIRF microscopy data of dynamic microtubules is presented and validated. This method makes use of direct rendering of Gaussians onto a pixel grid rather than the convolution of a high resolution image followed by down sampling. This new simulation method is used to analyse the accuracy and precision of a microtubule end tracking algorithm. It is shown that the main parameter in determining the precision in tracking is the signal to noise ratio which is normally distributed and has zero mean so the accuracy is still very good. I also derive a correction to the mean squared displacement to take account of the finite exposure time of the camera given the case of directed diffusion.
The report for this project can be downloaded here. The MATLAB script written for this project can be found on Bitbucket, here.
Mini Project 3 - Experimental investigation of the Photophysics of Oregon Green 488 and its suitability to biological research applications
Abstract from report.
A number of experiments were performed to determine the photophysical characteristics of Oregon Green 488 (Life Technologies, UK). The emission and absorption spectrum were determined. The fluorescence lifetime and anisotropy in water were measured. The fractional depletion, change in anisotropy and change in lifetime achieved by stimulated emission was determined for both single and two photon excitation. The quantum yield and a two photon cross section spectrum were determined by comparison to similar, well characterised fluorescent molecules, fluorescein and rhodamine B. The results of these experiments are discussed in the context of quantitative biological research applications which will be made possible by the understanding of these physical characteristics.
The report for this project can be downloaded here.
Mini Project 2 - The maintenance of cytoplasmic domains and nuclear independence
Abstract from report.
Anomalous diffusion in the eukaryotic cell cytoplasm and the maintenance of nuclear independence in polynucleated cell structures such as syncytia and coenocytes challenge the wide consensus of cytoplasmic continuity. Here I derive a model of nucleocytoplasmic protein shuttling in pairs of fused cells. The model implies that nuclear independence in syncytia could be maintained by large nuclear to cytoplasmic concentration gradients. In addition, new models of eukaryotic cell evolution suggest the endoplasmic reticulum (ER) may be responsible for forming cytoplasmic compartments affecting diffusion and maintaining independence in syncytia. Fluorescence recovery after photobleaching experiments on ER structure mutants and models of nucleocytoplasmic shuttling in cell-cell fusion experiments provide an insight into the ER mediated compartmentalisation hypothesis.
The report for this project can be downloaded here.
Mini Project 1 - QuickSOFI: High-Performance Superresolution Optical Fluctuation Imaging (SOFI) Plugin for ImageJ Using GPU Computing.
Abstract from report.
The field of superresolution microscopy holds the potential to truly visualise biological samples at the molecular scale – at resolution as low as 10nm, 20-fold greater than conventional microscopes. However these methods are still hindered by the need of large-data sampling and constrains such as a low densities of actively emitting fluorophores. Here we present an adaptation of a super resolution algorithm that tackles this limitation in the form of an easy to use plugin for ImageJ. This work holds the potential to simplify and greatly accelerate the acquisition of super-resolution data, potentially allowing live-cell superresolution to be achieved. This method is based on superresolution optical fluctuation imaging (SOFI) and accomplishes a 3D resolution enhancement by calculating the temporal or spatio-temporal cross cumulants of independent scholastically blinking fluorophores. SOFI analysis can be performed without extensive optimisation of fluorophore blinking characteristics, on conventional microscope setups and due to its simplicity will be available to a wide range of biological research applications.
The report for this project can be downloaded here.
While the aim of this project was to develop and make available an open source plugin for ImageJ/FIJI this plugin is not currently available due to a lack of rigorous performance testing in the time available during my CoMPLEX Mini Project. We hope to make this available at the earliest opportunity so please check again in the future if you are interested. In the mean time please see the original bSOFI authors' website where a MATLAB code has been made available for the calculation of 4th order bSOFI images.
MBA Poster - The Impact of Structured Stirring on the Success of External Fertilisation by Broadcast-Spawning
Abstract from poster.
The vast majority of sessile benthic invertebrates reproduce via external fertilisation by broadcast spawning. The worldwide demise of coral reefs and other sessile fauna highlights the need for better understanding of the physical-biological interactions which govern this little understood but important reproduction technique. Understanding the physical environment in which this takes place allows identification of adaptive strategies which increase fertilisation efficiency.
The poster can be downloaded here.