Micro-rotation fluorescence microscopy is a novel, optical imaging technique developed with a cell rotation system. The imaging system enables individual living cells to be rotated in suspension under microscopic dimensions, and allows us to acquire a series of images of the cells, simultaneously during the rotation. A challenging task in micro-rotation imaging is how to determine three-dimensional (3D) cell structure from the image series.
This thesis thus presents four alternative methods for reconstructing 3D objects from a series of micro-rotation images. The three former methods, the expectation maximisation (EM), the generalised Skilling-Bryan and the marginalisation methods, are iterative algorithms built on the Bayesian inversion theory, which is used to quantify uncertainties in data and model parameters, and also to utilise prior information about the unknown structure. The fourth method, dual filtered backprojection (DFBP), is a fast, non-iterative algorithm derived from the Fourier slice theorem in the classical computed tomography. Each method has its own features: the EM method serves as a basic tool for general usability; the Skilling-Bryan method is more flexible for modelling of noise and prior; the marginalisation method serves as a statistical treatment of the reconstruction that suffers from inaccurate image alignment; and the DFBP method beats the other methods by computational speed but restricts itself with a certain imaging condition. In general, selection of the reconstruction methods depends on imaging and data conditions, such as conventional widefield or confocal microscopy, the stability of cell rotation, and the quality of image alignment.
In conclusion, all the proposed reconstruction methods clearly increase capability to visualise 3D object structures, as shown by both simulations and experiments with real micro-rotation data. Two obvious messages from the results are that first, the quality of the reconstructed object highly depends on the accuracy of image alignment, and second, micro-rotation reconstructions with the current imaging system always contain poor resolution in the tangential direction of the rotation. Future interesting research is thus to combine the micro-rotation protocol with extended depth-of-focus microscopy that could strengthen the tangential resolution
