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Coherent Microscopy: New Analysis Methods for High Resolution 3D Imaging

Final Activity Report Summary - CMAM (Coherent Microscopy: New analysis methods for high resolution 3D imaging)

The main goals of the Intra-European Fellowship were the conversion of an existing inverted microscope for coherent imaging, the study of depth of field and new imaging modes of coherent microscopy, the computational modelling of scattering and the mapping of refractive index in 3D using weak and the strong scattering theories. All of the objectives of the project have been fulfilled, the results presented at several international conferences and submitted to relevant journals. In addition the fellow has organised (Co-chair) an International Workshop on Digital Holographic Reconstruction and Optical Tomography for Engineering Application and is Guest Editor of a special issue in Measurement Science and Technology to be published in 2008.

The main scientific achievements are:
1. New imaging modes of a Digital Holographic Microscope (DHM). DHM can be considered as a microscope with extended depth of field. From a single DHM recording the propagating component of the scattered field can be reconstructed in three-dimensions (3D). Conventional white light microscopy contrasting enhancing techniques can be applied to highlight characteristics of interest. If these techniques are used to enhance the reconstruction from a DHM then it is important to understand the characteristics of these techniques in 3D. Thus, the performance of Phase Contrast, Differential Interference Contrast, Hoffman and Spiral Phase Contrast visualisation methods was characterised in 3D.
2. Computational modelling. Several numerical methods have been studied to solve the Helmholtz equation for a given illumination and refractive index distribution (the forward problem). In particular, several forward problem solvers based on Finite Element Methods, Boundary Element Methods and iterative methods have been implemented in Matlab. These codes were the basis of algorithms to estimate the refractive index distribution of the object that best explains the scattering field measurements (this is the inverse problem in Optical Diffraction Tomography (ODT)).
3. Mapping of refractive index in 3D for weak and strong scattering problems.
* 3D images of the refractive index have been obtained using weak scattering theory. The limitations of the weak scattering approximation have been investigated using a simple object (a one-micron fibre on a glass plate).
* Several inverse methods have been considered and implemented in Matlab routines. It has been proved that in general Born Iteration Methods (BIM) cannot be used when multiple scattering effects are present, and that optimisation methods generally perform better. Computational experiments have been completed using a Conjugate Gradient Method (CGM) optimisation technique. The solution of the general problem is highly non-linear and requires regularisation. In microwave imaging different Tikhonov regularisation terms are generally used to modify the cost function. However the researcher's work shows that, in PIV applications, the use of certain a-priori information is more appropriate. A-priori knowledge of the refractive index and approximate seeding concentration of the particles can be enough to determine their distribution.