Feature-preserving image restoration and its application in biological fluorescence microscopy

This thesis presents a new investigation of image restoration and its application to fluorescence cell microscopy. The first part of the work is to develop advanced image denoising algorithms to restore images from noisy observations by using a novel featurepreserving diffusion approach. I have applied these algorithms to different types of images, including biometric, biological and natural images, and demonstrated their superior performance for noise removal and feature preservation, compared to several state of the art methods. In the second part of my work, I explore a novel, simple and inexpensive super-resolution restoration method for quantitative microscopy in cell biology. In this method, a super-resolution image is restored, through an inverse process, by using multiple diffraction-limited (low) resolution observations, which are acquired from conventional microscopes whilst translating the sample parallel to the image plane, so referred to as translation microscopy (TRAM). A key to this new development is the integration of a robust feature detector, developed in the first part, to the inverse process to restore high resolution images well above the diffraction limit in the presence of strong noise. TRAM is a post-image acquisition computational method and can be implemented with any microscope. Experiments show a nearly 7-fold increase in lateral spatial resolution in noisy biological environments, delivering multi-colour image resolution of ~30 nm

Translation Microscopy (TRAM) for super-resolution imaging

Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology

Mathematical Methods for the Quantification of Actin-Filaments in Microscopic Images

In cell biology confocal laser scanning microscopic images of the actin filament of human osteoblasts are produced to assess the cell development. This thesis aims at an advanced approach for accurate quantitative measurements about the morphology of the bright-ridge set of these microscopic images and thus about the actin filament. Therefore automatic preprocessing, tagging and quantification interplay to approximate the capabilities of the human observer to intuitively recognize the filaments correctly. Numerical experiments with random models confirm the accuracy of this approach

Upper airways segmentation using principal curvatures

Esta tesis propone una nueva técnica para segmentar las vías aéreas superiores. Esta propuesta permite la extracción de estructuras curvilíneas usando curvaturas principales. La propuesta permite la extracción de éstas estructuras en imágenes 2D y 3D. Entre las principales novedades se encuentra la propuesta de un nuevo criterio de parada en la propagación del algoritmo de realce de contraste (operador multi-escala de tipo sombrero alto). De la misma forma, el criterio de parada propuesto es usado para detener los algoritmos de difusión anisotrópica. Además, un nuevo criterio es propuesto para seleccionar las curvaturas principales que conforman las estructuras curvilíneas, que se basa en los criterios propuestos por Steger, Deng et. al. y Armande et. al. Además, se propone un nuevo algoritmo para realizar la supresión de nomáximos que permite reducir la presencia de discontinuidades en el borde de las estructuras curvilíneas. Para extraer los bordes de las estructuras curvilíneas, se utiliza un algoritmo de enlace que incluye un nuevo criterio de distancia para reducir la aparición de agujeros en la estructura final. Finalmente, con base en los resultados obtenidos, se utiliza un algoritmo morfológico para cerrar los agujeros y se aplica un algoritmo de crecimiento de regiones para obtener la segmentación final de las vías respiratorias superiores.This dissertation proposes a new approach to segment the upper airways. This proposal allows the extraction of curvilinear structures based on the principal curvatures. The proposal allows extracting these structures from 2D and 3D images. Among the main novelties is the proposal of a new stopping criterion to stop the propagation of the contrast enhancement algorithm (multiscale top-hat morphological operator). In the same way, the proposed stopping criterion is used to stop the anisotropic diffusion algorithms. In addition, a new criterion is proposed to select the principal curvatures that make up the curvilinear structures, which is based on the criteria proposed by Steger, Deng et. al. and Armande et. al. Furthermore, a new algorithm to perform the non-maximum suppression that allows reducing the presence of discontinuities in the border of curvilinear structures is proposed. To extract the edges of the curvilinear structures, a linking algorithm is used that includes a new distance criterion to reduce the appearance of gaps in the final structure. Finally, based on the obtained results, a morphological algorithm is used to close the gaps and a region growing algorithm to obtain the final upper airways segmentation is applied.Doctor en IngenieríaDoctorad

Directional edge and texture representations for image processing

An efficient representation for natural images is of fundamental importance in image processing and analysis. The commonly used separable transforms such as wavelets axe not best suited for images due to their inability to exploit directional regularities such as edges and oriented textural patterns; while most of the recently proposed directional schemes cannot represent these two types of features in a unified transform. This thesis focuses on the development of directional representations for images which can capture both edges and textures in a multiresolution manner. The thesis first considers the problem of extracting linear features with the multiresolution Fourier transform (MFT). Based on a previous MFT-based linear feature model, the work extends the extraction method into the situation when the image is corrupted by noise. The problem is tackled by the combination of a "Signal+Noise" frequency model, a refinement stage and a robust classification scheme. As a result, the MFT is able to perform linear feature analysis on noisy images on which previous methods failed. A new set of transforms called the multiscale polar cosine transforms (MPCT) are also proposed in order to represent textures. The MPCT can be regarded as real-valued MFT with similar basis functions of oriented sinusoids. It is shown that the transform can represent textural patches more efficiently than the conventional Fourier basis. With a directional best cosine basis, the MPCT packet (MPCPT) is shown to be an efficient representation for edges and textures, despite its high computational burden. The problem of representing edges and textures in a fixed transform with less complexity is then considered. This is achieved by applying a Gaussian frequency filter, which matches the disperson of the magnitude spectrum, on the local MFT coefficients. This is particularly effective in denoising natural images, due to its ability to preserve both types of feature. Further improvements can be made by employing the information given by the linear feature extraction process in the filter's configuration. The denoising results compare favourably against other state-of-the-art directional representations

Statistical Diffusion Tensor Imaging

Magnetic resonance diffusion tensor imaging (DTI) allows to infere the ultrastructure of living tissue. In brain mapping, neural fiber trajectories can be identified by exploiting the anisotropy of diffusion processes. Manifold statistical methods may be linked into the comprehensive processing chain that is spanned between DTI raw images and the reliable visualization of fibers. In this work, a space varying coefficients model (SVCM) using penalized B-splines was developed to integrate diffusion tensor estimation, regularization and interpolation into a unified framework. The implementation challenges originating in multiple 3d space varying coefficient surfaces and the large dimensions of realistic datasets were met by incorporating matrix sparsity and efficient model approximation. Superiority of B-spline based SVCM to the standard approach was demonstrable from simulation studies in terms of the precision and accuracy of the individual tensor elements. The integration with a probabilistic fiber tractography algorithm and application on real brain data revealed that the unified approach is at least equivalent to the serial application of voxelwise estimation, smoothing and interpolation. From the error analysis using boxplots and visual inspection the conclusion was drawn that both the standard approach and the B-spline based SVCM may suffer from low local adaptivity. Therefore, wavelet basis functions were employed for filtering diffusion tensor fields. While excellent local smoothing was indeed achieved by combining voxelwise tensor estimation with wavelet filtering, no immediate improvement was gained for fiber tracking. However, the thresholding strategy needs to be refined and the proposed model of an incorporation of wavelets into an SVCM needs to be implemented to finally assess their utility for DTI data processing. In summary, an SVCM with specific consideration of the demands of human brain DTI data was developed and implemented, eventually representing a unified postprocessing framework. This represents an experimental and statistical platform to further improve the reliability of tractography

Adaptive processing of thin structures to augment segmentation of dual-channel structural MRI of the human brain

This thesis presents a method for the segmentation of dual-channel structural magnetic resonance imaging (MRI) volumes of the human brain into four tissue classes. The state-of-the-art FSL FAST segmentation software (Zhang et al., 2001) is in widespread clinical use, and so it is considered a benchmark. A significant proportion of FAST’s errors has been shown to be localised to cortical sulci and blood vessels; this issue has driven the developments in this thesis, rather than any particular clinical demand. The original theme lies in preserving and even restoring these thin structures, poorly resolved in typical clinical MRI. Bright plate-shaped sulci and dark tubular vessels are best contrasted from the other tissues using the T2- and PD-weighted data, respectively. A contrasting tube detector algorithm (based on Frangi et al., 1998) was adapted to detect both structures, with smoothing (based on Westin and Knutsson, 2006) of an intermediate tensor representation to ensure smoothness and fuller coverage of the maps. The segmentation strategy required the MRI volumes to be upscaled to an artificial high resolution where a small partial volume label set would be valid and the segmentation process would be simplified. A resolution enhancement process (based on Salvado et al., 2006) was significantly modified to smooth homogeneous regions and sharpen their boundaries in dual-channel data. In addition, it was able to preserve the mapped thin structures’ intensities or restore them to pure tissue values. Finally, the segmentation phase employed a relaxation-based labelling optimisation process (based on Li et al., 1997) to improve accuracy, rather than more efficient greedy methods which are typically used. The thin structure location prior maps and the resolution-enhanced data also helped improve the labelling accuracy, particularly around sulci and vessels. Testing was performed on the aged LBC1936 clinical dataset and on younger brain volumes acquired at the SHEFC Brain Imaging Centre (Western General Hospital, Edinburgh, UK), as well as the BrainWeb phantom. Overall, the proposed methods rivalled and often improved segmentation accuracy compared to FAST, where the ground truth was produced by a radiologist using software designed for this project. The performance in pathological and atrophied brain volumes, and the differences with the original segmentation algorithm on which it was based (van Leemput et al., 2003), were also examined. Among the suggestions for future development include a soft labelling consensus formation framework to mitigate rater bias in the ground truth, and contour-based models of the brain parenchyma to provide additional structural constraints

Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm) and the bi-dimensional densities obtained with SDM (puncta/100 µm). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.This work was supported by grants from the following entities: the Spanish “Ministerio de Ciencia, Innovación y Universidades” (Grant PGC2018-094307-B-I00 and the Cajal Blue Brain Project [C080020-09; the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland]; the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 785907 (Human Brain Project, SGA2); the Wellcome Trust (Technology Development Grant 202932); and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (695568 SYNNOVATE). L.T.-R. is a recipient of grants from the EMBO Long-term fellowship 2016–2018 and the IBRO-PERC InEurope grants programme