RESUMO
Computer tomography (CT) techniques are the most widely applicable noninvasive methods for obtaining two- and three-dimensional insights into biological objects. They comprise CT for medical applications, as well as electron tomography used for investigating macromolecular and cellular specimens. Recent advances in the recording schemes improve the speed and resolution frontiers and provide new insights into structural organizations of different objects. However, many data sets suffer from a poor signal-to-noise ratio, which severely hinders the application of methods for automated data analysis, such as feature extraction, segmentation, and visualization. We propose the multidimensional implementation of two powerful signal reconstruction techniques, namely invariant wavelet filtering and nonlinear anisotropic diffusion. We establish quantitative measures to assess the signal reconstruction performance on synthetic data and biomedical images. The appropriate multidimensional implementations of wavelet and diffusion techniques allow for a superior performance over conventional noise-reduction methods. We derive the conditions for the choice between wavelet and diffusion techniques with respect to an optimal signal reconstruction performance. Results of applying the proposed methods in two very different imaging domains-molecular biology and clinical research-are provided.
Assuntos
Intensificação de Imagem Radiográfica/métodos , Tomografia Computadorizada por Raios X , Anisotropia , Microscopia Eletrônica de Transmissão e Varredura , Dinâmica não LinearRESUMO
Active zone material at the nervous system's synapses is situated next to synaptic vesicles that are docked at the presynaptic plasma membrane, and calcium channels that are anchored in the membrane. Here we use electron microscope tomography to show the arrangement and associations of structural components of this compact organelle at a model synapse, the frog's neuromuscular junction. Our findings indicate that the active zone material helps to dock the vesicles and anchor the channels, and that its architecture provides both a particular spatial relationship and a structural linkage between them. The structural linkage may include proteins that mediate the calcium-triggered exocytosis of neurotransmitter by the synaptic vesicles during synaptic transmission.
Assuntos
Junção Neuromuscular/ultraestrutura , Animais , Canais de Cálcio/fisiologia , Aumento da Imagem , Proteínas de Membrana/fisiologia , Proteínas de Membrana/ultraestrutura , Proteínas do Tecido Nervoso/fisiologia , Proteínas do Tecido Nervoso/ultraestrutura , Junção Neuromuscular/fisiologia , Organelas/fisiologia , Organelas/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Rana pipiens , Membranas Sinápticas/fisiologia , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestruturaRESUMO
The visualization of volume maps obtained by electron tomographic reconstruction is severely hampered by noise. As electron tomography is usually applied to individual, nonrepeatable structures, e.g., cell sections or cell organelles, the noise cannot be removed by averaging as is done implicitly in electron crystallography or explicitly in single particle analysis. In this paper, an approach for noise reduction is presented, based on a multiscale transformation, e.g., the wavelet transformation, in conjunction with a nonlinear filtration of the transform coefficients. After a brief introduction to the theoretical background, the effect of this type of noise reduction is demonstrated by test calculations as well as by applications to tomographic reconstructions of ice-embedded specimens. Regarding noise reduction and structure preservation, the method turns out to be superior to conventional filter techniques, such as the median filter or the Wiener filter. Results obtained with the use of different types of multiscale transformations are compared and the choice of suitable filter parameters is discussed.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Eletrônica/métodos , Actinas/ultraestrutura , Células/ultraestrutura , Gelo , Modelos Estruturais , Modelos Teóricos , Organelas/ultraestrutura , Tomografia/métodosRESUMO
After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or--by sophisticated pattern recognition methods--identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are presented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data.