Filtering, reconstruction, and measurement of the geometry of nuclei from hippocampal neurons based on confocal microscopy data

Abstract

The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium.

Synaptic Activity Induces Dramatic Changes in the Geometry of the Cell Nucleus: Interplay between Nuclear Structure, Histone H3 Phosphorylation, and Nuclear Calcium Signaling

“…The mathematical framework for the 3D image reconstruction of cell nuclei has been described (Queisser et al, 2008). See supplemental material, available at www.jneurosci.org, for a brief description of the algorithms used and for details of the mathematical modeling of nuclear calcium dynamics in nuclei with different geometries.…”

“…To visualize the structure of cell nuclei, we used a recently developed algorithm that allows 3D image reconstruction of cell organelles from optical sections generated using a laser-scanning confocal microscope (Queisser et al, 2008; see also supplemental material, available at www.jneurosci.org). For the microscopy analysis, cultured hippocampal neurons were fixed and subjected to immunocytochemistry using an antibody to lamin B that decorates the inner nuclear envelope (Fig.…”

Structural dynamics of the cell nucleus

“…3 In their function as 'flood gates' for nuclear calcium, an increased number of nuclear pore complexes will make the nuclear envelope more permeable. 4,5 Notably, the generation of signaling microdomains in the nucleus in conjunction with increased surface expression of nuclear pores will not only facilitate nuclear signaling of calcium-as outlined above-but it will also augment nuclear signaling of second messengers in general 3,6 (Fig. 6).…”

“…In order to investigate the influence of the nuclear morphology on calcium signals, we developed a computational model of calcium signaling in the nucleus, which takes into account the real nuclear morphology obtained from confocal images of cultured hippocampal neurons and reconstructed with a previously developed neuron reconstruction algorithm. 6,22 By reconstructing a large set of nuclei (examples in Fig. 1), we were able to perform in silico experiments of calcium signals in nuclei that provide a direct link between calcium and morphology (example of simulation in Fig.…”

Inertia based filtering of high resolution images using a GPU cluster

“…2 for details), introduced as nonlinear anisotropic diffusion in [3], is successfully used as a preprocessing step in the NeuRA algorithm for reconstructing the morphology of neuron cells [3] and nuclei from hippocampal neurons [19] from 2-photon or confocal microscopy images. Among other methods, like median filtering [8], wavelet methods [24], nonseparable filterbanks [20] or multiscale enhancement techniques [6], inertia based anisotropic filtering massively reduces the noise of these images while preserving existing structures [3,10,11,19]. The inertia based structure detection also turned out to be more powerful than the anisotropic diffusion filtering schemes of Perona-Malik [18] and Weickert [23], as shown in [19].…”