Tomographic brain imaging with nucleolar detail and automatic cell counting

Hieber, Simone; Bikis, Christos; Khimchenko, Anna; Schweighauser, Gabriel; Hench, Jürgen; Chicherova, Natalia; Schulz, Georg; Müller, Bert

doi:10.1038/srep32156

Cited by 63 publications

(58 citation statements)

References 69 publications

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“…After manually setting the filter parameters, cell segmentation was performed in an automatic fashion. The error of cell localization was determined to be to 5% by visual inspection [3]. Purkinje cell density was found to be 116 mm -3 , in agreement with values found in current literature [8].…”

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confidence: 87%

Visualization and Segmentation of Cells in Unstained Paraffin-Embedded Cerebral Tissue

et al. 2018

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The three-dimensional visualization of cerebral cells is a challenging task. The human brain with a weight of about 1 kg contains around 10 12 cells. Currently, brain cells are imaged by optical and electron microscopies. These imaging techniques yield only information on surface-near regions and volumes restricted to cubic micrometers. In the last decade, the tissue preparation and high-resolution hard X-ray tomography has been advanced to reveal individual unstained cells in cubic millimeter brain volumes [1,2] with phase contrast. More recently, sufficient contrast for cell imaging in single distance propagation mode [3] or even with lab-based conventional approaches [4] was obtained by embedding brain specimens in paraffin. This allows improving the spatial resolution from the regime of grating interferometry, limited by the grating period, usually in the range of a few micrometers, to subcellular length-scales. Such data lend themselves for (semi-)automatic cellular und subcellular structure segmentation. Several thousand Purkinje cells were visualized and segmented within a ~40 mm 3 cerebellum specimen. Individual dendritic trees of Purkinje cells were visualized from the same specimen with higher resolution scans.The cerebellum was excised from a donated body of a 73-year old male in accordance with the Guidelines of the Ethical committee Northwestern Switzerland. It was embedded in paraffin following fixation in 4% histological-grade buffered formalin and dehydration in ethanol. Two cylinders with diameters 2.6 and 6.0 mm were cut from the paraffin blocks by means of hollow punches. Singledistance propagation phase contrast micro computed tomography was performed at the beamlines ID19 (European Synchrotron Radiation Facility, Grenoble, France) and Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK) in local mode. Measurements at ID19 were performed with pink beam with a mean photon energy of 19.45 keV at 1.75 µm pixel length. 2004 equiangular projections were acquired over 360° with a FReLoN 2K CCD camera with 1 s exposure per projection after 80 cm propagation distance. At I13-2, 19 keV monochromatic beam energy and 0.45 µm pixel length were used. Projections were acquired with a pco.4000 camera (PCO AG, Kelheim, Germany) with 8 s exposure time at 2400 equiangular steps over 180° and 5 cm propagation distance. Phase retrieval was performed with the algorithm proposed [5] and implemented in the software package ANKAphase [6]. Reconstruction of the tomographic volumes was performed with the filtered backprojection routine implemented in MATLAB R2014b (Simulink, The MathWorks, Inc., USA). After tomographic measurements, the specimens were re-embedded into larger paraffin blocks for histological

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confidence: 87%

Visualization and Segmentation of Cells in Unstained Paraffin-Embedded Cerebral Tissue

et al. 2018

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“…Therefore, we must conclude that the use of contrast-enhancing techniques is imperative for high resolution imaging of mouse brain, as the native contrast in hydrated tissue is not sufficient to resolve single cells with the resolution needed to address the issue of neuronal connectivity. Embedding the tissue in materials with a different electron density than water, e.g., paraffin 31 or, as shown here, staining with metallic ions might be a decisive step in sample preparation in order to have the sufficient resolution and contrast to decipher neuronal connectivity with the help of x-ray phase-contrast tomography. To this end, specific staining procedures should be considered, as for example the immunogold labeling already used in x-ray diffraction 32 and soft x-ray microscopy, 33 staining of lipids with osmium, 34, 35 a standard protocol in transmission electron microscopy, as well as adaptations of protocols developed for bulk staining of FIB-SEM brain imaging.…”

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confidence: 95%

Phase-contrast tomography of neuronal tissues: from laboratory- to high resolution synchrotron CT

et al. 2016

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Assessing the three-dimensional architecture of neuronal tissues with sub-cellular resolution presents a significant analytical challenge. Overcoming the limitations associated with serial slicing, phase-contrast x-ray tomography has the potential to contribute to this goal. Even compact laboratory CT at an optimized liquid-metal jet microfocus source combined with suitable phase-retrieval algorithms and preparation protocols can yield renderings with single cell sensitivity in millimeter sized brain areas of mouse. Here, we show the capabilities of the setup by imaging a Golgi-Cox impregnated mouse brain. Towards higher resolution we extend these studies at our recently upgraded waveguide-based cone-beam holo-tomography instrument GINIX at DESY. This setup allows high resolution recordings with adjustable field of view and resolution, down to the voxel sizes in the range of a few ten nanometers. The recent results make us confident that important issues of neuronal connectivity can be addressed by these methods, and that 3D (virtual) histology with nanoscale resolution will become an attractive modality for neuroscience research.

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“…As propagation-based imaging is sensitive to relative changes in electron density, a medium with significantly smaller density with respect to the tissue of interest can lead to superior contrast in the reconstructed images [41,69,84,161].…”

Section: Embedding Mediamentioning

confidence: 99%

3d virtual histology of neuronal tissue by propagation-based x-ray phase-contrast tomography

Töpperwien¹

2018

Göttingen Series in X-Ray Physics

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Tomographic brain imaging with nucleolar detail and automatic cell counting

Cited by 63 publications

References 69 publications

Visualization and Segmentation of Cells in Unstained Paraffin-Embedded Cerebral Tissue

Visualization and Segmentation of Cells in Unstained Paraffin-Embedded Cerebral Tissue

Phase-contrast tomography of neuronal tissues: from laboratory- to high resolution synchrotron CT

3d virtual histology of neuronal tissue by propagation-based x-ray phase-contrast tomography

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