Clearing Specimens for the Demonstration of Bone

Cumley, R. W.; Crow, Jeff; Griffen, A. B.

doi:10.3109/10520293909110285

Cited by 29 publications

(8 citation statements)

References 4 publications

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“…2e,g). The use of index-matching solutions to reduce scattering in tissue has a seventy year history (Cumley et al, 1939) and enjoys contemporary use in combination with confocal laser scanning microscopy (CLSM) (Dickie et al, 2006; Wagner et al, 2006). More recently, aggressive clearing techniques have been reported on whole mouse brain (Dodt et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels

Tsai¹,

Kaufhold²,

Blinder³

et al. 2009

J. Neurosci.

520

542

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It is well known that the density of neurons varies within the adult brain. In neocortex, this includes variations in neuronal density between different lamina as well as between different regions. Yet the concomitant variation of the microvessels is largely uncharted. Here we present automated histological, imaging, and analysis tools to simultaneously map the locations of all neuronal and non-neuronal nuclei and the centerlines and diameters of all blood vessels within thick slabs of neocortex from mice. Based on total inventory measurements of different cortical regions (~ 107 cells vectorized across brains), these methods revealed: (1) In three dimensions, the mean distance of the center of neuronal somata to the closest microvessel was 14 μm. (2) Volume samples within lamina of a given region show that the density of microvessels does not match the strong laminar variation in neuronal density. This holds for both agranular and granular cortex. (3) Volume samples in successive radii from the midline to the ventral-lateral edge, where each volume summed the number of cells and microvessels from the pia to the white matter, show a significant correlation between neuronal and microvessel densities. These data show that while neuronal and vascular densities do not track each other on the 100 μm scale of cortical lamina, they do track each other on the 1 – 10 mm scale of the cortical mantle. The absence of a disproportionate density of blood vessels in granular lamina is argued to be consistent with the initial locus of functional brain imaging signals.

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Section: Discussionmentioning

confidence: 99%

Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels

Tsai¹,

Kaufhold²,

Blinder³

et al. 2009

J. Neurosci.

520

542

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“…Due to decreased light scattering in clearing solutions (Tuchin, 2005), detailed imaging of fluorescent markers was performed in thick tissue slices under LSCM. Tissue clearing solutions have a long history of use in imaging underlying biological structures (Cumley et al ., 1939), and have recently experienced a resurgence as a useful tool to investigate thick, intact tissue specimens via microscopy (Zucker, 2006; Dodt et al ., 2007; Fu and Tang, 2010; Clendenon et al ., 2011; Hama et al ., 2011). Though imaging depth is improved greatly with use of a clearing solution, the ramping of laser intensity and sensitivity of the microscope detectors also aids in producing highly detailed z-projections for quantification and morphological interpretation.…”

Section: Discussionmentioning

confidence: 99%

Chronic intracortical microelectrode arrays induce non-uniform, depth-related tissue responses

Woolley¹,

Desai²,

Otto³

2013

J. Neural Eng.

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Objective Brain-implanted microelectrode arrays show promise as future clinical devices. However, biological responses to various designs, compositions, and locations of these implants have not been fully characterized, and may impact the long term functionality of these devices. In order to improve our understanding of the tissue conditions at the interface of chronic brain-implanted microdevices, we proposed utilizing advanced histology and microscopy techniques to image implanted devices and surrounding tissue intact within brain slices. We then proposed utilizing these methods to examine whether depth within the cerebral cortex affected tissue conditions around implants. Approach Histological data was collected from rodent brain slices containing intact, intracortical microdevices 4 weeks after implantation surgery. Thick tissue sections containing the chronic implants were processed with fluorescent antibody labels, and imaged in an optical clearing solution using laser confocal microscopy. Main Results Tissue surrounding microdevices exhibited two major depth-related phenomena: a non-uniform microglial coating along the device length and a dense mass of cells surrounding the implant in cerebral cortical layers I and II. Detailed views of the monocyte-derived immune cells improve our understanding of the close and complex association that immune cells have with chronic brain-implants, and illuminated a possible relationship between cortical depth and the intensity of a chronic monocyte response penetrating microdevices. The dense mass of cells contained Vimentin, a protein typically expressed highly in non-CNS cells, evidence that non-CNS cells likely descended down the face of the penetrating devices from the pial surface. Significance Image data of highly non-uniform and depth-dependent biological responses along a device provides novel insight into the complexity of the tissue response to penetrating brain-implanted microdevices. The presented work also demonstrates the value of in situ histological collection of brain-implants for studying the complex tissue changes that occur, and the utility of pairing thick-tissue histology with appropriate optical clearing solutions.

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“…Nevertheless, this method leads to samples shrinkage, browning, and also to the damage of the biological samples outer layers (2-3 cm), due to the formation of bubbles and cell death prompted by hydrogen peroxide (Alnuami, Zeedi, Qadri, & Ashraf, 2008;Cumley, Crow, & Griffen, 1939;Steinke & Wolff, 2001). Additionally, the Spalteholz method can also affect the fluorescence emission by protein dyes (e.g., GFP loses its fluorescence upon exposure to oxyradicals that are produced by the hydrogen peroxide; Alnuami et al, 2008).…”

Section: Spalteholz's Techniquementioning

confidence: 99%

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Costa

Silva

Moreira

et al. 2019

Biotech & Bioengineering

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Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a lightscattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI;(b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted. K E Y W O R D Simaging, light scattering, optical clearing, optical microscopy, spheroids.

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Clearing Specimens for the Demonstration of Bone

Cited by 29 publications

References 4 publications

Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels

Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels

Chronic intracortical microelectrode arrays induce non-uniform, depth-related tissue responses

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

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