Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

Shomorony, Andre; Pfeifer, Charlotte R.; Aronova, Maria A.; Zhang, G.; Cai, Tao; Xu, Hongtao; Notkins, Abner Louis; Leapman, Richard D.

doi:10.1111/jmi.12276

Cited by 22 publications

(20 citation statements)

References 41 publications

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“…The total number of DCVs per β-cell was estimated by utilizing a combination of 3D volume measurements and two-dimensional vesicle density measurements 60 . The cellular, nuclear and mitochondrial volumes per β-cell were measured using Amira software, after which the nuclear and mitochondrial volumes were subtracted from the cell volume to determine the volume available to DCVs.…”

Section: Methodsmentioning

confidence: 99%

β-arrestin-2 is an essential regulator of pancreatic β-cell function under physiological and pathophysiological conditions

et al. 2017

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β-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the β-arrestin-2 gene, barr2, in β-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in β-arrestin-2 (barr2) deficient β-cells. In human β-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in β-cells. Importantly, overexpression of barr2 in β-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of β-cell function, which may serve as a new target to improve β-cell function.

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Section: Methodsmentioning

confidence: 99%

β-arrestin-2 is an essential regulator of pancreatic β-cell function under physiological and pathophysiological conditions

et al. 2017

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“…Platelets were stained as previously described [19–22]. The purified platelet pellet was fixed using 0.1 M cacodylate buffer containing 2.5% glutaraldehyde and 2 mM calcium chloride for 1 h in ice.…”

Section: Methodsmentioning

confidence: 99%

“…Each sample was then trimmed again to expose the opposite side, and sputter-coated with a 40 nm gold layer. The trimmed block was imaged at an accelerating voltage of 1.8 kV in a Zeiss SIGMA-VP (variable pressure) scanning electron microscope (SEM) equipped with a Gatan 3View serial block face (SBF) imaging system [21–22, 24]. The SEM was operated in the high vacuum mode with a 30 μm condenser aperture, and images containing 2000 × 2000 pixels were acquired using the Gatan DigitalMicrograph program with a pixel size of 5.5 nm in the x–y plane.…”

Section: Methodsmentioning

confidence: 99%

Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures

et al. 2016

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Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown. Here we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, wide field fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte. In conclusion, we suggest these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research.

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“…A new generation of imaging techniques in the electron microscope provide biologists with threedimensional nanoscale ultrastructure that helps to elucidate mechanisms for a wide range of important cellular processes. Two such techniques are serial block-face electron microscopy (SBEM) (Denk & Horstmann, 2004;Briggman et al, 2011;Helmstaedter et al, 2013;Shomorony et al, 2015;Porovskaya et al, 2018, Porovskaya et al, 2019, and focused ion beam scanning electron microscopy (FIB-SEM) (Hekking et al, 2009;Drobne, 2013;Narayan & Subramaniam, 2015;Glancy et al, 2015). In both these approaches, embedded blocks of cells or tissues, are imaged in a scanning electron microscope using the backscattered electron signal generated by scattering of electrons from heavy-atom stain incorporated into the biological specimens after fixation and prior to embedding.…”

Section: Introductionmentioning

confidence: 99%

Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

Fera

Leapman

2019

Preprint

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Stain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.

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Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

Cited by 22 publications

References 41 publications

β-arrestin-2 is an essential regulator of pancreatic β-cell function under physiological and pathophysiological conditions

β-arrestin-2 is an essential regulator of pancreatic β-cell function under physiological and pathophysiological conditions

Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures

Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

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