Primary cultured neuronal networks and type 2 diabetes model mouse fatty liver tissues in aqueous liquid observed by atmospheric SEM (ASEM): Staining preferences of metal solutions
“…4d–f). From their shape and distribution, these (arrowheads) could be oil droplets, that are reported to be present in COS7 cells 21 and fat liver tissue 31 .Figure 4Structures surrounding mineralization in osteoblast primary culture visualized by metal staining. ( a ) Mineralization imaged without staining using ASEM.…”
The malformation and disordered remodeling of bones induce various diseases, including osteoporosis. We have developed atmospheric SEM (ASEM) to directly observe aldehyde-fixed bone tissue immersed in radical scavenger buffer without thin sectioning. The short procedure realized the observation of bone mineralization surrounded by many cells and matrices in natural aqueous buffer, decreasing the risk of changes. In osteoblast primary cultures, mineralization was visible without staining. Correlative energy dispersive X-ray spectrometry indicated the formation of calcium phosphate mineral. Fixed bone was sectioned, and the section surface was inspected by ASEM. Mineralized trabeculae of talus spongy bone were directly visible. Associated large and small cells were revealed by phosphotungstic acid staining, suggesting remodeling by bone-absorbing osteoclasts and bone-rebuilding osteoblasts. In tibia, cortical bone layer including dense grains, was bordered by many cells with protrusions. Tissue immuno-EM performed in solution for the first time and anti-cathepsin-K antibody, successfully identified osteoclasts in femur spongy bone. A microfluidics chamber fabricated on the silicon nitride film window of an ASEM dish allowed mineralization to be monitored
in vitro
; calcium phosphate crystals as small as 50 nm were imaged. ASEM is expected to be widely applied to study bio-mineralization and bone-remodeling, and to help diagnose bone-related diseases.
“…4d–f). From their shape and distribution, these (arrowheads) could be oil droplets, that are reported to be present in COS7 cells 21 and fat liver tissue 31 .Figure 4Structures surrounding mineralization in osteoblast primary culture visualized by metal staining. ( a ) Mineralization imaged without staining using ASEM.…”
The malformation and disordered remodeling of bones induce various diseases, including osteoporosis. We have developed atmospheric SEM (ASEM) to directly observe aldehyde-fixed bone tissue immersed in radical scavenger buffer without thin sectioning. The short procedure realized the observation of bone mineralization surrounded by many cells and matrices in natural aqueous buffer, decreasing the risk of changes. In osteoblast primary cultures, mineralization was visible without staining. Correlative energy dispersive X-ray spectrometry indicated the formation of calcium phosphate mineral. Fixed bone was sectioned, and the section surface was inspected by ASEM. Mineralized trabeculae of talus spongy bone were directly visible. Associated large and small cells were revealed by phosphotungstic acid staining, suggesting remodeling by bone-absorbing osteoclasts and bone-rebuilding osteoblasts. In tibia, cortical bone layer including dense grains, was bordered by many cells with protrusions. Tissue immuno-EM performed in solution for the first time and anti-cathepsin-K antibody, successfully identified osteoclasts in femur spongy bone. A microfluidics chamber fabricated on the silicon nitride film window of an ASEM dish allowed mineralization to be monitored
in vitro
; calcium phosphate crystals as small as 50 nm were imaged. ASEM is expected to be widely applied to study bio-mineralization and bone-remodeling, and to help diagnose bone-related diseases.
“…[21][22][23] Environmental pollutants, such as metals, can cause toxicological effects which may result in central nervous system pathology. [1][2][3][4][5][6] Studies have shown that heavy metals produce ROS which causes DNA damage, lipid peroxidation and depletion of protein sulfhydryls. 24 Natural compounds, such as plant polyphenols, have been shown to have potential protective effects against neuronal system diseases.…”
Objectives To investigate the neuroprotective effects of six natural compounds (caffeine, gallic acid, resveratrol, epigallocatechin gallate [EGCG], L-ascorbic acid and alpha tocopherol [Vitamin E] on heavy metal-induced cell damage in rat PC12 cells. Methods In this in vitro experiment, rat PC12 cells were exposed to four heavy metals (CdCl2, HgCl2, CoCl2 and PbCl2) at different concentrations and cell apoptosis, necrosis and oxidative stress were assessed with and without the addition of the six natural compounds. Results The metals decreased cell viability but the natural compounds attenuated their effects on apoptosis, necrosis and reactive oxygen species (ROS) levels. Mitochondrial protein changes were involved in the regulation. Conclusion Overall, the natural compounds did provide protection against the metal-induced PC12 cell damage. These data suggest that natural compounds may have therapeutic potential against metal-induced neurodegenerative disease.
“…Examples include the microtome-integrated light microscope for TEM (Lemercier et al, 2017), microtome-integrated confocal and scanning probe (e.g. atomic force) microscope (Mochalov et al, 2017), cryofluorescence and cryo-TEM (Schorb et al, 2017), cryo-fluorescence and cryo-SEM tomography (Gorelick et al, 2019), and fluorescence imaging and environmental SEM of liquid-state cells (Peckys and de Jonge, 2015;Sato et al, 2019). Some of these integrated microscopes are commercially available and might achieve the goal of minimizing or eliminating the need for spatial registration.…”
Section: Methods For Spatial Registration In Correlative Microscopymentioning
confidence: 99%
“…In a broad sense, correlative microscopy refers to any combination of microscopy techniques (Karreman et al, 2016), microscopic X-ray computed tomography of excised tissue (Karreman et al, 2017), transmission electron microscopy (TEM) (Fuest et al, 2018), atmospheric or environmental scanning electron microscopy (SEM) (Peckys and de Jonge, 2015;Sato et al, 2019), cathodoluminescence of nanoparticles in SEM (Glenn et al, 2012), cryo-electron tomography (Tao et al, 2018), array tomography of electron microscopy (Burel et al, 2018;Collman et al, 2015;Micheva and Smith, 2007), X-ray holography, X-ray scanning diffraction (Bernhardt et al, 2018), X-ray fluorescence imaging and mass spectrometry imaging (Decelle et al, 2020). Some authors emphasized this integration of information from multiple imaging methods, by proposing a "morphomics" notation (Lucocq et al, 2015).…”
ABSTRACTBackgroundCorrelation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems.New methodHere we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets.ResultsWe imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level.Comparison with existing methodsThis method aligned images with minimal change or investment in the experimental systems.ConclusionsThis method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.
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