Confocal Microscopy
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2021 ◽  
Maximiliano Anzibar Fialho ◽  
Lucía Vázquez ◽  
Mariana Martínez ◽  
Miguel Calero ◽  
Jerome Baranger ◽  

Abstract The hippocampus plays an important role in learning and memory, requiring high-neuronal oxygenation. Understanding the relationship between blood flow and vascular structure – and how it changes with ageing – is physiologically and anatomically relevant. Ultrafast Doppler (µDoppler) and Scanning Laser Confocal Microscopy (SLCM) are powerful imaging modalities that can measure in-vivo Cerebral Blood Volume (CBV) and ex-vivo vascular structure, respectively. Here, we apply both imaging modalities to a cross-sectional and longitudinal study of hippocampi vasculature in wild-type mice brains. We introduce a segmentation of CBV distribution obtained from µDoppler and show that this mice-independent and mesoscopic measurement is correlated with the number of vessels and Vessel Volume Fraction (VVF) distributions obtained from SLCM – e.g., high CBV relates to fewer number of vessels but with large VVF. Moreover, we find significant changes in CBV distribution and vasculature due to ageing (5 vs. 21 month-old mice), highlighting the sensitivity of our approach. Overall, we are able to associate CBV with vascular structure – and track its longitudinal changes – at the artery-vein, venules, arteriole, and capillary levels. We believe that this correlative approach can be a powerful tool for studying other acute (e.g., brain injuries), progressive (e.g., neurodegeneration) or induced pathological changes.

2021 ◽  
Vol 11 (1) ◽  
Mirae Lee ◽  
Jiwon Woo ◽  
Doh-Hee Kim ◽  
Yu-Mi Yang ◽  
Eunice Yoojin Lee ◽  

AbstractThree-dimensional visualization of cellular and subcellular-structures in histological-tissues is essential for understanding the complexities of biological-phenomena, especially with regards structural and spatial relationships and pathologlical-diagnosis. Recent advancements in tissue-clearing technology, such as Magnified Analysis of Proteome (MAP), have significantly improved our ability to study biological-structures in three-dimensional space; however, their wide applicability to a variety of tissues is limited by long incubation-times and a need for advanced imaging-systems that are not readily available in most-laboratories. Here, we present optimized MAP-based method for paper-thin samples, Paper-MAP, which allow for rapid clearing and subsequent imaging of three-dimensional sections derived from various tissues using conventional confocal-microscopy. Paper-MAP successfully clear tissues within 1-day, compared to the original-MAP, without significant differences in achieved optical-transparency. As a proof-of-concept, we investigated the vasculature and neuronal-networks of a variety of human and rodent tissues processed via Paper-MAP, in both healthy and diseased contexts, including Alzheimer’s disease and glioma.

2021 ◽  
Vol 8 ◽  
Hui Wang ◽  
Zhenyu Zhong ◽  
Xiuli Wang ◽  
Liyun Zheng ◽  
Yifan Wang ◽  

Background: Amyloidosis cutis dyschromica (ACD) is a rare type of primary localized cutaneous amyloidosis. Non-invasive techniques can provide important clues for early diagnosis.Objectives: To highlight the characteristic imaging changes of ACD under dermoscopy and reflectance confocal microscopy (RCM), investigate gene mutations in a Chinese Han pedigree of ACD, and analyze the genotype–phenotype correlation.Methods: Dermoscopy and RCM examinations were completed together for the pedigree, and the imaging characteristics were described. The diagnosis of ACD was confirmed by pathological examination. Sequencing was performed followed by bioinformatics and genotype–phenotype correlation. ACD-related articles published on PubMed between January 1970 and March 2021 were reviewed and summarized.Results: In ACD, dermoscopy showed patchy white hypopigmentation and brownish spots, stripes, or hyperpigmented blotches and patches. RCM showed a highly refractive substance with clumpy, dotted, and linear structures inside the papillary dermis. Sequencing identified glycoprotein non-metastatic melanoma protein B (GPNMB) missense mutations [c.393T>G (p.Y131X; NM_001005340.2)] and a frameshift deletion mutation [c.719_720delTG (p.V240fs; NM_001005340.2)]. The ANNOtate VARiation (ANNOVAR) software predicted that c.393T>G is a pathogenic mutation. The literature review found 14 mutations, namely, 5 (35.7%) frameshift mutations, 4 (28.6%) non-sense mutations, 4 (28.6%) missense mutations, and 1 (7.1%) splice site mutation. Blisters and epidermolysis were observed in several cases, but there was no significant association between clinical manifestations and mutations in ACD.Conclusions: This study was the first to combine dermoscopy and RCM to describe ACD. Two GPNMB gene mutations were reported in a Chinese ACD pedigree. The genotype–phenotype correlation was analyzed for the first time; however, there was no significant correlation.

Agustina Belén Fernández Casafuz ◽  
María Cecilia De Rossi ◽  
Luciana Bruno

Abstract Uncovering the link between mitochondrial morphology, dynamics, positioning and function is challenging. Mitochondria are very flexible organelles that are subject to tension and compression within cells. Recent findings highlighted the importance of these mechanical aspects in the regulation of mitochondria dynamics, arising the question on which are the processes and mechanisms involved in their shape remodeling. In this work we explored in detail the morphological changes and spatio- temporal fluctuations of these organelles in living Xenopus laevis melanophores, a well- characterized cellular model. We developed an automatic method for the classification of mitochondria shapes based on the analysis of the curvature of the contour shape from confocal microscopy images. A persistence length of 2.1 μm was measured, quantifying, for the first time, the bending plasticity of mitochondria in their cellular environment. The shape evolution at the single organelle level was followed during a few minutes revealing that mitochondria can bend and unbend in the seconds timescale. Furthermore, the inspection of confocal movies simultaneously registering fluorescent mitochondria and microtubules suggests that the cytoskeleton network architecture and dynamics play a significant role in mitochondria shape remodeling and fluctuations. For instance changes from sinuous to elongated organelles related to transitions from confined behavior to fast directed motion along microtubule tracks were observed.

Ana Blasco ◽  
María José Coronado ◽  
Paula Vela ◽  
Paloma Martin ◽  
Jorge Solano ◽  

Aims: The mechanisms of coronary thrombosis can influence prognosis after STEMI and allow for different treatment groups to be identified; an association between neutrophil extracellular traps (NETs) and unfavorable clinical outcomes has been suggested. Our aim was to determine the role played by NETs in coronary thrombosis and their influence on prognosis. The role of other histological features in prognosis and the association between NETs and bacteria in the coronary thrombi were also explored. Methods and Results: We studied 406 patients with STEMI in which coronary thrombi were consecutively obtained by aspiration during angioplasty between 2012 and 2018. Analysis of NETs in paraffin-embedded thrombi was based on the colocalization of specific NET components by means of confocal microscopy. Immunohistochemistry stains were used to identify plaque fragments. Fluorescence in situ hybridization was used to detect bacteria. NETs were detected in 51% of the thrombi [NET density, median (IQR): 25% (17–38%)]. The median follow-up was 47 months (95% CI 43-51); 105 (26%) patients experienced major adverse cardiac events (MACE). A significant association was found between the presence of NETs in coronary aspirates and the occurrence of MACE in the first 30 days after infarction (HR 2.82; 95% CI 1.26–6.35, p=.012), mainly due to cardiac deaths and stent thrombosis. Conclusions: The presence of NETs in coronary thrombi was associated with a worse prognosis soon after STEMI. In some patients, NETs could be a treatment target and a feasible way to prevent reinfarction.

2021 ◽  
Vol 4 (1) ◽  
Katerina Kanevche ◽  
David J. Burr ◽  
Dennis J. Nürnberg ◽  
Pascal K. Hass ◽  
Andreas Elsaesser ◽  

AbstractAlthough techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically specific markers. To eliminate this flaw, we have developed a method of examining cellular cross sections using the imaging power of scattering-type scanning near-field optical microscopy and Fourier-transform infrared spectroscopy at a spatial resolution far beyond the diffraction limit. Herewith, nanoscale surface and volumetric chemical imaging is performed using the intrinsic contrast generated by the characteristic absorption of mid-infrared radiation by the covalent bonds. We employ infrared nanoscopy to study the subcellular structures of eukaryotic (Chlamydomonas reinhardtii) and prokaryotic (Escherichia coli) species, revealing chemically distinct regions within each cell such as the microtubular structure of the flagellum. Serial 100 nm-thick cellular cross-sections were compiled into a tomogram yielding a three-dimensional infrared image of subcellular structure distribution at 20 nm resolution. The presented methodology is able to image biological samples complementing current fluorescence nanoscopy but at less interference due to the low energy of infrared radiation and the absence of labeling.

2021 ◽  
Vol 9 (12) ◽  
pp. 2475
Guillaume Pillot ◽  
Oulfat Amin Ali ◽  
Sylvain Davidson ◽  
Laetitia Shintu ◽  
Yannick Combet-Blanc ◽  

Recent studies have shown the presence of an abiotic electrical current across the walls of deep-sea hydrothermal chimneys, allowing the growth of electroautotrophic microbial communities. To understand the role of the different phylogenetic groups and metabolisms involved, this study focused on electrotrophic enrichment with nitrate as electron acceptor. The biofilm density, community composition, production of organic compounds, and electrical consumption were monitored by FISH confocal microscopy, qPCR, metabarcoding, NMR, and potentiostat measurements. A statistical analysis by PCA showed the correlation between the different parameters (qPCR, organic compounds, and electron acceptors) in three distinct temporal phases. In our conditions, the Archaeoglobales have been shown to play a key role in the development of the community as the first colonizers on the cathode and the first producers of organic compounds, which are then used as an organic source by heterotrophs. Finally, through subcultures of the community, we showed the development of a greater biodiversity over time. This observed phenomenon could explain the biodiversity development in hydrothermal contexts, where energy sources are transient and unstable.

2021 ◽  
Adam Feliks Junka ◽  
Grzegorz Krasowski ◽  
Pawel Migdal ◽  
Marta Woroszylo ◽  
Karol Fijalkowski ◽  

The in vitro efficacy of locally applied antiseptic molecules against staphylococcal biofilm is frequently assessed by a set of standard quantitative and semi-quantitative methods. The development of software for parametric image processing allowed to obtain parametric data also from microscopic images of biofilm dyed with a variety of dyes, especially with propidium iodine and SYTO-9, differentiating dead from live cells. In this work, using confocal/epifluorescent microscopy, we analyzed such major properties of staphylococcal biofilm in vitro as its thickness, cellular density and share of Live/Dead cells within its individual parts. We also scrutinized the impact of sample preparation and antiseptic introduction on the outcome obtained. As a result of our analyses we developed a revelatory method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, in which the microscopic images are processed with the use of ABE formula (Antiseptics Biofilm Eradication) which implements all the data and phenomena detected and revealed within the course of this study. We tested ABE with regard to polyhexanide, povidone-iodine and hypochlorous antiseptics and found a high correlation between this parameter and the results obtained by means of traditional techniques. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool providing reliable, parametric data with this regard.

2021 ◽  
Vol 9 ◽  
Liliya Kotliarevski ◽  
Karthik Ananth Mani ◽  
Reut Amar Feldbaum ◽  
Noga Yaakov ◽  
Eduard Belausov ◽  

This study presents an individual encapsulation of fungal conidia in an oil-in-water Pickering emulsion at a single-conidium encapsulation yield of 44%. The single-conidium encapsulation yield was characterized by analysis of confocal microscopy micrographs. Mineral oil-in-water emulsions stabilized by amine-functionalized titania dioxide (TiO2-NH2 or titania-NH2) particles were prepared. The structure and the stability of the emulsions were investigated at different compositions by confocal microscopy and a LUMiSizer® respectively. The most stable emulsions with a droplet size suitable for single-conidium encapsulation were further studied for their individual encapsulation capabilities. The yields of individual encapsulation in the emulsions; i.e., the number of conidia that were individually encapsulated out of the total number of conidia, were characterized by confocal microscopy assay. This rapid, easy to use approach to single-conidium encapsulation, which generates a significantly high yield with eco-friendly titania-based emulsions, only requires commonly used emulsification and agitation methods.

eLife ◽  
2021 ◽  
Vol 10 ◽  
Yelena Y Bernadskaya ◽  
Haicen Yue ◽  
Calina Copos ◽  
Lionel Christiaen ◽  
Alex Mogilner

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

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