scholarly journals Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor

Sensors ◽  
2020 ◽  
Vol 20 (6) ◽  
pp. 1712 ◽  
Author(s):  
De-Ming Yang ◽  
Robeth Viktoria Manurung ◽  
Yu-Syuan Lin ◽  
Tai-Yu Chiu ◽  
Wei-Qun Lai ◽  
...  
Keyword(s):  
Heavy Metal ◽  
High Resolution ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Lead Level ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Blood Lead ◽  
Confocal Laser

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called “safe value” of the BLL seems to unreliably represent the secure threshold for children. In general, lead’s underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood–brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

scholarly journals Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

2003 ◽  
Vol 31 (5) ◽  
pp. 1020-1027 ◽  
Author(s):  
D.S. Lidke ◽  
P. Nagy ◽  
B.G. Barisas ◽  
R. Heintzmann ◽  
J.N. Post ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Laser Scanning ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

2010 ◽  
Vol 298 (5) ◽  
pp. L626-L636 ◽  
Author(s):  
Heike Schlenz ◽  
Wolfgang Kummer ◽  
Gitte Jositsch ◽  
Jürgen Wess ◽  
Gabriela Krasteva
Keyword(s):  
Smooth Muscle ◽  
Signaling Pathways ◽  
Laser Scanning ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Caveolin 1

Cholinergic bronchoconstriction is mediated by M2 and M3 muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R−/− and M3R−/− mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

scholarly journals Clinical Applications of In Vivo and Ex Vivo Confocal Microscopy

2021 ◽  
Vol 11 (5) ◽  
pp. 1979
Author(s):  
Stefania Guida ◽  
Federica Arginelli ◽  
Francesca Farnetani ◽  
Silvana Ciardo ◽  
Laura Bertoni ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Real Life ◽  
Laser Scanning Microscopy ◽  
Pathological Examination ◽  
Confocal Laser ◽  
Fluorescence Confocal Microscopy ◽  
Skin Cancers

Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis of skin cancers for both in vivo and ex vivo CLSM. In vivo CLSM involves reflectance mode, which is based on refractive index of cell structures serving as endogenous chromophores, reaching a depth of exploration of 200 μm. It has been proven to increase the diagnostic accuracy of skin cancers, both melanoma and non-melanoma. While histopathologic examination is the gold standard for diagnosis, in vivo CLSM alone and in addition to dermoscopy, contributes to the reduction of the number of excised lesions to exclude a melanoma, and to improve margin recognition in lentigo maligna, enabling tissue sparing for excisions. Ex vivo CLSM can be performed in reflectance and fluorescent mode. Fluorescence confocal microscopy is applied for “real-time” pathological examination of freshly excised specimens for diagnostic purposes and for the evaluation of margin clearance after excision in Mohs surgery. Further prospective interventional studies using CLSM might contribute to increase the knowledge about its application, reproducing real-life settings.

scholarly journals Design and Engineering of “Green” Nanoemulsions for Enhanced Topical Delivery of Bakuchiol Achieved in a Sustainable Manner: A Novel Eco-Friendly Approach to Bioretinol

2021 ◽  
Vol 22 (18) ◽  
pp. 10091
Author(s):  
Agnieszka Lewińska ◽  
Marta Domżał-Kędzia ◽  
Ewa Maciejczyk ◽  
Marcin Łukaszewicz ◽  
Urszula Bazylińska
Keyword(s):  
Human Skin ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Rapeseed Meal ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Biological Investigation ◽  
Vis Spectroscopy

In the present work, we establish novel “environmentally-friendly” oil-in-water nanoemulsions to enhance the transdermal delivery of bakuchiol, the so-called “bioretinol” obtained from powdered Psoralea corylifolia seeds via a sustainable process, i.e., using a supercritical fluid extraction approach with pure carbon dioxide (SC-CO2). According to Green Chemistry principles, five novel formulations were stabilized by “green” hybrid ionic surfactants such as coco-betaine—surfactin molecules obtained from coconut and fermented rapeseed meal. Preliminary optimization studies involving three dispersion stability tests, i.e., centrifugation, heating, and cooling cycles, indicated the most promising candidates for further physicochemical analysis. Finally, nanoemulsion colloidal characterization provided by scattering (dynamic and electrophoretic light scattering as well as backscattering), microscopic (transmission electron and confocal laser scanning microscopy), and spectroscopic (UV–Vis spectroscopy) methods revealed the most stable nanocarrier for transdermal biological investigation. In vitro, topical experiments provided on human skin cell line HaCaT keratinocytes and normal dermal NHDF fibroblasts indicated high cell viability upon treatment of the tested formulation with a final 0.02–0.2 mg/mL bakuchiol concentration. This excellent biocompatibility was confirmed by ex vivo and in vivo tests on animal and human skin tissue. The improved permeability and antiaging potential of the bakuchiol-encapsulated rich extract were observed, indicating that the obtained ecological nanoemulsions are competitive with commercial retinol formulations.

scholarly journals Meibomian Glands or Not? Identification of In Vivo and Ex Vivo Confocal Microscopy Features and Histological Correlates in the Eyelid Margin

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yu-jing Wang ◽  
Min Ke
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Meibomian Gland ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Eyelid Margin ◽  
Healthy Humans ◽  
Skin Appendages

Purpose. In vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. For the diagnostics of pathological eyelid margin lesions, the knowledge of the normal eyelid margin is essential. Methods. We examined 18 eyelid margins of healthy humans using the in vivo CLSM device and 10 samples of healthy eyelid margins from donor sites with ex vivo CLSM and compared the findings to the corresponding histological sections of donor sites. Cross-section images of different depths and depths of different skin appendages were measured. Results. The depth observed by in vivo CLSM is less than 150 μm into the eyelid. Images of the epidermis and superficial dermis skin, appendages including hair follicle, and sebaceous catheters can be captured associated with histopathology and ex vivo confocal microscopy. In correlation with histopathology, we identified different layers of the eyelid margin, different layers of the epidermis, and skin appendages by ex vivo confocal microscopy. Conclusions. The study offers an overview of the in vivo confocal microscopy human eyelid margin characteristics in comparison to the standard histological examination and confirms that in vivo CLSM could not observe the meibomian gland acini structure.

scholarly journals Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability

2018 ◽  
Vol 19 (9) ◽  
pp. 2722 ◽  
Author(s):  
Angela Fabiano ◽  
Elisa Brilli ◽  
Letizia Mattii ◽  
Lara Testai ◽  
Stefania Moscato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Laser Scanning ◽  
Time Course ◽  
Ex Vivo ◽  
Iron Bioavailability ◽  
Laser Scanning Microscopy ◽  
Raw Material ◽  
Confocal Laser ◽  
Ussing Chambers

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

scholarly journals Touching The Human Neuron: User-Centric Augmented Reality Viewing and Interaction of in-vivo Cellular Confocal Laser Scanning Microscopy (CLSM) Utilizing High Resolution zStack Data Sets

2017 ◽  
Vol 41 (1) ◽  
Author(s):  
Scott Riddle ◽  
Daniel Wasser ◽  
Michael McCarthy
Keyword(s):  
High Resolution ◽  
Augmented Reality ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Data Sets ◽  
Time Interaction ◽  
User Centric

This is the first report of a prototype that allows for real-time interaction with high-resolution cellular modules using GLASS© technology. The prototype was developed* using zStack data sets which allow for real-time interaction of low polygon and direct surface models exported from primary source imaging. This paper also discusses potential educational and clinical applications of a wearable, interactive, user-centric, augmented reality visualization of cellular structures.Full article title: Touching The Human Neuron: User-Centric Augmented Reality Viewing and Interaction of in-vivo Cellular Confocal Laser Scanning Microscopy (CLSM) Utilizing High Resolution zStack Data Sets for Applications in Medical Education and Clinical Medicine Using GLASS and Motion Tracking Technology

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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