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Nanomechanical Analysis of Cells from Cancer Patients
Published in Lajos P. Balogh, Nano-Enabled Medical Applications, 2020
Sarah E. Cross, Yu-Sheng Jin, Jianyu Rao, James K. Gimzewski
All studies were conducted using a Nanoscope IV Bioscope (Veeco Digital Instruments) with a combined inverted optical microscope (Nikon eclipse TE200). This combination permitted lateral positioning of the AFM tip over the nuclear region of the cell with micrometre precision (Fig. 18.1a, inset). The scan size for all measurements was set to 0 nm to maintain a constant position over the cell, and using the AFM software the tip was brought into contact with the central region of the cell [26]. Mechanical measurements were collected at 37◦C using sharpened silicon nitride cantilevers with experimentally determined [27] spring constants of 0.02 N m−1 and a tip radius of <20 nm.
Identifying Nanotoxicity at the Cellular Level Using Electron Microscopy
Published in Suresh C. Pillai, Yvonne Lang, Toxicity of Nanomaterials, 2019
Kerry Thompson, Alanna Stanley, Emma McDermott, Alexander Black, Peter Dockery
One must first consider the concept of resolution to truly understand the power of the electron microscope. Resolution can be defined as the ability of an optical microscope to distinguish detail and is related to the numerical aperture, or light gathering ability of the lens, and the wavelength of light used (Weakley, 1972). Unaided, the human eye can distinguish clearly two points that are approximately 0.2 millimetres (mm) apart from one another. If the points are closer to one another, they appear as a single blurry dot. Light microscopy affords a resolution of approximately 0.2 micrometres (µm), whilst electron microscopy allows objects to be resolved down to the nanometre (nm) scale (approximately 1 nm) or, in a biological context roughly the size of some cellular organelles (Perkins et al., 2009). It is predominantly for this reason that electron microscopy is one of the most useful techniques to study nanotoxicity at the cellular level. The high-magnification, high-resolution images allow the observer to analyse the intricacies of tissue, cellular, and subcellular environments in detail.
Single-Molecule Kinetics
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
Single-molecule measurements are conducted using small volumes and usually involve some form of optical microscopy. The resolution of an optical microscope is limited by the wavelength of light, which is conventionally defined by the Abbe limit of λ/2NA, where NA is the limiting numerical aperture (NA) (Section 7.5.1). For a high numerical lens and visible light, this limit is of the order of 200 to 300 nm. That is not to say objects smaller than 200 nm cannot be detected, but that two objects separated by this distance cannot be resolved without using special tricks [283,655,656]. In most in vitro single-molecule kinetic measurements, fluorescently labeled molecules are diluted to the point where they are separated, on average, by at least several micrometers, so that each acts as a point source that can be imaged. Each molecule appears as a disk of light with a Gaussian intensity profile of half-width around 200 nm (Figure 9.2b).
Thoughts on digital microscopes
Published in British Journal of Neurosurgery, 2021
I will look at features of these systems in turn starting with their basic resolution. This is a digital imaging application where resolution is particularly important and current products use ‘4K’ that is the standard high resolution available from most digital cine cameras and television screens. It means about 4000 pixels from one side of the screen to the other equating to an image resolution of about 8–9 MP. This is not however the best available. Apple’s 32” retinal screen has over 20MP and ‘8K’ systems with image resolution of 33MP are manufactured, though these are more recent not yet available on clinical microscopes. 8K would be an advantage though because resolution is noticeably limiting compared to an optical microscope with those we have tried and it cannot be long in coming - something to consider when deciding whether to purchase on now or ‘next time’.
Chronic exposure to ethanol alters the expression of miR-155, miR-122 and miR-217 in alcoholic liver disease in an adult zebrafish model
Published in Biomarkers, 2021
Amanda Pasqualotto, Raquel Ayres, Larisse Longo, Diego Del Duca Lima, Diogo Losch de Oliveira, Mário Reis Alvares-da-Silva, Themis Reverbel da Silveira, Carolina Uribe-Cruz
Hepatic tissue samples were collected and fixed in Tissue Tek® O.T.C. Compound (Sakura, California, USA), cryosectioned (Leica Biosvystems, Wetzlar, Germany) (3–5 μm) and stained with H&E and Oil Red (5 mg/mL in propylene glycol) to evaluate the presence of lipids. All sections were examined under an optical microscope. Microphotographs were used to record the samples (n = 5). The sections stained with Oil Red were quantified using the Image J software for the amount of lipids (n = 5). Lipid deposits in the liver were quantified using a modified Gómez-Lechon protocol (Gómez-Lechón et al. 2007). Hepatic tissue had been previously homogenized with PBS (20 mg tissue/ml) and incubated with 1 μl of Nile Red solution (1 mg/ml in acetone) (Sigma-Aldrich, WGK Germany) for 15 min at 37 °C. Fluorescence was measured at 488 nm of excitation and 550 nm of emission (SpectraMax M3). The results were normalized for total proteins present in the homogenate (n = 8), which were obtained by the Bradford method (Bradford 1976).
CAT25 defines microsatellite instability in colorectal cancer by high-resolution melting PCR
Published in British Journal of Biomedical Science, 2020
AG Sánchez, I Juaneda, H Eynard, AL Basquiera, E Palazzo, P Calafat, V Palla, PA Romagnoli, T Alvarellos
Immunohistochemistry was performed on 9 biopsy samples classified as MSI-High. Samples were fixed in 10% pH 7 buffered formaldehyde before being embedded in paraffin, and then 5 microns sections were made from selected blocks. The primary antibodies used were: MSH2 (CELL MARQUE, clone 6219–1129); MSH6 (CELL MARQUE, clone 44); MLH1 (Ventana, clone M1) and PMS2 (Ventana, clone ERP3947). The technique was carried out on Benchmark GX – VENTANA platforms (Roche, Mannheim, Germany) and revealed with Optiview DAB Detection Kit (Roche, Mannheim, Germany) in all cases. For the markings of MLH1 and PMS2, Optiview Amplification Kit (Roche, Mannheim, Germany) was also used according to protocols suggested by the manufacturer. Subsequently, the slides with the samples were dehydrated and mounted for visualization and evaluation under an optical microscope. A sample was considered negative when no tumour cell nucleus with positive staining for the protein under study (MSH2, MSH6, MLH1 or PMS2) was observed. Positive staining from the proteins studied observed in the cell nuclei of infiltrating lymphocytes, stromal cells and normal mucosa was considered as positive internal controls. “CAT25, BAT25 or BAT26 instability” was considered when a different amplicon size for each marker by capillary electrophoresis analysis was found comparing tumour DNA vs. peripheral blood DNA of the same patient. In HRM-PCR analysis, CAT25, BAT25 and BAT26 instability was considered to be present when HRM curves (Normalized and Different plot) were discordant between the samples previously described for capillary electrophoresis analysis.