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Role of Mitochondrial Injury During Oxidative Injury to Hepatocytes: Evidence of a Mitochondrial Permeability Transition by Laser Scanning Confocal Microscopy
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Anna-Liisa Nieminen, Roberto Imberti, Alice K. Saylor, Samuel A. Tesfai, Brian Herman, John J. Lemasters
To monitor the permeability of mitochondria inside intact cultured hepatocytes, the cytosol was loaded with calcein, a fluorophore whose fluorescence is not influenced by pH, Ca2+, or other environmental parameter that might be expected to change during cell injury. Images of the cells were then collected using laser scanning confocal microscopy. The advantage of confocal microscopy over conventional microscopy is that confocal microscopy creates thin optical slices of less than 1 μm in thickness. Theses slices exclude light from other planes of focus that would otherwise degrade image quality. In such thin optical slices, it is possible to distinguish mitochondrial and cytosolic cell volumes.30–32 In confocal images from calcein-loaded hepatocytes, cytosolic spaces were filled with diffuse fluorescence (Figure 7A). Individual mitochondria were dark round voids. Co-loading experiments with TMRM, a cationic fluorophore that accumulates into mitochondria in response to mitochondrial ΔΨ,33 confirmed that the holes in the calcein images were individual mitochondria (Figure 7A). Calcein has a molecular weight of 623 and should move through the permeability transition pore when it opens. During normal aerobic incubations, no redistribution of calcein fluorescence into mitochondria occurred, even after more than an hour. Thus, we conclude that the mitochondrial permeability transition pore remains fully closed inside living hepatocytes under normal conditions.
Bacteriocins as Anticancer Peptides: A Biophysical Approach
Published in Ananda M. Chakrabarty, Arsénio M. Fialho, Microbial Infections and Cancer Therapy, 2019
Filipa D. Oliveira, Miguel A.R.B. Castanho, Diana Gaspar
Different studies show the importance of microscopy techniques for detailing bacteriocins’ mechanism(s) of action. Chen et al. relied on confocal microscopy to investigate the distribution of KL15 in SW480 cells [96]. Confocal microscopy allows imaging fixed or living cells and tissues previously labeled with fluorescent probes, with an increased lateral and axial resolution when compared to epifluorescence microscopy [113]. In this study, the bacteriocin-derived peptide KL15 was labeled with iV-hydroxysuccinimide (NHS)-fluorescein, a green fluorescent dye, while the cell membrane was labeled with the red fluorescent dye di-8-ANEPPS and the blue fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) was applied to label the cell nucleus [96]. Evaluating the colocalization of the dyes it was possible to infer KL15 localization inside the cell. Confocal microscopy images indicated that KL15 enters the cells and causes significant changes in their morphology [96]. Nonetheless, red fluorescence was detected and found to be colocalized with DAPI, used for nuclei staining. This observation was in agreement with the results obtained from SEM, as it was demonstrated that KL15 may damage treated cells by cell membrane penetration and consequent intrusion into the cells [96].
Routine and Special Techniques in Toxicologic Pathology
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Daniel J. Patrick, Matthew L. Renninger, Peter C. Mann
Confocal microscopy, or confocal laser scanning microscopy, is a type of optical sectioning microscopy that provides high-resolution images. It shares many of the same principles of conventional wide-field fluorescent microscopy except that excitation and detection are both in focus. To achieve this, the excited light comes from a laser beam and is focused on one point in the specimen (the illumination spot, or Airy disk), the return emitted light from that spot is also focused, and a small pinhole over the detector screens out almost all of the undesirable emitted light outside the plane of focus. Use of these two focal points for illumination (excited light) and detection (emitted light) almost completely eliminates background fluorescence, which markedly increases contrast (Conchello and Lichtman 2005). Typically, to create an image, the illumination spot is moved in a raster fashion (like reading a book) over a thin focal plane section of the specimen, and the two-dimensional image is generated by adding all of the information together. Three-dimensional images can be generated by computationally combining the image data from a stack of two-dimensional images. Extremely fine detail of fluorescently labeled structures near or below the limit of resolution can be visualized, such as cytoskeletal microtubules, organelles, inorganic metallic ions, and receptors.
Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies
Published in OncoImmunology, 2022
Ángel Ramírez-Fernández, Óscar Aguilar-Sopeña, Laura Díez-Alonso, Alejandro Segura-Tudela, Carmen Domínguez-Alonso, Pedro Roda-Navarro, Luis Álvarez-Vallina, Belén Blanco
For 19-CAR and CD3 localization studies, J-NT-T, J-CAR-T19, or J-STAb-T19 cells (1 × 105) were co-cultured for 2 hours with CMAC-labeled NALM6 cells at a 2:1 E:T ratio in U-bottom 96-well plates. Co-cultures were then incubated on poly-L-lysine-coated coverslips at 37°C, 5% CO2 and fixed and permeabilized as described above. 19-CAR localization at the lysosomal compartment was assessed by staining with GAMIgG F(ab’)2-biotin (Jackson ImmunoResearch) followed by streptavidin-Alexa Fluor 594 (Life Technologies-Thermo Fisher Scientific, Carlsbad, CA, USA) and mouse anti-CD107a (IDB4 clone)-Alexa Fluor 647 (Biolegend, San Diego, CA, USA). CD3 localization was determined by staining with mouse anti-CD3ε (T3b clone) followed by GAM-Alexa-488. All samples were mounted with Mowiol (Sigma-Aldrich) as described above. Confocal sections were acquired using the SP-8 scanning laser confocal microscopy equipped as described. CMAC, Alexa 488, Alexa 594, and Alexa 647 were excited by 405, 488, 594, or 633 nm laser lines, respectively. Image acquisition was automatically optimized with the Leica software to get an image resolution of 58 nm/pixel. In the case of 19-CAR localization, Z-stacks through the cell were acquired every 0.8 μm. Colocalization was estimated by Pearson correlation coefficients obtained in complete stacks of cells (Figure 2b, c). CD3ε uptake by target cells shown in Figure 3e was estimated as the ratio of the signal of CD3ε in NALM6 cells and JK cells after subtracting the background. Analysis was implemented in ImageJ freeware.
Optimisation of ethosomal nanogel for topical nano-CUR and sulphoraphane delivery in effective skin cancer therapy
Published in Journal of Microencapsulation, 2020
Kriti Soni, Ali Mujtaba, Md. Habban Akhter, Ameeduzzafar Zafar, Kanchan Kohli
To corroborate the drug delivery from ethosome formulation into distinguished layer of skin rhodamine B, a fluorescent dye was used to put in place of drug to predict the distribution of drug using CLSM. Confocal microscopy is a powerful tool for generating high-resolution images and 3-D reconstructions of a specimen. Laser Confocal Microscope with Fluorescence Correlation Spectroscopy (FCS)-Olympus FluoView™ FV1000 with FLIP and FCS PicoQuant was used employed applying software Olympus fluoview ver.1.7a. The ethosome formulation and control labelled with Rhodamine B (0.02% w/v) were prepared and applied to skin which was mounted on donor compartment of Franz diffusion cell and experiment was carried out for 24 h. The receptor compartment was filled with PBS of pH 7.4 and maintained the temperature 32 ± 0.5 °C with circulating water bath. At the end of 24 h, skin was carefully removed, prevents contamination and developed for observation under confocal microscope for measuring the distribution of rhodamine in different skin layers. The wavelength of excitation, λex was set at 542 nm and emission, λem at 625 nm using argon laser beam and 65× objective lens (EC-Plan Neofluar 65×/01.40 Oil DICM27). The optical scanning of skin tissue was done through z-axis of confocal microscope to analyse the depth of fluorescent permeation through the layers of skin.
Mnb/Dyrk1A orchestrates a transcriptional network at the transition from self-renewing neurogenic progenitors to postmitotic neuronal precursors
Published in Journal of Neurogenetics, 2018
Mirja N. Shaikh, Francisco J. Tejedor
For quantitative image analysis, mutant and control specimens were processed in parallel for IHC analysis in the same experiment. Furthermore, all samples of each experiment were analyzed within the same confocal microscope work session using equal optical and image acquisition parameters. Quantitative determination of immune labeling intensity was carried out using the Image J program on unmodified digital images of confocal sections covering equivalent regions (X, Y, Z axis) of a minimum of four brains of each genotype. Final data was obtained as labeling intensity per area unit (10,000 μm2) and were analyzed for Student’s t-test with the GraphPad Prism software. Data are presented as means values ± Standard Error of Mean (SEM). Statistical significance was attributed for p < 0.05 (*), p < 0.005 (**) or p < 0.0005 (***).