Routine and Special Techniques in Toxicologic Pathology
Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard in Toxicologic Pathology, 2018
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.
Role of Mitochondrial Injury During Oxidative Injury to Hepatocytes: Evidence of a Mitochondrial Permeability Transition by Laser Scanning Confocal Microscopy
John J. Lemasters, Constance Oliver in Cell Biology of Trauma, 2020
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.
Fluorescent Analysis Technique
Victoria Vladimirovna Roshchina in Fluorescence of Living Plant Cells for Phytomedicine Preparations, 2020
The first basic method of fluorimetry for living cells was luminescence microscopy. The first steps in the creation of this type of microscopy took place at the beginning of the 20th century. The first observation of chlorophyll fluorescence in plant cells under a microscope was made by the Russian scientist Tswett (1911). Today, he is mainly known as a pioneer of chromatography. The improvement of fluorescence microscopy led to the construction of microspectrofluorimeters for the registration of the emission spectra (Chance and Thorell 1959). The development of microspectrofluorimetry as a method with patents (Karnaukhov et al. 1982, 1983) brought new possibilities for the analysis of intact animal and plant cells, as described in a monograph (Karnaukhov 1978). However, the industrial application range of the apparatuses has been narrow up to now, notwithstanding most of their advantages. This technique is awaiting new employers who are able to make these apparatuses on an industrial scale. Similar non-invasive apparatuses, such as a luminescence microscope in combination with a transmitting microscope, are widespread in practice but cannot provide spectral data, unlike microspectrofluorimetry and spectrofluorimetry. Laser-scanning confocal microscopy apparatuses were developed parallel to the above-mentioned techniques and have been mainly used for fluorescent dyes and probes. Beginning from the year 2000, this technique has been included in many studies on plant systems due to the volume of images of the fluorescing sample structure received by laser excitation in the form of optical slices (then combined as a stack). However, some laser-scanning confocal microscopy apparatuses may record fluorescence spectra.
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.
Microencapsulation of fish oil by casein-pectin complexes and gum arabic microparticles: oxidative stabilisation
Published in Journal of Microencapsulation, 2019
Arianne Cunha dos Santos Vaucher, Patrícia C. M. Dias, Pablo T. Coimbra, Irina dos Santos Miranda Costa, Ricardo Neves Marreto, Gisela Maria Dellamora-Ortiz, Osvaldo De Freitas, Mônica F. S. Ramos
Confocal laser scanning microscopy (CSLM): Analyses were carried out according to the procedure described by Lamprecht et al. (2000), with modifications. For P1, P2, and P3 preparations 250 μL of fluorescein isothiocyanate (FITC (Sigma-Aldrich, St. Louis, MO); 1 mg/mL in DMSO) were added in aqueous GA dispersions, maintained under constant stirring (300 rpm) for 40 min. NR was dispersed in FO (NR/FO; 0.05% (w/w)) and poured over the dispersion of GA-FITC. The dispersion was kept under stirring (300 rpm) for 10 min, followed by homogenisation in Turrax (13 500 rpm) for 2 min. MD was then added slowly to the dispersion, and in the end the mixture was kept under stirring for 5 min, followed by atomisation. For P4, P5, and P6, 250 μL of FITC (1 mg/mL in DMSO) were added to the CP dispersion (pH 8.0) and NR to FO (NR/FO; 0.05% (w/w)). The mixture FO-NR was poured over the dispersion CP-FITC. The dispersion was maintained under stirring at 400 rpm for 10 min, and homogenised in Turrax (13 500 rpm) for 2 min. The medium was acidified with 1 M HCl until pH 3.5 ± 0.3. MD was then added slowly to the dispersion until total solubilisation, followed by atomisation. All of the steps (sample preparation and atomisation) were carried out in the absence of light. Images were acquired using a CSLM Fluoview version 3.3 BX51 (Olympus Corporation, Tokyo, Japan) with an excitation wavelength of 488 nm, using emission filters at 510–530 nm for FITC, and laser excitation wavelength of 530 nm, with emission filters at 560–600 nm for NR (Lamprecht et al.2000).
The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery
Published in Drug Delivery, 2018
Lijuan Jiang, Xin Liang, Gan Liu, Yun Zhou, Xinyu Ye, Xiuli Chen, Qianwei Miao, Li Gao, Xudong Zhang, Lin Mei
MCF-7 cells were seeded into 12-well plates at a density of 2 × 104 cells/well in DMEM containing 10% FBS and allowed to adhere overnight. FITC was used as a model fluorescent molecule and was formulated in nBSA and LA-nBSA. Non-transfected or DsRed-Rab7 and DsRed-Rab34 were incubated with 1 mg/mL FITC-labeled LA-nBSA and nBSA at 37 °C for 4 h. For lysosome detection, the cells were incubated with Lyso-Tracker Red for 1 h. After incubation, the MCF-7 cells in each group were washed with PBS twice. Subsequently, the MCF-7 cells were fixed in 4% formaldehyde for 10 min, and stained cell nuclei with DAPI for 10 min. Then, confocal microscopy was performed with a FLUO-VIEW laser scanning confocal microscope (Olympus, FV1000, Olympus Optical, Tokyo, Japan) in sequential scanning mode using a 60–100× objective. The operation processes were similar to those reported in the literature (Zhang et al., 2014).
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