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.
Introduction to optical imaging
Ahmad Fadzil Mohamad Hani, Dileep Kumar in Optical Imaging for Biomedical and Clinical Applications, 2017
Confocal microscopy is a technique in which both the object and the corresponding image generated are in focus, as depicted by the schematic representation of confocal microscopy set-up in Figure 1.8. The out-focal data that are generated in the case of normal microscopy is eliminated by the use of confocal pinholes. These confocal pinholes act as spatial filters, which filter out the out-focal beams. Only in-focal light beams are used to create the images, whereas above-focal and below-focal light beams are filtered out using the pinholes. Lasers are used as the light source instead of arc lamps as in the case of general fluorescence imaging because lasers form very bright and sharp wavelength light sources. Thus, confocal images are the sharper but of lesser brightness as compared to fluorescence images. Highly sensitive PMTs are used as photon detectors in order to pick up very low-intensity signals due to filtered out-focal data by the pinhole filters.
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.
Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells
Published in OncoImmunology, 2018
Francesca Tosetti, Roberta Venè, Caterina Camodeca, Elisa Nuti, Armando Rossello, Cristina D'Arrigo, Denise Galante, Nicoletta Ferrari, Alessandro Poggi, Maria Raffaella Zocchi
Confocal Microscopy. 2.5 × 104 MSC (either MSC16412 or MSC773) seeded on 0.2 mm thin round glass slides were incubated for 24 h with Rab5GFP (CellLight Reagents BacMam 2.0, Thermo Fisher, 2 µL) or 1 h with LysoTracker DND99 (Thermo Fisher, 50 nM). After extensive washes, slides were stained with CAM36 or CAM50 at 5 µM for 60 min at RT. Some samples were treated with 10 µM nocodazole (Sigma-Aldrich) for 1 h at 37°C prior to exposure to CAM36. In other experiments, L428 or MSC16412, labelled with either Rab5-GFP or LysoTracker DND99, were exposed to CAM36-ExoV (15μg/105 cells) as described above and incubated 4 h (to optimize images) at 37°C before performing confocal microscopy. Samples were then analyzed by FV500 (FluoView confocal Laser Scanning Microscope System, Olympus Europe GMBH, Hamburg, Germany) equipped with an Argon laser to excite carboxyfluoscein and a He-Neon red laser at 633 nm to excite cyanine 5 dye associated to a IX81 motorized microscope (Olympus). Samples were observed with PlanApo 40x NA1.00 or 60x NA1.40 oil objectives and data analyzed with FluoView 4.3b computer program (Olympus). Each image has been taken in sequence mode to avoid cross-contribution of each fluorochrome. Results are shown as bright field or pseudocolor images.
Effects of N-terminal and C-terminal modification on cytotoxicity and cellular uptake of amphiphilic cell penetrating peptides
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Mehdi Soleymani-Goloujeh, Ali Nokhodchi, Mehri Niazi, Saeedeh Najafi-Hajivar, Javid Shahbazi-Mojarrad, Nosratollah Zarghami, Parvin Zakeri-Milani, Ali Mohammadi, Mohammad Karimi, Hadi Valizadeh
Confocal microscopy. MCF-7 cells were seeded on coverslips in a six-well plate (5 × 105 cells per well) and cultured for ∼22 h in serum-containing RPMI-1640 and incubated at 37 ºC with 5% CO2. The next day, medium was removed from the plate and the peptide solutions were added to each well. The cells were incubated for 2 h at 37 °C before the peptide solutions were removed. The cells were washed three times with PBS buffer and fixed with 4% paraformaldehyde for 20 min at room temperature. Afterwards, the cells were washed twice with PBS buffer and treated with RNase (DNase free) enzyme to cleave probably double strand molecules except DNA. Propidium iodide was added to each sample for 5 min at room temperature to stain the dead cells. The cells were washed twice with PBS and the coverslips were placed cell-side-down on cytoslides with 4 μL mounting medium. After drying, coverslips were fixed with colorless nail polish. Samples were stored protected from light at 4 °C until measurements using a confocal laser scanning microscope (CLSM) (TCS-SP5 II, Leica, Wetzlar, Germany) equipped with a 20 × and 63 × objective lens were carried out.
Methods for Assessing Corneal Opacity
Published in Seminars in Ophthalmology, 2019
Thomas H. Dohlman, Jia Yin, Reza Dana
In confocal microscopy, a pinhole aperture is used to block out-of-focus light rays from reaching a detector. As only in-focus light rays are detected, higher resolution images can be obtained as compared to standard microscopy. As with OCT and Scheimpflug, these high-resolution images of the cornea can then be analyzed to quantify light scatter as an indicator of corneal opacity. This technique has been used to quantify corneal opacity in transgenic murine models of corneal haze,35 a rabbit model of photorefractive keratectomy (PRK),36 and in the clinical setting to quantify corneal haze after refractive surgery.37 The potential utility of confocal microscopy in quantifying opacity is supported by two animal studies which examined post-PRK haze and showed a strong correlation between confocal-measured light scatter and both subjective clinical opacity score38 and subepithelial scar-tissue thickness.36 However, compared to OCT and Scheimpflug, confocal image acquisition requires more time for image processing and can be uncomfortable for patients, given that it requires physical contact with the cornea. In addition, while confocal microscopes are commercially available, they are generally only found at academic medical centers.
Related Knowledge Centers
- Contrast
- Fluorescence
- Micrograph
- Optical Sectioning
- Fluorescence Microscope
- Camera
- Point Spread Function
- Cardinal Point
- Avalanche Photodiode
- Signal-to-Noise Ratio