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Methods of Identifying Microbiological Hazards in Indoor Environments
Published in Rafał L. Górny, Microbiological Corrosion of Buildings, 2020
Particles in samples can be observed in transmitted light (for transparent specimens) or reflected light (for opaque specimens), in light field or dark field or in polarised light to enhance the contract and precisely visualise the details of observed elements. Specimens can be stained using e.g. methylene blue, crystal violet, safranin, fuchsine or other differentiating stains in order to classify microbiological particles into appropriate groups. On the other hand, when the observed particles are almost invisible and observation of the specimen in another medium is impossible or unacceptable, a phase-contrast microscope is used. Fluorescence microscopy is a variant of light microscopy; it uses an ultraviolet or near-ultraviolet light source which causes particles with fluorescent properties to emit light.
Approaches to the Measurement of Biological Pollutants
Published in Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus, Environmental Chemical Analysis, 2018
Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus
A fluorescence microscope (often referred to as epifluorescence microscope) is an optical microscope which uses the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. Here, the organism of interest in the specimen is labeled specifically with a fluorescent molecule or a dye. The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, leading to emission at longer wavelengths. A typical fluorescence microscope is shown in Figure 10.3. The excitation beam is separated from the weaker fluorescent beam using a spectral emission filter. Typical components of a fluorescence microscope are the light source (e.g., xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter. The filters and the dichroic are chosen to match the spectral features of the excitation and emission.
Biomedical Imaging
Published in Mohammad E. Khosroshahi, Applications of Biophotonics and Nanobiomaterials in Biomedical Engineering, 2017
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of (or in addition to) reflection and absorption to study properties of organic or inorganic material where it uses fluorescence to generate an image. The specimen is illuminated with light of a specific wavelength which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope are a light source (xenon arc lamp or mercury-vapor lamp or more advanced forms of high-power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beam splitter), and the emission filter. The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. In this manner, the distribution of a single fluorophore (i.e., color) is imaged at a time. Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e., through the objective). These microscopes are widely used in biology and are the basis for more advanced microscope designs, such as the confocal microscope and the total internal reflection fluorescence microscope.
Comparison of STED, confocal and optical microscopy of ultra-short pitch cholesterics
Published in Liquid Crystals, 2020
J. Pišljar, G. Posnjak, S. Pajk, A. Godec, R. Podlipec, B. Kokot, I. Muševič
In polarised light microscopy, the acquired image is a result of phase and amplitude contrast of birefringent sample under observation. Although LC director fields are most often observed using polarised light microscopy, fluorescent imaging methods often prove as beneficial because of their different principle of operation. In contrast to ordinary light microscopy, fluorescence microscopy acquires image of the sample by exciting fluorescent molecules in the sample to radiate fluorescent light. In this case, fluorescent dye molecules which are designed to align their radiative moment along (or perpendicular) to the local director, are added in small amounts to the LC. Since excitation of the anisotropic dye molecule is dependent on the polarisation of the excitation beam, the fluorescent emission intensity distribution is anisotropic and the amount of acquired fluorescent light depends on the local alignment of the radiative dipole moment [2]. This contrast in fluorescent light is used to acquire an image of the sample in fluorescent microscopy and it is quite clear that the image contrast depends on the degree of ordering of dye molecules with respect to the director.
3D printed porous β-Ca2SiO4 scaffolds derived from preceramic resin and their physicochemical and biological properties
Published in Science and Technology of Advanced Materials, 2018
Shengyang Fu, Wei Liu, Shiwei Liu, Shichang Zhao, Yufang Zhu
rBMSCs were first seeded onto the scaffolds at a density of 2 × 105 cells per scaffold. After cultured for 3 days, the scaffolds were rinsed by phosphate buffer saline (PBS) three times. Subsequently, 2.5–3.0% glutaraldehyde was added for 15 min for fixation, and alcohol at concentration of 50%, 75%, 80%, 90%, 95%, and 100% were used for dehydration for 10 min twice. Finally, the samples were treated with isoamyl acetate overnight. The scaffolds were sprayed with gold for the observation with scanning electron microscope. On the other hand, the cell distributions on the surface of scaffold were also observed by fluorescence microscope. After cultured for 3 days, the culture medium was removed and the live cells were fixed in 4% paraformaldehyde before washing three times with PBS. Next, cells were treated with 0.5% Triton X-100 for 15 min. Afterward, the cell actin and nuclei were labeled with 0.6 ml of 5 mg/ml phalloidin (Yeasen, Shanghai) and 0.6 ml of 10 mg/ml 4′,6-diamidino-2-phenylindole (DAPI; Yeasen, Shanghai) for 5 min for staining, respectively. Fluorescence images were taken with a fluorescence microscope (DMI6000B, Leica, Germany) and analyzed by using Image J software.
A review of microfluidic concepts and applications for atmospheric aerosol science
Published in Aerosol Science and Technology, 2018
Andrew R. Metcalf, Shweta Narayan, Cari S. Dutcher
By far, the majority of microfluidic experiments are performed on a microscope where the most basic on-chip measurement available is visual microscopy; that is, taking an image of the device and fluid flow with an attached camera. Brightfield and darkfield microscopy images rely on a refractive index contrast between immiscible liquid phases or between suspended particles and the surrounding liquid. Brightfield imaging is used to determine interfacial boundaries for physical property measurements. Fluorescence microscopy, on the other hand, typically excites the sample with a pulse of light at one (range of) wavelength(s) and then images at a shorter (range of) wavelength(s) to measure the excitation from the sample. Fluorescence is typically used in biological agent detection; however, a recent study used fluorescence microscopy to image phase-separated atmospheric aerosol (You et al. 2012). In either imaging scenario, high-speed cameras, with frame rates in excess of 10,000 images per second, allow capture of fast, dynamic phenomena, such as interfacial deformation and relaxation.