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Published in Bethe A. Scalettar, James R. Abney, Cyan Cowap, Introductory Biomedical Imaging, 2022
Bethe A. Scalettar, James R. Abney, Cyan Cowap
Contrast generation is a rich topic when discussed in the context of optical microscopy because many commonly imaged specimens, such as cells and tissues, are intrinsically clear and colorless and thus tend (under standard illumination and detection conditions) to generate images with very poor contrast. One way to circumvent this problem is experimental and involves altering the sample by labeling cellular constituents with absorbing stains or fluorescent molecules that generate contrast via variations in the amplitude or color of the electric field (Box 5.1). A second approach is more optics based and gives rise to contrast-enhancing techniques such as darkfield, phase-contrast, polarization, and differential interference contrast (DIC) microscopies. We discuss these four techniques after introducing standard “brightfield” microscopy and highlighting the contrast problem that often plagues this approach. Fluorescence will be the focus of Chapters 6–8.
Optical Interference
Published in Rajpal S. Sirohi, Optical Methods of Measurement, 2018
where θ is the angle of either wedge forming the Wollaston prism. The Nomarski interferometer can be employed in two distinctly different modes. With an isolated microscopic object, it is convenient to use a lateral shear that is larger than the dimensions of the object. Two images of the object are then seen, covered with fringes that contour the phase changes due to the object. Often, the Nomarski interferometer is used with smaller shear than the dimensions of a microscopic object. The interference pattern then shows the phase gradients. This mode of operation is known as differential interference contrast (DIC) microscopy. The Nomarski interferometer can also be used with white light; the phase changes are then decoded as color changes. Biological objects, usually phase objects, can also be studied using microscopes.
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Published in P. Dakin John, G. W. Brown Robert, Handbook of Optoelectronics, 2017
Constantinos Pitris, Tuan Vo-Dinh, R. Eugene Goodson, Susie E. Goodson
In dark-field microscopy, contrast is generated in the image by altering the illumination pattern so as to reject the light directly passing through the sample. In phase contrast and differential interference contrast (DIC) microscopy, the illumination is also modified. However, in these cases, complementary optical accessories (e.g., filters or prisms) condition the light before it strikes the specimen and manipulate the light after it has interacted with the specimen. These alterations provide contrast based on the phase difference of light passing through the sample versus through air and result in desirable imaging features such as high resolution and reduced artifacts [48]. Fluorescence microscopy incorporates excitation and emission wavelength-selecting filters to visualize the fluorescence emission from tissue samples. The florescence can originate from either endogenous or exogenous fluorophores. It, thus, provides tissue-specific contrast and even functional characterization because the exogenous fluorophores can be designed to bind to specific cellular moieties.
Impact of activated sludge ozonation on filamentous bacteria viability and possible added benefits
Published in Environmental Technology, 2019
Filip Nilsson, Åsa Davidsson, Per Falås, Simon Bengtsson, Kai Bester, Karin Jönsson
A Zeiss Axioskop 2 plus epifluorescence microscope was used for the characterization of the samples. Phase contrast, differential interference contrast and dark field light microscopy were applied with magnifications between 100× and 630×. The samples were characterized with respect to morphology and semi-quantitatively assessed according to a previously established methodology [16]. The abundance of protozoa and metazoan (mL-1) were determined by enumeration. The samples were stained by ‘LIVE/DEAD® BacLight™ Bacterial Viability Kit, L7007’ according to the manufacturer’s protocol. In brief, equivolumes of component A (SYTO® 9 dye, 3.34 mM) and B (Propidium iodide, 20 mM) were mixed and 3 μL of this mixture was added to 1 mL of sample which was incubated for 15 min in darkness. The samples were examined under the epifluorescence microscope equipped with filters suitable for the excitation and emission wavelengths associated with ‘Live’ (Omega filter XF22) and ‘Dead’ (Omega filter XF32) response. The presented images comprise overlays in pairs of pictures taken on the positive (green) and negative (red) response. A similar semi-quantitative assessment was conducted for the Live/Dead response as for the morphology.
Industrial lubricant removal using an ultrasonically activated water stream, with potential application for Coronavirus decontamination and infection prevention for SARS-CoV-2
Published in Transactions of the IMF, 2020
M. Malakoutikhah, C. N. Dolder, T. J. Secker, M. Zhu, C. C. Harling, C. W. Keevil, T. G. Leighton
A second set of measurements was conducted using the PAO and the MO lubricant. Visualisation of the grease on the stainless steel tokens was carried out using sensitive Episcopic Differential Interference Contrast (EDIC) microscopy (Nikon Eclipse LV100, custom modified by Best Scientific, UK57,58). The EDIC microscope uses the fundamentals of Nomarski DIC (Differential Interference Contrast) but using episcopic light as opposed to the transmitted light of Nomarski DIC. This allows the visualisation of translucent samples directly on opaque surfaces, creating a pseudo-3D image of the sample with high resolution, in the z-plane. Long Working Distance (LWD) lenses utilised with EDIC microscopy allows samples to be visualised with minimal sample preparation.57
Enhancement of growth and biomolecules (carbohydrates, proteins, and chlorophylls) of isolated Chlorella thermophila using optimization tools
Published in Preparative Biochemistry & Biotechnology, 2022
Sambit Sarkar, Jaivik Mankad, Nitin Padhihar, Mriganka Sekhar Manna, Tridib Kumar Bhowmick, Kalyan Gayen
Pure axenic culture of isolated strain (BTA 9035) was used for microscopic identification.[25] A small portion of the culture was used for the analysis of morphological characteristics using an optical microscope. Image of the isolated strain was captured at 100× magnification in Differential Interference Contrast (DIC) mode using an upright and inverted optical microscope (Olympus, BX53) for the study of morphological properties and preliminary identification.