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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
In this imaging mode, spatially varying phase shifts in light that are induced by a transparent specimen are converted into amplitude variations and thereby into contrast. The method was developed by Frits Zernike, earning him a Nobel Prize in Physics in 1953, and involves manipulation of the illumination profile in the FFP of the condenser, spatial filtering in the BFP of the objective, and interference. Advantages of phase-contrast imaging include its relatively low cost, its ease of implementation, and its ability to produce high-quality images of “phase” objects, most notably living, unstained cells. Unlike DIC, phase is not subject to orientation-dependent artifacts, and it is insensitive to polarization and birefringence effects, permitting examination of cultured cells grown in plastic dishes.
Advanced Widefield Microscopy
Published in John Girkin, A Practical Guide to Optical Microscopy, 2019
The key concept in phase contrast microscopy is to separate out light that passes through the area around the sample and that, which passes through the area of interest. In the sample, where there are differences in the local refractive index, the light is diffracted. The light from the two different paths is then recombined on the detector. Figure 4.2a illustrates the main features for the method as implemented in a standard microscope. The light that passes around the sample is normally attenuated so that it does not dominate the final image, and a known phase advancement, or delay, of λ/4 is added to this light to further help improve the final image contrast.. To achieve this in an optical microscope two extra components are added to the basic optical system described previously. The first is an optical annulus placed such that it is re-imaged onto the back aperture of the objective (aperture conjugate planes), while the second, a phase plate, is placed at the back aperture of the objective lens. In undertaking phase contrast microscopy the light source must be well adjusted for Köhler illumination to ensure high contrast images.
Microscopic Tissue Imaging
Published in Vadim Backman, Adam Wax, Hao F. Zhang, A Laboratory Manual in Biophotonics, 2018
Vadim Backman, Adam Wax, Hao F. Zhang
The phase-contrast microscope is based on phase-shifting diffracted light to improve the contrast of the observed image upon interference with the undeviated light. Figure 4.7 shows how this is implemented in a microscopy scheme. The illumination train is modified to include a condenser annulus adjacent to the condenser aperture, located at the back focal plane of the condenser. The annulus masks the illuminating light to create parallel rays incident on the sample. The illumination travels at an angle relative to the optical axis, so the typical description as a “cone of light” is not entirely accurate but is a good analogy to help with intuitive understanding of this scheme. The combination of the condenser and objective image the annulus onto the back focal plane of the object where the phase plate is located. The phase plate is an annular ring of phase-shifting material that imparts a + or - 90° shift to the undeviated light. Schemes with a +90° shift are termed positive phase contrast, while those with a −90° shift are termed negative phase contrast. In practice, the phase-shifting material is usually included in the housing of the objective.
Assays and enumeration of bioaerosols-traditional approaches to modern practices
Published in Aerosol Science and Technology, 2020
Maria D. King, Ronald E. Lacey, Hyoungmook Pak, Andrew Fearing, Gabriela Ramos, Tatiana Baig, Brooke Smith, Alexandra Koustova
Phase-contrast microscopy is a variant of bright-field microscopy that takes advantage of the variation in the refractive index between a microorganism and its surrounding medium to enhance contrast and provide easier viewing (Zernike 1942; Morris 1995). Phase-contrast microscopy can be used to observe live microorganisms, but is particularly useful for imaging low-contrast specimens. Fluorescence is a powerful tool that can be used in microscopy to examine particular structures or molecules in a microorganism or its surroundings. Fluorescence can be achieved either with fluorescent dyes or with cells that are naturally or artificially fluorescent. A variant of fluorescent microscopy is confocal microscopy, which allows very precise resolution and location of fluorescent molecules in a cell. Epifluorescence microscopy has been used to determine viral abundance in bioaerosols (Michaud et al. 2018).