Phase Contrast Without Phase Plates and Phase Rings-Optical Solutions for Improved Imaging of Phase Structures (original) (raw)
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Conventional, Apodized, and Relief Phase-Contrast Microscopy
Neuromethods
Non-absorbing colorless specimens (phase objects) can be visualized either by chemical staining (histology) or by the so-called optical contrasting (staining). The latter is achieved by converting optical phase shifts within the specimen, invisible to human eye, to intensity differences in the microscopic image. Out of several available modes of microscopic phase visualization, conventional, apodized, and relief phase contrast are described in detail. A comparison with relief contrast alone, that is, the off-axis (schlieren) illumination mode is presented as well. Images of various phase specimens of biological origin are shown, demonstrating the strong and weak points of each mode. Physiological aspects of image comprehension, facilitated by shading and other visual cues to depth structure are briefly discussed. A phase-contrast microscope equipped with an objective hosting no phase annulus is presented; the latter is located in a pupil projection (optically relayed) plane, in an external attachment unit. This setup enables phase-contrast and, for example, conventional epi-fluorescence and total internal reflection fluorescence (TIRF) images to be acquired with a single objective lens. Such modality is demonstrated in growth cones of neuroblastoma-glioma hybrid mouse-rat cells and touch receptor neurons of Caenorhabditis elegans. Examples of phase-contrast imaging in electron and X-ray (synchrotron radiation) microscopy are also presented.
Phase-shifting Zernike phase contrast microscopy for quantitative phase measurement
Optics Letters, 2011
Zernike phase contrast microscopy is extended and combined with a phase-shifting mechanism to perform quantitative phase measurements of microscopic objects. Dozens of discrete point light sources on a ring are constructed for illumination. For each point light source, three different levels of point-like phase steps are designed, which are alternatively located along a ring on a silica plate to perform phase retardation on the undiffracted (dc) component of the object waves. These three levels of the phase steps are respectively selected by rotating the silica plate. Thus, quantitative evaluation of phase specimens can be performed via phase-shifting mechanism. The proposed method has low "halo" and "shade-off" effects, low coherent noise level, and high lateral resolution due to the improved illumination scheme.
Journal of Biomedical Optics, 2008
Microscopic images of biological phase specimens of various optical thickness, acquired under off-axis illumination and apodized/conventional phase-contrast are compared. The luminance profiles in appropriately filtered apodized phase-contrast images compare well with those in the original off-axis illumination images. The two unfiltered image types also yield similar results in terms of quasithree-dimensional surface ͑pseudo-relief͒ rendering, and thus are comparable in terms of the information contents ͑optical thickness map͒. However, the overall visual impression is very different as the visual cues to depth structure are present in the off-axis illumination images only. The comparison demonstrated in the present paper was made possible owing to apodization, which substantially reduces the "halo"/shade-off artifacts in the phase-contrast images. The results imply the possibility of combining the off-axis illumination and apodized phase-contrast imaging to examine specimens of medium optical thickness, in which the phase visualization capability of the two imaging modes substantially overlaps ͑e.g., larger cells or cell clusters͒.
Relief Phase Contrast:A New Technique for Phase-Contrast Light Microscopy
2007
Relief phase contrast is a new modification of conventional phase contrast which leads to visible improvements of image quality in light microscopy. In particular, the following parameters can be improved: contrast, focal depth, sharpness, three dimensionality, planeness, and halo artifacts. These effects can be achieved when the ring-shaped masks in the condenser are replaced by crescent- or punctate-shaped masks. Several solutions are described which are suitable to create this modification. The achievable improvements of image quality are relevant for all quality levels of objectives. The new technique can be used for phase contrast objectives from different manufacturers, so that the usual limitations of compatibility are eliminated.
Quantitative Phase Microscopy: how to make phase data meaningful
Proceedings - Society of Photo-Optical Instrumentation Engineers, 2014
The continued development of hardware and associated image processing techniques for quantitative phase microscopy has allowed superior phase data to be acquired that readily shows dynamic optical volume changes and enables particle tracking. Recent efforts have focused on tying phase data and associated metrics to cell morphology. One challenge in measuring biological objects using interferometrically obtained phase information is achieving consistent phase unwrapping and -dimensions and correct for temporal discrepanices using a temporal unwrapping procedure. The residual background shape due to mean value fluctuations and residual tilts can be removed automatically using a simple object characterization algorithm. Once the phase data are processed consistently, it is then possible to characterize biological samples such as myocytes and myoblasts in terms of their size, texture and optical volume and track those features dynamically. By observing optical volume dynamically it is p...
X-ray phase-contrast microscopy and microtomography
Optics Express, 2003
In-line phase contrast enables weakly absorbing specimens to be imaged successfully with x-rays, and greatly enhances the visibility of fine scale structure in more strongly absorbing specimens. This type of phase contrast requires a spatially coherent beam, a condition that can be met by a microfocus x-ray source. We have developed an x-ray microscope, based on such a source, which is capable of high resolution phase-contrast imaging and tomography. Phase retrieval enables quantitative information to be recovered from phase-contrast microscope images of homogeneous samples of known composition and density, and improves the quality of tomographic reconstructions.
IAR Consortium, 2022
Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark. Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image. In this review, we explained the Principles, Applications, Advantages & Limitations of Dark-field microscopy, Phase-contrast microscopy fluorescence microscopy in detail.
Phase-contrast microscopy principle and applications in Materials sciences
Phase-contrast microscopy defines a process or technique that converts phase shifts in the light passing through a transparent sample that can be utilized to produce high-contrast images. In this paper, the author explains the basic principles of Phase-contrast microscopy, its applications, and the advantages and disadvantages of such an approach.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2008
Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by π /2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by π . By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phas...