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Applied and Environmental Microbiology Oct 2016Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple...
UNLABELLED
Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with l-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spore's inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly.
IMPORTANCE
Much research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this study, Raman spectroscopy and phase-contrast microscopy were used to analyze CaDPA levels and kinetics of nutrient- and non-nutrient-germinant-induced germination of multiple individual spores of Bacillus subtilis that were treated by a planar CAP device. The roles of different plasma species involved in spore inactivation were also investigated. The knowledge obtained in this study will aid in understanding the mechanism(s) of spore inactivation by CAP and potentially facilitate the development of more effective and efficient plasma sterilization techniques in various applications.
Topics: Bacillus subtilis; Bacteriological Techniques; Microbial Viability; Microscopy, Phase-Contrast; Picolinic Acids; Plasma Gases; Spectrum Analysis, Raman; Spores, Bacterial
PubMed: 27422840
DOI: 10.1128/AEM.01669-16 -
Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC).PloS One 2017We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial...
We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification-an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various in vitro cell samples and c. elegans in a micro-fluidic channel.
Topics: Animals; Caenorhabditis elegans; Calibration; Color; Image Processing, Computer-Assisted; Microfluidics; Microscopy, Phase-Contrast; Models, Theoretical
PubMed: 28152023
DOI: 10.1371/journal.pone.0171228 -
Molecular Vision 2012Images from cultured lens cells do not convey enough spatial information, and imaging of fixed lens specimens cannot reveal dynamic changes in the cells. As such, a...
PURPOSE
Images from cultured lens cells do not convey enough spatial information, and imaging of fixed lens specimens cannot reveal dynamic changes in the cells. As such, a real-time, convenient approach for monitoring label-free imaging of dynamic processes of living cells within the whole lens is urgently needed.
METHODS
Female Wistar rat lenses were kept in organ culture. Insulin-like growth factor-I was added to the culture medium to induce cell mitosis. A novel method of ultraviolet (UV) irradiation was used to induce cell apoptosis and fiber damage. The cellular morphological dynamics within the whole lens were monitored by inverted phase contrast microscopy. Apoptosis was assessed using a commercial kit with Hoechst 33342/YO-PRO®-1/propidium iodide (PI).
RESULTS
The intrinsic transparency and low-light scattering property of the rat lens permitted direct imaging of the lens epithelial cells (LECs) and the superficial fiber cells. We visualized the processes of mitosis and apoptosis of the LECs, and we obtained dynamic images of posterior fiber cells following UVA irradiation.
CONCLUSIONS
This method opens a new window for observing lens cells in their physiologic location, and it can be readily applied in studies on lens physiology and pathology.
Topics: Animals; Apoptosis; Epithelial Cells; Female; Insulin-Like Growth Factor I; Lens, Crystalline; Microscopy, Phase-Contrast; Mitosis; Molecular Imaging; Organ Culture Techniques; Rats; Rats, Wistar; Time-Lapse Imaging; Ultraviolet Rays
PubMed: 22879736
DOI: No ID Found -
Nano Letters Feb 2019Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery...
Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery systems. However, to track these materials and their cellular interactions, current studies often depend on surface-bound fluorescent labels, which have the potential to alter native cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions independent of surface modification. Addressing these concerns, here we show how scatter enhanced phase contrast (SEPC) microscopy can be extended to work as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. To determine which materials can be studied using SEPC, we turn to Lorenz-Mie theory, which predicts that individual particles down to ∼35 nm can be observed. We confirm this experimentally, demonstrating that SEPC works for a variety of metal and metal oxides, including Au, Ag, TiO, CeO, AlO, and FeO nanoparticles. We then demonstrate that SEPC microscopy can be used in a quantitative, time-dependent fashion to discriminate between distinct modes of active cellular transport, including intracellular transport and membrane-assisted transport. Finally, we combine this technique with microcontact printing to normalize transport dynamics across multiple cells, allowing for a careful study of ensemble TiO nanoparticle uptake. This revealed three distinct regions of particle transport across the cell, indicating that membrane dynamics play an important role in regulating particle flow. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state and their role in endocytosis and cellular transport.
Topics: Biological Transport; Endocytosis; Equipment Design; Human Umbilical Vein Endothelial Cells; Humans; Metals; Microscopy, Phase-Contrast; Nanoparticles; Oxides; Surface Properties
PubMed: 30616354
DOI: 10.1021/acs.nanolett.8b03903 -
PloS One 2015X-ray phase-contrast imaging is a novel technology that achieves high soft-tissue contrast. Although its clinical impact is still under investigation, the technique may...
X-ray phase-contrast imaging is a novel technology that achieves high soft-tissue contrast. Although its clinical impact is still under investigation, the technique may potentially improve clinical diagnostics. In conventional attenuation-based X-ray computed tomography, radiological diagnostics are quantified by Hounsfield units. Corresponding Hounsfield units for phase-contrast imaging have been recently introduced, enabling a setup-independent comparison and standardized interpretation of imaging results. Thus far, the experimental values of few tissue types have been reported; these values have been determined from fixated tissue samples. This study presents phase-contrast Hounsfield units for various types of non-fixated human soft tissues. A large variety of tissue specimens ranging from adipose, muscle and connective tissues to liver, kidney and pancreas tissues were imaged by a grating interferometer with a rotating-anode X-ray tube and a photon-counting detector. Furthermore, we investigated the effects of formalin fixation on the quantitative phase-contrast imaging results.
Topics: Connective Tissue; Humans; Kidney; Liver; Microscopy, Phase-Contrast; Muscles; Pancreas; Photons; Tomography, X-Ray Computed; X-Ray Diffraction
PubMed: 26322638
DOI: 10.1371/journal.pone.0137016 -
Nature Communications Dec 2022Phase contrast microscopy has played a central role in the development of modern biology, geology, and nanotechnology. It can visualize the structure of translucent...
Phase contrast microscopy has played a central role in the development of modern biology, geology, and nanotechnology. It can visualize the structure of translucent objects that remains hidden in regular optical microscopes. The optical layout of a phase contrast microscope is based on a 4 f image processing setup and has essentially remained unchanged since its invention by Zernike in the early 1930s. Here, we propose a conceptually new approach to phase contrast imaging that harnesses the non-local optical response of a guided-mode-resonator metasurface. We highlight its benefits and demonstrate the imaging of various phase objects, including biological cells, polymeric nanostructures, and transparent metasurfaces. Our results showcase that the addition of this non-local metasurface to a conventional microscope enables quantitative phase contrast imaging with a 0.02π phase accuracy. At a high level, this work adds to the growing body of research aimed at the use of metasurfaces for analog optical computing.
Topics: Microscopy, Phase-Contrast; Microscopy; Image Processing, Computer-Assisted; Geology; Nanostructures
PubMed: 36543788
DOI: 10.1038/s41467-022-34197-6 -
Computational and Mathematical Methods... 2015Imaging techniques for visualizing cerebral vasculature and distinguishing functional areas are essential and critical to the study of various brain diseases. In this...
Imaging techniques for visualizing cerebral vasculature and distinguishing functional areas are essential and critical to the study of various brain diseases. In this paper, with the X-ray phase-contrast imaging technique, we proposed an experiment scheme for the ex vivo mouse brain study, achieving both high spatial resolution and improved soft-tissue contrast. This scheme includes two steps: sample preparation and volume reconstruction. In the first step, we use heparinized saline to displace the blood inside cerebral vessels and then replace it with air making air-filled mouse brain. After sample preparation, X-ray phase-contrast tomography is performed to collect the data for volume reconstruction. Here, we adopt a phase-retrieval combined filtered backprojection method to reconstruct its three-dimensional structure and redesigned the reconstruction kernel. To evaluate its performance, we carried out experiments at Shanghai Synchrotron Radiation Facility. The results show that the air-tissue structured cerebral vasculatures are highly visible with propagation-based phase-contrast imaging and can be clearly resolved in reconstructed cross-images. Besides, functional areas, such as the corpus callosum, corpus striatum, and nuclei, are also clearly resolved. The proposed method is comparable with hematoxylin and eosin staining method but represents the studied mouse brain in three dimensions, offering a potential powerful tool for the research of brain disorders.
Topics: Air; Algorithms; Animals; Brain; Computational Biology; Imaging, Three-Dimensional; Mice; Microscopy, Phase-Contrast; Models, Anatomic; Models, Animal; Phantoms, Imaging; Radiographic Image Interpretation, Computer-Assisted; Tomography, X-Ray Computed
PubMed: 26576198
DOI: 10.1155/2015/530580 -
Applied and Environmental Microbiology Dec 1985A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and...
A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other microorganisms and debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observing characteristic internal morphological features with phase-contrast microscopy. With this method, Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished surface water supplies during several recent outbreaks.
Topics: Fluorescent Antibody Technique; Giardia; Microscopy, Phase-Contrast; Water Microbiology; Water Supply
PubMed: 3911906
DOI: 10.1128/aem.50.6.1434-1438.1985 -
Cytometry Aug 1994Reliable autofocus is required to obtain accurate measurements of fluorescent stained cellular components from a system capable of scanning multiple microscope fields.... (Comparative Study)
Comparative Study
Reliable autofocus is required to obtain accurate measurements of fluorescent stained cellular components from a system capable of scanning multiple microscope fields. Autofocus could be performed directly with fluorescence images, but due to photobleaching and destructive fluorescence by-products, it is best to minimize fluorescence exposure for photosensitive specimens and live cells. This exposure problem could be completely avoided by using phase-contrast microscopy, implemented through the same optics as fluorescence microscopy. The purpose of this work was to evaluate functions for both phase-contrast and fluorescence autofocus and determine the suitability of phase-contrast autofocus for fluorescence microscopy. Eleven autofocus functions were independently evaluated for fluorescence and phase-contrast microscopy. The most suitable functions were then chosen from these and phase-contrast and fluorescence autofocus were compared on scans each comprising more than 1,000 microscope fields. Autofocus standard deviation (S.D.) of better than 100 nm was achieved for both phase contrast and fluorescence. There was a measurable difference between the best focus positions in the two modes, but the difference was constant enough to be measured and corrected, suggesting the possibility of using phase contrast to predict best focus in fluorescence microscopy. The scanning experiments also showed that autofocus can be performed at least as fast as 0.25 s/field without loss of precision.
Topics: 3T3 Cells; Animals; Automation; Fluorescence; Fourier Analysis; Image Processing, Computer-Assisted; Mice; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Photochemistry
PubMed: 7988291
DOI: 10.1002/cyto.990160402 -
Computational and Mathematical Methods... 2019In the field of cell and molecular biology, green fluorescent protein (GFP) images provide functional information embodying the molecular distribution of biological...
In the field of cell and molecular biology, green fluorescent protein (GFP) images provide functional information embodying the molecular distribution of biological cells while phase-contrast images maintain structural information with high resolution. Fusion of GFP and phase-contrast images is of high significance to the study of subcellular localization, protein functional analysis, and genetic expression. This paper proposes a novel algorithm to fuse these two types of biological images via generative adversarial networks (GANs) by carefully taking their own characteristics into account. The fusion problem is modelled as an adversarial game between a generator and a discriminator. The generator aims to create a fused image that well extracts the functional information from the GFP image and the structural information from the phase-contrast image at the same time. The target of the discriminator is to further improve the overall similarity between the fused image and the phase-contrast image. Experimental results demonstrate that the proposed method can outperform several representative and state-of-the-art image fusion methods in terms of both visual quality and objective evaluation.
Topics: Algorithms; Cell Biology; Computational Biology; Deep Learning; Green Fluorescent Proteins; Image Processing, Computer-Assisted; Microscopy, Phase-Contrast; Models, Biological
PubMed: 31885682
DOI: 10.1155/2019/5450373