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Journal of Microscopy Sep 2018Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the...
Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy-based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.
Topics: Automation, Laboratory; Cell Count; Cell Line, Tumor; Cell Proliferation; Epithelial Cells; Humans; Image Processing, Computer-Assisted; Microscopy, Phase-Contrast
PubMed: 29999527
DOI: 10.1111/jmi.12726 -
Biophysical Journal Feb 2021Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native,...
Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.e., segmentation. Typical approaches to segmenting DIC images either involve converting images to those resembling phase contrast, mimicking the optics of DIC object formation, or using the morphological properties of objects. Here, we describe MATLAB based, single-particle tracking tool with a GUI for mobility analysis of objects from in vitro and in vivo DIC time-series microscopy. The tool integrates contrast enhancement with multiple modified Gaussian filters, automated threshold detection for segmentation and minimal distance-based two-dimensional single-particle tracking. We compare the relative performance of multiple filters and demonstrate the utility of the tool for DIC object tracking (DICOT). We quantify subcellular dynamics of a time series of Caenorhabditis elegans embryos in the one-celled stage by detecting birefringent yolk granules in the cytoplasm with high precision. The resulting two-dimensional map of oscillatory dynamics of granules quantifies the cytoplasmic flows driven by anaphasic spindle oscillations. The frequency of oscillations across the anterior-posterior (A-P) and transverse axes of the embryo correspond well with the reported frequency of spindle oscillations. We validate the quantitative accuracy of our method by tracking the in vitro diffusive mobility of micron-sized beads in glycerol solutions. Estimates of the diffusion coefficients of the granules are used to measure the viscosity of a dilution series of glycerol. Thus, our computational method is likely to be useful for both intracellular mobility and in vitro microrheology.
Topics: Animals; Caenorhabditis elegans; Image Processing, Computer-Assisted; Microscopy, Interference; Microscopy, Phase-Contrast; Reproducibility of Results
PubMed: 33359170
DOI: 10.1016/j.bpj.2020.12.013 -
BioEssays : News and Reviews in... May 2012The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an... (Review)
Review
The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an instrument that can access all scales relevant to modern biology - from individual molecules all the way to whole tissues and organisms and from single snapshots to time-lapse recordings sampling from milliseconds to days. As these and more new technologies appear, the challenges of delivering them to the community grows as well. I discuss some of these challenges, and the examples where openly shared technology have made an impact on the field.
Topics: Animals; Biological Science Disciplines; High-Throughput Screening Assays; Humans; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Microscopy, Polarization
PubMed: 22408015
DOI: 10.1002/bies.201100168 -
Magnetic Resonance in Medicine Apr 2011The strain values extracted from steady-state free-precession (SSFP) and phase contrast (PC) images acquired with a 1.5T scanner on a compliant flow phantom and within...
The strain values extracted from steady-state free-precession (SSFP) and phase contrast (PC) images acquired with a 1.5T scanner on a compliant flow phantom and within the thoracic aorta of 52 healthy subjects were compared. Aortic data were acquired perpendicular to the aorta at the level of the pulmonary artery bifurcation. Cross sectional areas were obtained by using an automatic and robust segmentation method. While a good correlation (r = 0.99) was found between the aortic areas extracted from SSFP and PC sequences, a lower correlation (r = 0.71) was found between the corresponding aortic strain values. Strain values estimated using SSFP and PC sequences were equally correlated with age. Interobserver reproducibility was better for SSFP than for PC. Strain values in the ascending and descending aorta were better correlated for SSFP (r = 0.8) than for PC (r = 0.65) and fitted with the expectation of a larger strain in the ascending aorta when using SSFP. The spatial and temporal resolutions of the acquisitions had a minor influence upon the estimated strain values. Thus, if PC acquisitions can be used to estimate both pulse wave velocity and aortic strain, an additional SSFP sequence may be useful to improve the accuracy in estimating the aortic strain.
Topics: Adolescent; Adult; Aged; Aorta, Thoracic; Elastic Modulus; Elasticity Imaging Techniques; Female; Humans; Image Enhancement; Image Interpretation, Computer-Assisted; Magnetic Resonance Angiography; Male; Microscopy, Phase-Contrast; Middle Aged; Phantoms, Imaging; Reproducibility of Results; Sensitivity and Specificity; Young Adult
PubMed: 21413062
DOI: 10.1002/mrm.22678 -
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 -
Journal of Synchrotron Radiation Sep 2018X-ray imaging allows biological cells to be examined at a higher resolution than possible with visible light and without some of the preparation difficulties associated... (Comparative Study)
Comparative Study
X-ray imaging allows biological cells to be examined at a higher resolution than possible with visible light and without some of the preparation difficulties associated with electron microscopy of thick samples. The most used and developed technique is absorption contrast imaging in the water window which exploits the contrast between carbon and oxygen at an energy of around 500 eV. A variety of phase contrast techniques are also being developed. In general these operate at a higher energy, enabling thicker cells to be examined and, in some cases, can be combined with X-ray fluorescence imaging to locate specific metals. The various methods are based on the differences between the complex refractive indices of the cellular components and the surrounding cytosol or nucleosol, the fluids present in the cellular cytoplasm and nucleus. The refractive indices can be calculated from the atomic composition and density of the components. These in turn can be obtained from published measurements using techniques such as chemical analysis, scanning electron microscopy and X-ray imaging at selected energies. As examples, the refractive indices of heterochromatin, inner mitochondrial membranes, the neutral core of lipid droplets, starch granules, cytosol and nucleosol are calculated. The refractive index calculations enable the required doses and fluences to be obtained to provide images with sufficient statistical significance, for X-ray energies between 200 and 4000 eV. The statistical significance (e.g. the Rose criterion) for various requirements is discussed. The calculations reveal why some cellular components are more visible by absorption contrast and why much greater exposure times are required to see some cellular components. A comparison of phase contrast as a function of photon energy with absorption contrast in the water window is provided and it is shown that much higher doses are generally required for the phase contrast measurements. This particularly applies to those components with a high carbon content but with a mass density similar to the surrounding cytosol or nucleosol. The results provide guidance for the most appropriate conditions for X-ray imaging of individual cellular components within cells of various thicknesses.
Topics: Cell Nucleus; Cytosol; Heterochromatin; Lipids; Microscopy, Electron, Scanning; Microscopy, Phase-Contrast; Mitochondrial Membranes; Photons; Radiation Dosage; Starch; X-Rays
PubMed: 30179189
DOI: 10.1107/S1600577518009566 -
Cytometry. Part a : the Journal of the... Aug 2022The current classical blood smear technique to observe the morphology of single red blood cells (RBCs) for classification is a laborious and error-prone process. To...
The current classical blood smear technique to observe the morphology of single red blood cells (RBCs) for classification is a laborious and error-prone process. To objectively evaluate the morphology of blood cells, we established a method of computational imaging based on a programmable light emitting diode array. By using quantitative differential phase contrast (qDPC), we characterized the morphology of unlabeled RBCs as well as blood smears. By focusing on comparing the difference of imaging between unlabeled RBCs and stained RBCs under multimode microscopic imaging technology, we demonstrated that qDPC could clearly differentiate discocytes and spherocytes in both unlabeled RBCs and blood smears. The phase map provided by quantitative phase imaging further enhanced the classification accuracy. According to statistical analysis from morphological indexes, the qDPC imaging has a significantly improvement in non-circularity, texture inhomogeneity and equivalent diameters of cells. Thus, this method has a significant superiority in the capability to analyze the morphology of RBCs and could be applied to clinical assays for determining morphological, functional, and structural deterioration of RBCs.
Topics: Erythrocyte Count; Erythrocytes; Microscopy, Phase-Contrast
PubMed: 35243761
DOI: 10.1002/cyto.a.24546 -
The Journal of Cell Biology Jun 1975This paper describes the development of a miniature, temperature-controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is...
A new miniature hydrostatic pressure chamber for microscopy. Strain-free optical glass windows facilitate phase-contrast and polarized-light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature.
This paper describes the development of a miniature, temperature-controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is directly adaptable to standard phase-contrast and polarized-light microscopes and requires a minimum amount of equipment to generate and measure pressure. Birefringence retardation (BR) og 0.1 nm up to 3,000 psi, 0.4 nm up to 5,000 psi and 1.0 nm up to 10,000 psi can be detected over a 0.75-mm central field with two strain-free Leitz 20 times UM objectives, one used as a condenser. In phase-contrast studies a Nikon DML 40 times phase objective and Zeiss model IS long working-distance phase condenser were used, with little deterioration of image quality or contrast at pressures as high as 12,000 psi. The actual design process required a synthesis of various criteria which may be categorized under four main areas of consideration: (a) specimen physiology; (b) constraints imposed by available optical equipment and standard microscope systems; (c) mechanical strength and methods for generating pressure; and (d) optical requirements of the chamber windows. Procedures for using the chambers, as well as methods for shifting and controlling the temperature within the chamber, are included.
Topics: Atmosphere Exposure Chambers; Birefringence; Cell Biology; Cell Survival; Hydrostatic Pressure; Microscopy, Phase-Contrast; Microscopy, Polarization; Optics and Photonics; Pressure; Specimen Handling; Temperature
PubMed: 1094021
DOI: 10.1083/jcb.65.3.587 -
Computational and Mathematical Methods... 2015The paper proposes an improved active contour model for segmenting and tracking accurate boundaries of the single lymphocyte in phase-contrast microscopic images. Active...
The paper proposes an improved active contour model for segmenting and tracking accurate boundaries of the single lymphocyte in phase-contrast microscopic images. Active contour models have been widely used in object segmentation and tracking. However, current external-force-inspired methods are weak at handling low-contrast edges and suffer from initialization sensitivity. In order to segment low-contrast boundaries, we combine the region information of the object, extracted by morphology gray-scale reconstruction, and the edge information, extracted by the Laplacian of Gaussian filter, to obtain an improved feature map to compute the external force field for the evolution of active contours. To alleviate initial location sensitivity, we set the initial contour close to the real boundaries by performing morphological image processing. The proposed method was tested on live lymphocyte images acquired through the phase-contrast microscope from the blood samples of mice, and comparative experimental results showed the advantages of the proposed method in terms of the accuracy and the speed. Tracking experiments showed that the proposed method can accurately segment and track lymphocyte boundaries in microscopic images over time even in the presence of low-contrast edges, which will provide a good prerequisite for the quantitative analysis of lymphocyte morphology and motility.
Topics: Animals; Cell Movement; Cell Shape; Cell Tracking; Computational Biology; Image Processing, Computer-Assisted; Lymphocytes; Mice; Microscopy, Phase-Contrast
PubMed: 26089973
DOI: 10.1155/2015/693484 -
Journal of Biomedical Optics Nov 2020Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We...
SIGNIFICANCE
Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We propose simultaneous multiplane DPC imaging using volume holographic microscopy (VHM).
AIM
To design and implement a new configuration of DPC-VHM for multiplane imaging.
APPROACH
The angularly multiplexed volume holographic gratings (AMVHGs) and the wavelength-coded volume holographic gratings (WC-VHGs) are used for this purpose. To obtain asymmetric illumination for DPC images, a dynamic illumination system is designed by modifying the regular Köhler illumination using a thin film transistor panel (TFT-panel).
RESULTS
Multidepth DPC images of standard resolution chart and biosamples were used to compare imaging performance with the corresponding bright-field images. An average contrast enhancement of around three times is observed for target resolution chart by DPC-VHM. Imaging performance of our system is studied by modulation transfer function analysis, which suggests that DPC-VHM not only suppresses the DC component but also enhances high-frequency information.
CONCLUSIONS
Proposed DPC-VHM can acquire multidepth-resolved DPC images without axial scanning. The illumination part of the system is adjustable so that the system can be adapted to bright-field mode, phase contrast mode, and DPC mode by controlling the pattern on the TFT-panel.
Topics: Diagnostic Tests, Routine; Holography; Lighting; Microscopy; Microscopy, Phase-Contrast
PubMed: 33247561
DOI: 10.1117/1.JBO.25.12.123704