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Scientific Reports Mar 2017We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric...
We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric module, which generates sufficient data for reversing the halo artifact. Acquiring four independent intensity images, our approach first measures haloed phase maps of the sample. We solve for the halo-free sample transmission function by using a physical model of the image formation under partial spatial coherence. Using this halo-free sample transmission, we can numerically generate artifact-free PC images. Furthermore, this transmission can be further used to obtain quantitative information about the sample, e.g., the thickness with known refractive indices, dry mass of live cells during their cycles. We tested our hfPC method on various control samples, e.g., beads, pillars and validated its potential for biological investigation by imaging live HeLa cells, red blood cells, and neurons.
Topics: Artifacts; Erythrocytes; HeLa Cells; Humans; Image Processing, Computer-Assisted; Microscopy, Phase-Contrast; Models, Theoretical; Neurons; Refractometry
PubMed: 28338086
DOI: 10.1038/srep44034 -
Optics Express Mar 2022Panoramic and long-term observation of nanosized organelle dynamics and interactions with high spatiotemporal resolution still hold great challenge for current imaging...
Panoramic and long-term observation of nanosized organelle dynamics and interactions with high spatiotemporal resolution still hold great challenge for current imaging platforms. In this study, we propose a live-organelle imaging platform, where a flat-fielding quantitative phase contrast microscope (FF-QPCM) visualizes all the membrane-bound subcellular organelles, and an intermittent fluorescence channel assists in specific organelle identification. FF-QPCM features a high spatiotemporal resolution of 245 nm and 250 Hz and strong immunity against external disturbance. Thus, we could investigate several important dynamic processes of intracellular organelles from direct perspectives, including chromosome duplication in mitosis, mitochondrial fusion and fission, filaments, and vesicles' morphologies in apoptosis. Of note, we have captured, for the first time, a new type of mitochondrial fission (entitled mitochondrial disintegration), the generation and fusion process of vesicle-like organelles, as well as the mitochondrial vacuolization during necrosis. All these results bring us new insights into spatiotemporal dynamics and interactions among organelles, and hence aid us in understanding the real behaviors and functional implications of the organelles in cellular activities.
Topics: Microscopy; Microscopy, Phase-Contrast; Mitochondria; Organelles
PubMed: 35299377
DOI: 10.1364/OE.454023 -
Philosophical Transactions of the Royal... Jun 2008Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is... (Review)
Review
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 pi/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 pi. 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. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.
Topics: Cells; Cryoelectron Microscopy; Microscopy, Electron, Transmission; Microscopy, Phase-Contrast; Proteins; Viruses
PubMed: 18339604
DOI: 10.1098/rstb.2008.2268 -
Cytometry. Part a : the Journal of the... May 2017
Topics: Animals; Flow Cytometry; Humans; Microscopy, Phase-Contrast
PubMed: 28544798
DOI: 10.1002/cyto.a.23130 -
PLoS Computational Biology Nov 2023Reliable detection and classification of bacteria and other pathogens in the human body, animals, food, and water is crucial for improving and safeguarding public...
Reliable detection and classification of bacteria and other pathogens in the human body, animals, food, and water is crucial for improving and safeguarding public health. For instance, identifying the species and its antibiotic susceptibility is vital for effective bacterial infection treatment. Here we show that phase contrast time-lapse microscopy combined with deep learning is sufficient to classify four species of bacteria relevant to human health. The classification is performed on living bacteria and does not require fixation or staining, meaning that the bacterial species can be determined as the bacteria reproduce in a microfluidic device, enabling parallel determination of susceptibility to antibiotics. We assess the performance of convolutional neural networks and vision transformers, where the best model attained a class-average accuracy exceeding 98%. Our successful proof-of-principle results suggest that the methods should be challenged with data covering more species and clinically relevant isolates for future clinical use.
Topics: Humans; Deep Learning; Microscopy, Phase-Contrast; Neural Networks, Computer; Bacteria; Bacterial Infections
PubMed: 37956197
DOI: 10.1371/journal.pcbi.1011181 -
Scientific Reports Jul 2019An optical microscope enables image-based findings and diagnosis on microscopic targets, which is indispensable in many scientific, industrial and medical settings. A...
An optical microscope enables image-based findings and diagnosis on microscopic targets, which is indispensable in many scientific, industrial and medical settings. A standard benchtop microscope platform, equipped with e.g., bright-field and phase-contrast modes, is of importance and convenience for various users because the wide-field and label-free properties allow for morphological imaging without the need for specific sample preparation. However, these microscopes never have capability of acquiring molecular contrast in a label-free manner. Here, we develop a simple add-on optical unit, comprising of an amplitude-modulated mid-infrared semiconductor laser, that is attached to a standard microscope platform to deliver the additional molecular contrast of the specimen on top of its conventional microscopic image, based on the principle of photothermal effect. We attach this unit, termed molecular-contrast unit, to a standard phase-contrast microscope, and demonstrate high-speed label-free molecular-contrast phase-contrast imaging of silica-polystyrene microbeads mixture and molecular-vibrational spectroscopic imaging of HeLa cells. Our simple molecular-contrast unit can empower existing standard microscopes and deliver a convenient accessibility to the molecular world.
Topics: HeLa Cells; Humans; Lasers, Semiconductor; Light; Microscopy, Phase-Contrast; Microspheres; Molecular Imaging; Polystyrenes; Silicon Dioxide; Spectrophotometry, Infrared
PubMed: 31316091
DOI: 10.1038/s41598-019-46383-6 -
The International Journal of... Mar 2017Label-free imaging uses inherent contrast mechanisms within cells to create image contrast without introducing dyes/labels, which may confound results. Quantitative... (Review)
Review
Label-free imaging uses inherent contrast mechanisms within cells to create image contrast without introducing dyes/labels, which may confound results. Quantitative phase imaging is label-free and offers higher content and contrast compared to traditional techniques. High-contrast images facilitate generation of individual cell metrics via more robust segmentation and tracking, enabling formation of a label-free dynamic phenotype describing cell-to-cell heterogeneity and temporal changes. Compared to population-level averages, individual cell-level dynamic phenotypes have greater power to differentiate between cellular responses to treatments, which has clinical relevance e.g. in the treatment of cancer. Furthermore, as the data is obtained label-free, the same cells can be used for further assays or expansion, of potential benefit for the fields of regenerative and personalised medicine.
Topics: Cell Cycle; Cell Lineage; Cell Movement; Cell Tracking; Cytological Techniques; Humans; Microscopy, Interference; Microscopy, Phase-Contrast; Phenotype; Single-Cell Analysis
PubMed: 28111333
DOI: 10.1016/j.biocel.2017.01.004 -
Ultramicroscopy Nov 2020Phase plates (PPs) are beneficial devices to improve the phase contrast of life-science objects in cryo-transmission electron microscopy (TEM). The development of the... (Comparative Study)
Comparative Study
Phase plates (PPs) are beneficial devices to improve the phase contrast of life-science objects in cryo-transmission electron microscopy (TEM). The development of the hole-free (HF) PP, which consists of a thin carbon film, has led to impressive results due to its ease in fabrication, implementation and application. However, the phase shift of the HFPP can be controlled only indirectly. The electrostatic Zach PP uses a strongly localized and adjustable electrostatic potential to generate well-defined and variable phase shifts between scattered and unscattered electrons. However, artifacts in phase-contrast TEM images are induced by the presence of the PP rod in the diffraction plane. We present a detailed analysis and comparison of the contrast-enhancing capabilities of both PP types and their emerging artifacts. For this purpose, cryo-TEM images of a standard T4-bacteriophage test sample were acquired with both PP types. Simulated images reproduce the experimental images well and substantially contribute to the understanding of contrast formation. An electrostatic Zach PP was used in this work to acquire cryo-electron tomograms with enhanced contrast, which are of similar quality as tomograms obtained by HFPP TEM.
Topics: Artifacts; Bacteriophage T4; Computer Simulation; Cryoelectron Microscopy; Electrons; Histocytological Preparation Techniques; Microscopy, Electron, Transmission; Microscopy, Phase-Contrast
PubMed: 32781400
DOI: 10.1016/j.ultramic.2020.113086 -
Magnetic Resonance in Medical Sciences... Mar 2022To extract the status of hydrocephalus and other cerebrospinal fluid (CSF)-related diseases, a technique to characterize the cardiac- and respiratory-driven CSF motions...
PURPOSE
To extract the status of hydrocephalus and other cerebrospinal fluid (CSF)-related diseases, a technique to characterize the cardiac- and respiratory-driven CSF motions separately under free breathing was developed. This technique is based on steady-state free precession phase contrast (SSFP-PC) imaging in combination with a Stockwell transform (S-transform).
METHODS
2D SSFP-PC at 3 T was applied to measure the CSF velocity in the caudal-cranial direction within a sagittal slice at the midline (N = 3) under 6-, 10-, and 16-s respiratory cycles and free breathing. The frequency-dependent window width of the S-transform was controlled by a particular scaling factor, which then converted the CSF velocity waveform into a spectrogram. Based on the frequency bands of the cardiac pulsation and respiration, as determined by the electrocardiogram (ECG) and respirator pressure sensors, Gaussian bandpass filters were applied to the CSF spectrogram to extract the time-domain cardiac- and respiratory-driven waveforms.
RESULTS
The cardiac-driven CSF velocity component appeared in the spectrogram clearly under all respiratory conditions. The respiratory-driven velocity under the controlled respiratory cycles was observed as constant frequency signals, compared to a time-varying frequency signal under free breathing. When the widow width was optimized using the scale factor, the temporal change in the respiratory-driven CSF component was even more apparent under free breathing.
CONCLUSION
Velocity amplitude variations and transient frequency changes of both cardiac- and respiratory-driven components were successfully characterized. These findings indicated that the proposed technique is useful for evaluating CSF motions driven by different cyclic forces.
Topics: Cerebrospinal Fluid; Heart; Magnetic Resonance Imaging; Microscopy, Phase-Contrast; Motion; Respiration
PubMed: 35173115
DOI: 10.2463/mrms.mp.2021-0126 -
Journal of Visualized Experiments : JoVE Aug 2008Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942,...
Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
Topics: Microscopy, Interference; Microscopy, Phase-Contrast
PubMed: 19066508
DOI: 10.3791/844