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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 -
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 -
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 -
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 -
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 -
Journal of Microscopy Jan 2015Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample....
Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4-100×), numerical apertures (0.13-1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared.
Topics: Animals; Cell Line; Humans; Light; Mice; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Plant Cells
PubMed: 25226859
DOI: 10.1111/jmi.12181 -
Journal of Hazardous Materials Aug 2023The PCM (phase contrast microscopy) method for asbestos counting needs special sample treatments, hence it is time consuming and rather expensive. As an alternative, we...
The PCM (phase contrast microscopy) method for asbestos counting needs special sample treatments, hence it is time consuming and rather expensive. As an alternative, we implemented a deep learning procedure on images directly acquired from the untreated airborne samples using standard Mixed Cellulose Ester (MCE) filters. Several samples with a mix of chrysotile and crocidolite with different concentration loads have been prepared. Using a 20x objective lens coupled with a backlight illumination system a number of 140 images were collected from these samples, which along with additional 13 highly fibre loaded artificial images constituted the database. About 7500 fibres were manually recognised and annotated following the National Institute for Occupational Safety and Health (NIOSH) fibre counting Method 7400 as input for the training and validation of the model. The best trained model provides a total precision of 0.84 with F1-Score of 0.77 at a confidence of 0.64. A further post-detection refinement to ignore detected fibres < 5 µm in length improves the final precision. This method can be considered as a reliable and competent alternative to conventional PCM.
Topics: United States; Deep Learning; Asbestos; Asbestos, Serpentine; Microscopy, Phase-Contrast; Asbestos, Crocidolite; Occupational Exposure
PubMed: 37178531
DOI: 10.1016/j.jhazmat.2023.131590 -
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 -
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 -
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