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Journal of Visualized Experiments : JoVE Feb 2018Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is...
Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 10 cell morphological measurements.
Topics: Cell Culture Techniques; Humans; Microscopy, Video
PubMed: 29553497
DOI: 10.3791/56580 -
Microcirculation (New York, N.Y. : 1994) Oct 2022The aim of this study was to describe possible remodeling (i.e., dilatation and elongation) of papillary capillaries induced by increased oxygen demand for the repair...
OBJECTIVE
The aim of this study was to describe possible remodeling (i.e., dilatation and elongation) of papillary capillaries induced by increased oxygen demand for the repair process following a skin wound.
METHODS
Computer-assisted video microscopy was used to examine 10 healthy volunteers before (baseline) and after (≈1 h and ≈24 h) an incision (5 mm long and 1 mm deep) on the forearm, 0-1 mm and 30 mm (control site) from the incision. We defined categories from 0 (low) to 3 (high) to grade dilatation and elongation of the nutritive papillary capillaries, as well as the visibility of the superficial vascular plexus. Approximately 10 000 capillaries from 200 films were scored.
RESULTS
The nutritive papillary capillaries were dilated and elongated (p < 0.01) after ≈24 h; that is, elongation (score 1.9 ± 0.9) vs baseline (score 0.9 ± 0.6), p < 0.01 and dilatation (score 2.2 ± 0.7) vs baseline (score 0.3 ± 0.3), p < 0.01. Superficial plexus visibility increased (p < 0.01) after ≈1 h (score 2.0 ± 0.7) and ≈24 h (score 2.7 ± 0.3) vs baseline (score 0.8 ± 0.4).
CONCLUSION
The superficial vascular skin plexus showed enhanced visibility already ≈1 h after the skin trauma. Morphological remodeling in the nutritive papillary capillaries-dilatation and elongation after ≈24 h-facilitate increased O supply.
Topics: Humans; Microcirculation; Capillaries; Skin; Microscopy, Video; Forearm
PubMed: 35231135
DOI: 10.1111/micc.12755 -
Physiological Reviews Oct 1999To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing... (Review)
Review
To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.
Topics: Animals; Calcium; Fluorescent Dyes; Humans; Intracellular Fluid; Microscopy, Confocal; Microscopy, Fluorescence; Microscopy, Video
PubMed: 10508230
DOI: 10.1152/physrev.1999.79.4.1089 -
Scientific Reports Jul 2022Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a...
Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a single-particle characterization technology that determines the size and index of refraction of individual particles using the Lorenz-Mie theory of light scattering. Although assessment of cell viability is a challenge in many applications, including biologics manufacturing, traditional approaches often include unreliable labeling with dyes and/or time consuming methods of manually counting cells. In this work we measured the viability of Saccharomyces cerevisiae yeast in the presence of various concentrations of isopropanol as a function of time. All THC measurements were performed in the native environment of the sample with no dilution or addition of labels. Holographic measurements were made with an in-line holographic microscope using a 40[Formula: see text] objective lens with plane wave illumination. We compared our results with THC to manual counting of living and dead cells as distinguished with trypan blue dye. Our findings demonstrate that THC can effectively distinguish living and dead yeast cells by the index of refraction of individual cells.
Topics: Coloring Agents; Holography; Microscopy; Microscopy, Video; Saccharomyces cerevisiae
PubMed: 35882977
DOI: 10.1038/s41598-022-17098-y -
Experimental Eye Research Nov 2022Non-invasive imaging techniques are increasingly used to objectively quantify anterior segment structures of the eye. In this study, we apply the novel oxygen delivery...
Non-invasive imaging techniques are increasingly used to objectively quantify anterior segment structures of the eye. In this study, we apply the novel oxygen delivery index (ODIN) concept that, quantifies microvascular capacity for oxygen delivery, to the ocular surface in healthy humans. The purpose of the study was to test the applicability of the technologies used for data acquisition from the human ocular surface. We also validated whether the ODIN concept has sufficient sensitivity to detect and differentiate between microvascular structure and function in limbal and bulbar conjunctiva. Multiple ocular surface measurements using computer-assisted video microscopy (field of view: 1.6 mm × 0.9 mm) and diffuse reflectance spectroscopy (measuring volume: ∼0.1 mm3) were obtained from limbal and bulbar conjunctiva in 20 healthy volunteers. Three parameters were extracted during analyses: Functional capillary density, capillary flow velocity, and microvascular oxygen saturation. Functional capillary density was higher at limbus than in bulbar conjunctiva (11.2 ± 1.8 c/mm versus 5.2 ± 1.2 c/mm, p < 0.01), and microvascular oxygen saturation was lower at limbus (77 ± 8%) as compared to bulbar conjunctiva (89 ± 6%), p < 0.01. More than 80% of scored capillaries had continuous blood flow and no difference was seen between the recording sites (p = 0.68). In conclusion, the ODIN concept is applicable for the assessment of human ocular surface microvascular function and has sufficient sensitivity to detect increased capillary density and oxygen extraction at limbus as compared with bulbar conjunctiva.
Topics: Humans; Microcirculation; Microscopy, Video; Conjunctiva; Spectrum Analysis; Oxygen; Computers
PubMed: 36055389
DOI: 10.1016/j.exer.2022.109232 -
Experimental Eye Research Dec 2020In piglets we tested the applicability of digital video microscopy and diffuse reflectance spectroscopy for non-invasive assessments of limbal and bulbar conjunctival...
In piglets we tested the applicability of digital video microscopy and diffuse reflectance spectroscopy for non-invasive assessments of limbal and bulbar conjunctival microcirculation. A priori we postulated that the metabolic rate is higher in limbal as compared to bulbar conjunctiva, and that this difference is reflected in microvascular structure or function between the two locations. Two study sites, Oslo University Hospital (OUH), Norway and Cleveland Clinic (CC), USA, used the same video microscopy and spectroscopy techniques to record limbal and bulbar microcirculation in sleeping piglets. Recordings were analyzed with custom-made software to quantify functional capillary density, capillary flow velocity and microvascular oxygen saturation in measuring volumes of approximately 0.1 mm. The functional capillary density was higher in limbus than in bulbar conjunctiva at both study sites (OUH: 18.1 ± 2.9 versus 12.2 ± 2.9 crossings per mm line, p < 0.01; CC: 11.3 ± 3.0 versus 7.1 ± 2.8 crossings per mm line, p < 0.01). Median categorial capillary blood flow velocity was higher in bulbar as compared with limbal recordings (CC: 3 (1-3) versus 1 (0-3), p < 0.01). Conjunctival microvascular oxygen saturation was 88 ± 5.9% in OUH versus 94 ± 7.5% in CC piglets. Non-invasive digital video microscopy and diffuse reflectance spectroscopy can be used to obtain data from conjunctival microcirculation in piglets. Limbal conjunctival microcirculation has a larger capacity for oxygen delivery as compared with bulbar conjunctiva.
Topics: Animals; Blood Flow Velocity; Conjunctiva; Female; Image Processing, Computer-Assisted; Male; Microcirculation; Microscopy, Video; Microvessels; Models, Anatomic; Models, Animal; Spectrum Analysis; Swine
PubMed: 33157128
DOI: 10.1016/j.exer.2020.108312 -
Bioinformatics (Oxford, England) Oct 2023Reliable label-free methods are needed for detecting and profiling apoptotic events in time-lapse cell-cell interaction assays. Prior studies relied on fluorescent...
MOTIVATION
Reliable label-free methods are needed for detecting and profiling apoptotic events in time-lapse cell-cell interaction assays. Prior studies relied on fluorescent markers of apoptosis, e.g. Annexin-V, that provide an inconsistent and late indication of apoptotic onset for human melanoma cells. Our motivation is to improve the detection of apoptosis by directly detecting apoptotic bodies in a label-free manner.
RESULTS
Our trained ResNet50 network identified nanowells containing apoptotic bodies with 92% accuracy and predicted the onset of apoptosis with an error of one frame (5 min/frame). Our apoptotic body segmentation yielded an IoU accuracy of 75%, allowing associative identification of apoptotic cells. Our method detected apoptosis events, 70% of which were not detected by Annexin-V staining.
AVAILABILITY AND IMPLEMENTATION
Open-source code and sample data provided at https://github.com/kwu14victor/ApoBDproject.
Topics: Humans; Microscopy, Video; Time-Lapse Imaging; Neural Networks, Computer; Extracellular Vesicles; Annexins
PubMed: 37773981
DOI: 10.1093/bioinformatics/btad584 -
The Journal of Biological Chemistry Jun 1999
Review
Topics: Cholesterol Oxidase; Computer Simulation; Enzymes; Fluorescence; Kinetics; Microscopy, Video; Models, Chemical; Numerical Analysis, Computer-Assisted; Protein Conformation; Structure-Activity Relationship
PubMed: 10347141
DOI: 10.1074/jbc.274.23.15967 -
The Biological Bulletin Aug 2016In 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research... (Review)
Review
In 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research at the Marine Biological Laboratory (MBL), which quickly became Inoué's scientific home. Primed by his Japanese mentor, Katsuma Dan, Inoué followed Dan's mantra to work with healthy, living cells, on a fundamental problem (mitosis), with a unique tool set that he refined for precise and quantitative observations (polarized light microscopy), and a fresh and brilliant mind that was unafraid of challenging current dogma. Building on this potent combination, Inoué contributed landmark observations and concepts in cell biology, including the notion that there are dynamic, fine structures inside living cells, in which molecular assemblies such as mitotic spindle fibers exist in delicate equilibrium with their molecular building blocks suspended in the cytoplasm. In the late 1970s and 1980s, Inoué and others at the MBL were instrumental in conceiving video microscopy, a groundbreaking technique which married light microscopy and electronic imaging, ushering in a revolution in how we know and what we know about living cells and the molecular mechanisms of life. Here, we recount some of Inoué's accomplishments and describe how his legacy has shaped current activities in polarized light imaging at the MBL.
Topics: Cell Biology; Cell Physiological Phenomena; Cells; History, 20th Century; History, 21st Century; Image Processing, Computer-Assisted; Microscopy, Polarization; Microscopy, Video; Microtubules; Mitosis
PubMed: 27638697
DOI: 10.1086/689593 -
Immunity Sep 2004Recent advances in photonics, particularly multi-photon microscopy (MPM) and new molecular and genetic tools are empowering immunologists to answer longstanding... (Review)
Review
Recent advances in photonics, particularly multi-photon microscopy (MPM) and new molecular and genetic tools are empowering immunologists to answer longstanding unresolved questions in living animals. Using intravital microscopy (IVM) investigators are dissecting the cellular and molecular underpinnings controlling immune cell motility and interactions in tissues. Recent IVM work showed that T cell responses to antigen in lymph nodes are different from those observed in vitro and appear dictated by factors uniquely relevant to intact organs. Other IVM models, particularly in the bone marrow, reveal how different anatomic contexts regulate leukocyte development, immunity, and inflammation. This article will discuss the current state of the field and outline how IVM can generate new discoveries and serve as a "reality check" for areas of research that were formerly the exclusive domain of in vitro experimentation.
Topics: Animals; Cell Communication; Cell Movement; Dendritic Cells; Diagnostic Imaging; Humans; Image Processing, Computer-Assisted; Microscopy; Microscopy, Video; Models, Immunological; T-Lymphocytes
PubMed: 15357943
DOI: 10.1016/j.immuni.2004.08.006