-
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 -
Cytometry. Part a : the Journal of the... May 2017They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated...
They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm ) over a large field of view of 29.4 mm . Combined with the compactness of the system which fits altogether inside an incubator, lens-free microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry.
Topics: Cell Count; Cell Culture Techniques; Cell Movement; Epithelial Cells; Holography; Humans; Lenses; Microscopy, Video
PubMed: 28240818
DOI: 10.1002/cyto.a.23079 -
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 -
Journal of Cell Science Feb 2011Metastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fully understood. Although histological... (Review)
Review
Metastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fully understood. Although histological techniques have provided important information, they give only a static image and thus compromise interpretation of this dynamic process. New advances in intravital microscopy (IVM), such as two-photon microscopy, imaging chambers, and multicolor and fluorescent resonance energy transfer imaging, have recently been used to visualize the behavior of single metastasizing cells at subcellular resolution over several days, yielding new and unexpected insights into this process. For example, IVM studies showed that tumor cells can switch between multiple invasion strategies in response to various densities of extracellular matrix. Moreover, other IVM studies showed that tumor cell migration and blood entry take place not only at the invasive front, but also within the tumor mass at tumor-associated vessels that lack an intact basement membrane. In this Commentary, we will give an overview of the recent advances in high-resolution IVM techniques and discuss some of the latest insights in the metastasis field obtained with IVM.
Topics: Basement Membrane; Cell Movement; Extracellular Matrix; Humans; Microscopy, Fluorescence, Multiphoton; Microscopy, Video; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic
PubMed: 21242309
DOI: 10.1242/jcs.072728 -
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 -
Optics Express Mar 2010We introduce optical solenoid beams, diffractionless solutions of the Helmholtz equation whose diffraction-limited in-plane intensity peak spirals around the optical...
We introduce optical solenoid beams, diffractionless solutions of the Helmholtz equation whose diffraction-limited in-plane intensity peak spirals around the optical axis, and whose wavefronts carry an independent helical pitch. Unlike other collimated beams of light, appropriately designed solenoid beams have the noteworthy property of being able to exert forces on illuminated objects that are directed opposite to the direction of the light's propagation. We demonstrate this through video microscopy observations of a colloidal sphere moving upstream along a holographically projected optical solenoid beam.
Topics: Algorithms; Colloids; Holography; Light; Microscopy, Video; Models, Statistical; Optical Tweezers; Optics and Photonics; Physics; Software
PubMed: 20389718
DOI: 10.1364/OE.18.006988 -
Journal of Cell Science Feb 2023Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes....
Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.
Topics: Microscopy; Imaging, Three-Dimensional; Microscopy, Video
PubMed: 36727532
DOI: 10.1242/jcs.260728 -
Journal of Visualized Experiments : JoVE Feb 2015Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical...
Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.
Topics: Animals; Blood Vessels; Disease Models, Animal; Hemostasis; Microscopy, Video; Time-Lapse Imaging; Wound Healing; Zebrafish
PubMed: 25742284
DOI: 10.3791/52460 -
BMC Bioinformatics Nov 2018Large-scale pairwise drug combination analysis has lately gained momentum in drug discovery and development projects, mainly due to the employment of advanced...
BACKGROUND
Large-scale pairwise drug combination analysis has lately gained momentum in drug discovery and development projects, mainly due to the employment of advanced experimental-computational pipelines. This is fortunate as drug combinations are often required for successful treatment of complex diseases. Furthermore, most new drugs cannot totally replace the current standard-of-care medication, but rather have to enter clinical use as add-on treatment. However, there is a clear deficiency of computational tools for label-free and temporal image-based drug combination analysis that go beyond the conventional but relatively uninformative end point measurements.
RESULTS
COMBImage is a fast, modular and instrument independent computational framework for in vitro pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies. Jointly with automated analyses of temporal changes in cell morphology and confluence, it performs and displays conventional cell viability and synergy end point analyses. The image processing algorithms are parallelized using Google's MapReduce programming model and optimized with respect to method-specific tuning parameters. COMBImage is shown to process time-lapse microscopy movies from 384-well plates within minutes on a single quad core personal computer. This framework was employed in the context of an ongoing drug discovery and development project focused on glioblastoma multiforme; the most deadly form of brain cancer. Interesting add-on effects of two investigational cytotoxic compounds when combined with vorinostat were revealed on recently established clonal cultures of glioma-initiating cells from patient tumor samples. Therapeutic synergies, when normal astrocytes were used as a toxicity cell model, reinforced the pharmacological interest regarding their potential clinical use.
CONCLUSIONS
COMBImage enables, for the first time, fast and optimized pairwise drug combination analyses of temporal changes in label-free video microscopy movies. Providing this jointly with conventional cell viability based end point analyses, it could help accelerating and guiding any drug discovery and development project, without use of cell labeling and the need to employ a particular live cell imaging instrument.
Topics: Algorithms; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Survival; Drug Discovery; Drug Therapy, Combination; Glioblastoma; Humans; Image Processing, Computer-Assisted; Microscopy, Video; Motion Pictures
PubMed: 30477419
DOI: 10.1186/s12859-018-2458-x -
Journal of Visualized Experiments : JoVE Sep 2011The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation(1,2). In the postcapillary venules of...
The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation(1,2). In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling(3). Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)(4). Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment(2,5). In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm(3) size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.
Topics: Animals; Cell Migration Assays, Leukocyte; Cell Movement; Chemokine CXCL2; Chemotactic Factors; Chemotaxis, Leukocyte; Endothelium, Vascular; Male; Mice; Microscopy, Video; Neutrophils; Oligopeptides; Sepharose
PubMed: 21968530
DOI: 10.3791/3296