-
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 -
Journal of Critical Care Feb 2021To assess the presence of sublingual microcirculatory and skin perfusion alterations in COVID-19 pneumonia. (Observational Study)
Observational Study
PURPOSE
To assess the presence of sublingual microcirculatory and skin perfusion alterations in COVID-19 pneumonia.
MATERIALS AND METHODS
This is a preliminary report of a prospective observational study performed in four teaching intensive care units. We studied 27 mechanically ventilated patients with acute respiratory distress syndrome secondary to COVID-19. Sublingual microcirculation was assessed by hand-held videomicroscopy. A software-assisted analysis of videos was performed. We also measured capillary refill time.
RESULTS
Patients were hemodynamically stable with normal lactate (1.8 [1.6-2.5] mmol/L) and high D-dimer (1.30 [0.58-2.93] μg/mL). Capillary refill time was prolonged (3.5 [3.0-5.0] s). Compared to previously reported normal values, total and perfused vascular density (21.9 ± 3.9 and 21.0 ± 3.5 mm/mm) and heterogeneity flow index (0.91 ± 0.24) were high; and the proportion of perfused vessels (0.96 ± 0.03), microvascular flow index (2.79 ± 0.10), and red blood cell velocity (1124 ± 161 μm/s) were reduced. The proportion of perfused vessels was inversely correlated with total vascular density (Pearson r = -0.41, P = 0.03).
CONCLUSIONS
COVID-19 patients showed an altered tissue perfusion. Sublingual microcirculation was characterized by decreases in the proportion of perfused vessel and flow velocity along with high vascular densities. This last finding might be related to enhanced angiogenesis or hypoxia-induced capillary recruitment.
Topics: Aged; COVID-19; Capillaries; Critical Care; Female; Hemodynamics; Humans; Hypoxia; Intensive Care Units; Male; Microcirculation; Microscopy, Video; Middle Aged; Mouth Floor; Perfusion; Prospective Studies; Respiration, Artificial; Respiratory Distress Syndrome; Skin; Software
PubMed: 33096349
DOI: 10.1016/j.jcrc.2020.10.002 -
Medicina Intensiva 2017Microcirculatory alterations play a pivotal role in sepsis-related morbidity and mortality. However, since the microcirculation has been a "black box", current... (Review)
Review
Microcirculatory alterations play a pivotal role in sepsis-related morbidity and mortality. However, since the microcirculation has been a "black box", current hemodynamic management of septic patients is still guided by macrocirculatory parameters. In the last decades, the development of several technologies has shed some light on microcirculatory evaluation and monitoring, and the possibility of incorporating microcirculatory variables to clinical practice no longer seems to be beyond reach. The present review provides a brief summary of the current technologies for microcirculatory evaluation, and attempts to explore the potential role and benefits of their integration to the resuscitation process in critically ill septic patients.
Topics: Arterioles; Blood Gas Analysis; Capillaries; Carbon Dioxide; Hemodynamics; Humans; Hypoxia; Microcirculation; Microscopy, Video; Monitoring, Physiologic; Oximetry; Oxygen; Partial Pressure; Point-of-Care Systems; Resuscitation; Sepsis; Spectroscopy, Near-Infrared
PubMed: 28104277
DOI: 10.1016/j.medin.2016.11.011 -
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 -
Annals of the American Thoracic Society Aug 2016Primary ciliary dyskinesia (PCD) is a rare, heterogeneous, recessive, genetic disorder of motile cilia, leading to chronic upper and lower respiratory symptoms.... (Review)
Review
Primary ciliary dyskinesia (PCD) is a rare, heterogeneous, recessive, genetic disorder of motile cilia, leading to chronic upper and lower respiratory symptoms. Prevalence is estimated at around 1:10,000, but many patients remain undiagnosed, whereas others receive the label incorrectly. Proper diagnosis is complicated by the fact that the key symptoms, such as wet cough, chronic rhinitis, and recurrent upper and lower respiratory infection, are common and nonspecific. There is no single gold standard test to diagnose PCD. Currently, the diagnosis is made in patients with a compatible medical history after a demanding combination of tests including nasal nitric oxide, high-speed video microscopy, and transmission electron microscopy and genetic and ciliary culture testing. These tests are costly and need sophisticated equipment and experienced staff, restricting use to highly specialized centers. Therefore, it would be desirable to have a screening test for identifying those patients who should undergo detailed diagnostic testing. Three recent studies focused on potential screening tools: one study assessed the validity of nasal nitric oxide for screening, and two studies developed new symptom-based screening tools. These simple tools are welcome, and it is hoped that they will assist physicians in determining whom to refer for definitive testing. However, they have been developed in tertiary care settings, where 10 to 50% of tested patients have PCD. The sensitivity and specificity of the tools are reasonable, but positive and negative predictive values may be poor in primary or secondary care settings. Although these studies are an important step toward an earlier diagnosis of PCD, more remains to be done before we have tools tailored to different health care settings.
Topics: Cilia; Diagnostic Techniques, Respiratory System; Early Diagnosis; Genetic Testing; Humans; Kartagener Syndrome; Microscopy, Electron, Transmission; Microscopy, Video; Nitric Oxide; Sensitivity and Specificity
PubMed: 27258773
DOI: 10.1513/AnnalsATS.201605-331PS -
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 -
Journal of Immunological Methods Dec 2022Although parallel plate flow chamber assays are widely performed, extraction of kinetic parameters is limited to specialized labs with mathematical expertise and...
Although parallel plate flow chamber assays are widely performed, extraction of kinetic parameters is limited to specialized labs with mathematical expertise and customized video-microscopy tracking tools. The recent development of Trackmate has increased researcher accessibility to tracking particles in video-microscopy experiments; however, there is a lack of tools that analyze this tracking information. We report a software tool, compatible with Trackmate, that extracts Receptor Ligand Non-Equilibrium Kinetic (RLNEK) parameters from video-microscopy data. This software should be of particular interest to the community of researchers and scientists interrogating the target-specific binding and release of immune cells.
Topics: Ligands
PubMed: 36341963
DOI: 10.1016/j.jim.2022.113381 -
Nature Medicine Feb 2020Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends...
Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.
Topics: Adolescent; Adult; Cell Separation; Child; Ciliopathies; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Exome; Female; Flow Cytometry; HEK293 Cells; Homozygote; Humans; Microscopy, Phase-Contrast; Microscopy, Video; Mucociliary Clearance; Mutation; NIMA-Related Kinases; Phenotype; Proteome; Respiratory System; Tomography, X-Ray Computed; X-Ray Microtomography; Young Adult
PubMed: 31959991
DOI: 10.1038/s41591-019-0730-x -
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