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Westmoreland D.,University College London | Westmoreland D.,Endothelial Cell Biology Laboratory | Shaw M.,National Physical Laboratory United Kingdom | Grimes W.,University College London | And 9 more authors.
Journal of Thrombosis and Haemostasis | Year: 2016

Background: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. Objective: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. Methods: Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. Results: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. Conclusions: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders. © 2016 International Society on Thrombosis and Haemostasis.


LoBiondo J.,Nikon Instruments | Friedman M.M.,AccuMed International
Current Protocols in Cytometry | Year: 2011

The objective is the most crucial image-forming component of a microscope. A knowledge of the many types of objectives available and their characteristics is critical to the selection of appropriate objectives for image cytometry. This unit discusses aberrations in image formation and their correction, construction, and types of objectives, and objectives for other microscopy applications, explaining the advantages and limitations of each one. © 2011 by John Wiley & Sons, Inc.


LoBiondo J.,Nikon Instruments
Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] | Year: 2011

The objective is the most crucial image-forming component of a microscope. A knowledge of the many types of objectives available and their characteristics is critical to the selection of appropriate objectives for image cytometry. This unit discusses aberrations in image formation and their correction, construction, and types of objectives, and objectives for other microscopy applications, explaining the advantages and limitations of each one.


Davis M.A.,Nikon Instruments | Prazsky O.,Laboratory Imaging | Sysko L.R.,Nikon Instruments
Current Protocols in Cytometry | Year: 2015

Time-lapse imaging is a rich data source offering potential kinetic information of cellular activity and behavior. Tracking and extracting measurements of objects from time-lapse datasets are challenges that result from the complexity and dynamics of each object's motion and intensity or the appearance of new objects in the field of view. A wide range of strategies for proper data sampling, object detection, image analysis, and post-analysis interpretation are available. Theory and methods for single-particle tracking, spot detection, and object linking are discussed in this unit, as well as examples with step-by-step procedures for utilizing semi-automated software and visualization tools for achieving tracking results and interpreting this output. © 2015 by John Wiley & Sons, Inc.


Drent P.,Nikon Instruments
Biochemist | Year: 2010

Optical light microscopy is set to enter a new era of super-resolution with the development of technologies that overcome the resolution limit of traditional light microscopes. Ideal for a variety of disciplines within the biological sciences, these new technologies enable the study of cell structure at the nanoscale, revealing cellular features previously impossible to see. Whereas nanoscale imaging has been possible for many years using electron microscopy methods, the new super-resolution optical technologies enable two-dimensional and three-dimensional imaging of fixed and/or living specimens. In this article, we provide a brief overview of the development of super-resolution microscopy and Nikon's offering for super-resolution fluorescence imaging, N-SIM, with lateral resolution twice that of conventional light microscopes and suitable for fixed and live cell imaging, and N-STORM, which achieves a remarkable lateral resolution of approximately 20 nm and axial resolution of approximately 50 nm in fixed specimens. © 2010 The Biochemical Society.

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