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University of New South Wales Australia

6 ARTICLES PUBLISHED IN JoVE

Biology

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

¹Department of Developmental Biology, University of Pittsburgh

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Neuroscience

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Huazheng Liang^1,2, Emma Schofield¹, George Paxinos^1,2

¹Brain Structure and Function Group, Neuroscience Research Australia, ²School of Medical Sciences, The University of New South Wales

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Immunology and Infection

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

George Ashdown¹, Elvis Pandžić³, Andrew Cope², Paul Wiseman⁴, Dylan Owen¹

¹Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, ²Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Disease, King's College London, ³ARC Centre for Advanced Molecular Imaging, Australian Centre for NanoMedicine, University of New South Wales Australia, ⁴Departments of Chemistry and Physic, McGill University

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Bioengineering

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

Murray C. Killingsworth^1,2,3,4, Yuri V. Bobryshev^3,4,5

¹South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales Australia, ²School of Medicine, Western Sydney University, ³Correlative Microscopy Group, Ingham Institute for Applied Medical Research, ⁴Electron Microscopy Laboratory, Department of Anatomical Pathology, Sydney South West Pathology Service, New South Wales Health Pathology, ⁵School of Medical Sciences, Faculty of Medicine, University of New South Wales Australia

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Biology

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

Huazheng Liang^*1,2, Bassem Akladios^*1, Cesar P. Canales^*1, Richard Francis³, Edna H. Hardeman¹, Annemiek Beverdam¹

¹The School of Medical Sciences, University of New South Wales Australia, ²Neuroscience Research Australia, ³Biomedical Imaging Facility, University of New South Wales Australia

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Medicine

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Katelin M. Allan^1,2, Sharon L. Wong^1,2, Laura K. Fawcett^1,2,3, Alexander Capraro^1,2, Adam Jaffe^1,2,3, Cristan Herbert⁴, Elvis Pandzic⁵, Shafagh A. Waters^1,2,3

¹School of Women's and Children's Health, Faculty of Medicine and Health, University of New South Wales, ²Molecular and Integrative Cystic Fibrosis Research Centre (miCF RC), Faculty of Medicine and Health, University of New South Wales, ³Department of Respiratory Medicine, Sydney Children's Hospital, ⁴Department of Pathology, School of Medical Sciences, University of New South Wales Australia, ⁵Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, University of New South Wales

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.