Bitplane Imaris 4D Software helps the Centre for Microvascular Research to understand regulation of transendothelial migration of neutrophils in vivo.
Image visualization and analysis software offers researchers a stable platform to examine long term microstructural changes in 3D, in real time
Belfast 27/10/11. The migration of neutrophils from the vascular lumen into inflamed tissues is a crucial component of the normal immune response and a significant factor in the development of inflammatory pathologies, where inappropriate inflammation contributes to tissue damage and disease progression.
The researchers used the mouse cremaster model, and in all the images the endothelial junctional protein PECAM-1 is immunofluorescently labeled (red). Leukocytes, predominantly neutrophils, express green fluorescent protein (green). Each green cell is approximately 7 µm in diameter. In the left image, an inflamed post-capillary venule is seen from the abluminal side. Leukocytes migrate through the vessel wall and into the surrounding tissues. The right image shows a luminal view of a leukocyte migrating through the junction between three endothelial cells to the sub-endothelial space. Other leukocytes can be seen crawling on the luminal surface of the venule.
A decisive step in this process is the passage of the blood neutrophils through the endothelial cells lining the lumen – transendothelial migration (TEM).
The complex structure of the vessel wall and the contribution of environmental factors, such as blood flow and local chemokine production, have limited the value of in vitro studies into the mechanisms governing this process. Now, advanced Imaris – 4D image visualization and analysis software from Bitplane is enabling in vivo analysis of the dynamics of leukocyte migration with a high degree of spatial and temporal resolution, leading to a new understanding of TEM regulation highlighting the role of junctional adhesion molecule C (JAM-C).
The work is reported in a paper by Dr Abigail Woodfin and her colleagues at the Centre for Microvascular Research, which investigates the molecular and cellular events within the microvasculature focusing on the regulation of vascular integrity, morphology and function. The Centre has a strong and internationally acknowledged expertise in the application of specialised imaging methods, including confocal intravital microscopy which allows in vivo observation of events within the microcirculation, in 3D, in real time.
“After investigating several 3D modelling platforms we chose Imaris from Bitplane. As well as a comprehensive feature set and user-friendly interface, it proved to be the most stable when analysing very large files, which was essential to our work,” says Dr Woodfin. “Because of this, Imaris enabled us to track the movement of leukocytes relative to the endothelial cells lining the vessel wall over many sequential time points.
“During long periods of analysis there is always a degree of movement with living tissue and we used the Imaris automatic Drift Correction function of ImarisTrack module to account for this. The crucial thing was that Imaris allowed us to convert our data into a virtual 3D object that could be fully manipulated in terms of rotation position, zoom, and the intensity at which each channel is displayed. This is not possible using 2D projections of Z-stacks, which is how confocal microscopy images are traditionally presented.”
“Fast, precise and easy-to-use, Imaris is a uniquely powerful and versatile solution for the visualization, analysis and interpretation of 3D and 4D images,” says Marcin Barszczewski at Bitplane. “For additional flexibility, Imaris also offers a range of several specialist modules, including MeasurementPro, Track, Cell and FilamentTracer which give Imaris users a set of powerful tools to tackle even the toughest and most complex image data sets”
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