Abstract
In order to unravel rapid mechano-chemical feedback mechanisms in sprouting angiogenesis, we combine selective plane illumination microscopy (SPIM) and tailored image registration algorithms - further referred to as SPIM-based displacement microscopy - with an in vitro model of angiogenesis. SPIM successfully tackles the problem of imaging large volumes while upholding the spatial resolution required for the analysis of matrix displacements at a subcellular level. Applied to in vitro angiogenic sprouts, this unique methodological combination relates subcellular activity - minute to second time scale growing and retracting of protrusions - of a multicellular systems to the surrounding matrix deformations with an exceptional temporal resolution of 1 minute for a stack with multiple sprouts simultaneously or every 4 seconds for a single sprout, which is 20 times faster than with a conventional confocal setup. Our study reveals collective but non-synchronised, non-continuous activity of adjacent sprouting cells along with correlations between matrix deformations and protrusion dynamics.
Publication types
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Research Support, Non-U.S. Gov't
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Video-Audio Media
MeSH terms
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Algorithms
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Cell Culture Techniques / methods
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Collagen Type I
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Fiducial Markers
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Human Umbilical Vein Endothelial Cells
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Humans
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Hydrogels
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Imaging, Three-Dimensional / methods*
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Intravital Microscopy / methods*
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Microscopy, Fluorescence / methods
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Microspheres
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Neovascularization, Physiologic / physiology*
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Time-Lapse Imaging*
Substances
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Collagen Type I
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Hydrogels
Grants and funding
The authors are grateful for funding support from the Research Foundation Flanders (FWO,
https://www.fwo.be/en/) (postdoctoral fellowship to C.S., FWO grants G.0821.13, G0B9615N, G087018N, G.0B49.15, ZW15_09 GOH6316N) and from the European Research Council (ERC,
https://erc.europa.eu/) under the European Union's Seventh Framework Program (FP7/2007-2013)/ ERC Grant Agreement n°308223) to H.V.O. J.H. acknowledges support of the Flemish government through long term structural funding Methusalem (CASAS2, Meth/15/04) (KU Leuven internal funding,
https://www.kuleuven.be/english/research/support/if/methusalem). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.