At the cell - extracellular matrix interface, physiologically important traction forces exerted by angiogenic sprouts can be investigated indirectly by mapping the consecutive matrix deformations. In this paper we present an approach to study these forces in three dimensions and with high time resolution. The technique employs lightsheet microscopy, in which a sheet of light is used to illuminate the sample - resulting in z-sectioning capability, superior image recording speed and reduced phototoxicity.
For this study, human umbilical vein endothelial cells (HUVEC) are transduced with a LifeAct adenoviral vector to visualize the actin cytoskeleton during live sprouting into a collagen type I hydrogel. The calculation of the matrix deformations is formulated as a B-spline-based 3D non-rigid image registration process that warps the image of beads inside the stressed gel to match the image after stress relaxation.
Using this approach we study the role of fast moving actin filaments for filopodia- and tip-cell dynamics in 3D under chemically defined culture conditions such as inhibited acto-myosin force generation. With a time resolution in the range of ten seconds, we find that our technique is at least 20 times faster than conventional traction force microscopy based on confocal imaging. Ultimately, this approach will shed light on rapid mechano-chemical feedback mechanisms important for sprouting angiogenesis.
Christian Steuwe, Marie-Mo Vayens, Alvaro Jorge Peñas, Bartosz Krajnik, Hans Van Oosterwyck, and Maarten B. J. Roeffaers, "Three-dimensional rapid visualization of matrix deformations around angiogenic sprouts (Conference Presentation)," Proc. SPIE 10067, Optical Elastography and Tissue Biomechanics IV, 100670X (Presented at SPIE BiOS: January 30, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2253795.5380018842001.
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Study of self-shadowing effect as a simple means to realize nanostructured thin films and layers with special attentions to birefringent obliquely deposited thin films and photo-luminescent porous silicon