Feeling for Filaments

BPJ_109_4.c1.inddThe cover image on the Biophysical Journal issue released August 18, 2015 features a live cultured endothelial cell that was imaged using two different techniques: The green fluorescence part was recorded with an optical confocal microscope and displays actin filaments inside the cell in a maximum intensity projection.  The greyscale square shows a shaded visualization of the sample surface as probed by atomic force microscopy (AFM).

The AFM is capable of ‘feeling’ the rigid structures of the cortical cytoskeleton through the cell membrane, hence the rough appearance of the cell’s surface. We developed a technique to image this cortical cytoskeleton web with high resolution and quantify its density. This enables us to observe dynamic behavior and study the effects of pharmacological treatments. Simultaneous confocal fluorescence microscopy enhances the method in terms of molecular labeling; it both allows correlation with AFM data and completes the three-dimensional view of the cell towards the basal side.

Our aim is to elucidate the link between the structure of the cortical actin cytoskeleton, cell mechanics, and physiology. The research in vascular endothelium is inspired by findings on how cell mechanics determine endothelial function. Thus it deals with the pathophysiological background of hypertension and other cardiovascular diseases. Another application can be found in oncogenesis and metastasis, where cell mechanics are also altered.

Our paper is also, hopefully, an interesting read for anyone using AFM in other cell imaging applications because of the methodological issues it addresses: Force stability with uncoated cantilevers, contact-mode versus newer, fast force mapping modes, image processing to extract and segment features on large scale objects, and more.

More information on the endothelial physiology research, AFM mechanobiology and microscopy applications can be found on our website.

–Cornelius Kronlage, Marco Schäfer-Herte, Daniel Böning, Hans Oberleithner, and Johannes Fels

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