In recent decades, investigation of the filaments of the cytoskeleton, such as actin, vimentin, and microtubules, has become increasingly important for our understanding of cellular functions. [1][2][3][4] For example, the spatial organization of the cytoskeletal network has an important role in cell migration, 5,6 cancer metastasis, 7,8 and cellular mechanics. 1,[9][10][11][12] The methods used to detect and image these structures vary from low-resolution fluorescence imaging, through high-resolution fluorescence imaging, to electron microscopy. 3,6,[13][14][15][16] The resolution of conventional light microscopy allows imaging down to 200 nm, and superresolution microscopy can now detect features with a

The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.

The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional (2D) methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the Mesh Hole Size, and Filament Length and Connectivity (also known as Coordination Number. In addition, the method allows sub-networks to be distinguished in 2D images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of 2D images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.

The cellular cortex is a 200-nm-thick actin network that lies beneath the cell membrane. It is responsible for the mechanical properties of the cell and is involved in many cellular processes, such as cell migration and interactions with the environment. To develop a clear view of the structure of this meshwork, high resolution imaging is essential, such as electron microscopy. This technique requires complex sample preparation that can lead to artifacts like shrinkage or hole formation. We present a preparation method that reduces artifacts significantly. Here, the final drying step that is typically performed by critical point drying is replaced by hexamethyldisilazane drying. We quantitatively investigated sample integrity after both preparation methods, and show that there are significant advantages of hexamethyldisilazane drying compared to critical point drying. Furthermore, automated analysis of a network is classically performed by thresholding-based software programs, which are sensitive to noise and uneven brightness of images. The here presented analysis that we have developed is based on a vectorial node algorithm. It reproduces all kinds of networks sufficiently to allow derivation of quantitative network-specific parameters, such as mesh hole size. We use this analysis to compare the network structure of cells prepared by these two drying methods, and show that hexamethyldisilazane drying leads to fewer artificial mesh holes compared to critical point drying. We thus present here a significantly improved method to quantitatively investigate the actin cortex of cells, and show that hexamethyldisilazane drying leads to more accurate imaging compared to critical point drying. Insight BoxThe highest resolution for imaging the cellular actin cortex is provided by electron microscopy. Scanning electron microscopy samples require a drying process, usually 3 achieved by critical point drying, which is critical for the sample integrity. We compare the structural defects in the actin cortex of hTert RPE1 cells after critical point drying and a chemical based method, namely hexamethyldisilazane drying. In order to characterize the actin network, we also developed a new vectorial based tracing software. We bring here new tool, both experimental and analytical, which will help to streamline studies of the actin cortex.