Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts

Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by α-actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.

Super-resolved live-cell imaging using Random Illumination Microscopy

SummarySuper-resolution fluorescence microscopy has been instrumental to progress in biology. Yet, the photo-induced toxicity, the loss of resolution into scattering samples or the complexity of the experimental setups curtail its general use for functional cell imaging. Here, we describe a new technology for tissue imaging reaching a 114nm/8Hz resolution at 30 µm depth. Random Illumination Microscopy (RIM) consists in shining the sample with uncontrolled speckles and extracting a high-fidelity super-resolved image from the variance of the data using a reconstruction scheme accounting for the spatial correlation of the illuminations. Super-resolution unaffected by optical aberrations, undetectable phototoxicity, fast image acquisition rate and ease of use, altogether, make RIM ideally suited for functional live cell imaging in situ. RIM ability to image molecular and cellular processes in three dimensions and at high resolution is demonstrated in a wide range of biological situations such as the motion of Myosin II minifilaments in Drosophila.

Active-modulated, random-illumination, super-resolution optical fluctuation imaging

Super-resolution optical fluctuation imaging (SOFI) provides subdiffraction resolution based on the analysis of temporal stochastic intensity fluctuations.

Aphid Identification and Counting Based on Smartphone and Machine Vision

Exact enumeration of aphids before the aphids outbreak can provide basis for precision spray. This paper designs counting software that can be run on smartphones for real-time enumeration of aphids. As a first step of the method used in this paper, images of the yellow sticky board that is aiming to catch insects are segmented from complex background by using GrabCut method; then the images will be normalized by perspective transformation method. The second step is the pretreatment on the images; images of aphids will be segmented by using OSTU threshold method after the effect of random illumination is eliminated by single image difference method. The last step of the method is aphids’ recognition and counting according to area feature of aphids after extracting contours of aphids by contour detection method. At last, the result of the experiment proves that the effect of random illumination can be effectively eliminated by using single image difference method. The counting accuracy in greenhouse is above 95%, while it can reach 92.5% outside. Thus, it can be seen that the counting software designed in this paper can realize exact enumeration of aphids under complicated illumination which can be used widely. The design method proposed in this paper can provide basis for precision spray according to its effective detection insects.