2014, Walde, M., Monypenny, J., Heintzmann, R., Jones, G.E., Cox, S.

Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.

2020, Waithe, Dominic, Brown, Jill M., Reglinski, Katharina, Diez-Sevilla, Isabel, Roberts, David, Eggeling, Christian

Object detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines. © 2020 Waithe et al.

2020, Kässner F., Kirstein A., Händel N., Schmid G.L., Landgraf K., Berthold A., Tannert A., Schaefer M., Wabitsch M., Kiess W., Körner A., Garten A.

Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

2016, Lu-Walther, Hui-Wen, Hou, Wenya, Kielhorn, Martin, Arai, Yoshiyuki, Nagai, Takeharu, Kessels, Michael M., Qualmann, Britta, Heintzmann, Rainer

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.