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land § These two authors contributed equally ABSTRACT. Measuring forces inside cells is particularly challenging. With the development of quantitative microscopy, fluorophores which allow the measurement of forces became highly desirable. We have previously introduced a mechanosensitive flipper probe, which responds to the change of plasma membrane tension by changing fluorescence lifetime and thus allows tension imaging by FLIM. Herein, we describe the design, synthesis, and evaluation of flipper probes that selectively label intracellular organelles, i.e., lysosomes, mitochondria, and the endoplasmic reticulum. The probes respond uniformly to osmotic shocks applied extracellularly, thus confirming sensitivity toward changes in membrane tension.At rest, different lifetimes found for different organelles relate to known differences in membrane organization rather than membrane tension and allow co-labeling in the same cells. At the organelle scale, lifetime heterogeneity provides unprecedented insights on ER tubules and sheets, and nuclear membranes.Examples on endosomal trafficking or increase of tension at mitochondrial constriction sites outline the potential of intracellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.The importance of mechanical forces in biological processes is only starting to emerge. 1-3 Plasma membrane tension is a topic of particular current interest because mounting evidence suggests its involvement in regulating various biochemical processes in cells. 2 Although membrane tension should also regulate membranous organelles' functions, standard techniques of force measurements, such as optical tweezers or force microscopes are difficult to apply inside of cells. 3 Therefore, the role of membrane tension in

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Super-resolution microscopies, which allow features below the diffraction limit to be resolved, have become an established tool in biological research. However, imaging throughput remains a major bottleneck in using them for quantitative biology, which requires large datasets to overcome the noise of the imaging itself and to capture the variability inherent to biological processes. Here, we develop a multi-focal flat illumination for field independent imaging (mfFIFI) module, and integrate it into an instant structured illumination microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 µm 2 without compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching multiple adjacent images together to perform fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at an effective resolution of ~35 nm. We apply classification and particle averaging to these large datasets, allowing us to map the 3D organization of post-translational modifications of centriolar microtubules, revealing differences in their coverage and positioning.