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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.

Highlights d Mitochondria constricted by canonical fission factors do not invariably divide d The average tension for mitochondria that divide is higher than for those that do not d Depolymerizing microtubules or inhibiting myosin II reduces mitochondrial tension d Probability of division, but not of constriction, depends on mitochondrial tension

A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition (EDA) framework, in which neural networkbased recognition of specific biological events triggers real-time control in an instant structured illumination microscope (iSIM). Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because EDA allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content.

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2Mitochondria rely on cellular machinery for their division, which is an essential component of metabolic response of the cell. Many division factors have been identified; however, a framework accounting for the energetic requirements of the mitochondrial fission process is lacking. We report that the presence of an active constriction does not ensure fission. Instead, by measuring constrictions down to ~100 nm with time-lapse super-resolution microscopy, we found that 34% of constrictions failed to divide and 'reversed' to an unconstricted state. Higher local curvaturesreflecting an increased bending energy -made constriction sites more likely to divide, but often with a significant residual energy barrier to fission. Our data suggest that membrane tension, largely arising from pulling forces, could account for this missing energy. These results lead us to propose that mitochondrial fission is probabilistic, and can be modeled as arising from bending energy complemented by a fluctuating membrane tension.Mitochondria are highly dynamic organelles, transported through the cytoplasm along cytoskeletal networks as they change in size and shape. Mitochondrial morphologies can range from a filamentous, connected network to a fragmented collection of individuals. Underlying such morphological changes are altered equilibria between fusion and division 1,2 . These transformations have been linked to an adaptive response to cellular energy requirements, for example in response to stress [3][4][5] or the cell cycle 6 . As a vestige of their bacterial origins, mitochondria cannot be generated de novo, but must multiply by division, or fission, of existing mitochondria 7 . Division has also been suggested to act as part of a quality control mechanism 8,9 and an intracellular signal for mitophagy 10,11 .All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/255356 doi: bioRxiv preprint first posted online 3 In bacterial division systems, internal assembly of the fission machinery is tightly regulated and a series of cell cycle checkpoints ensure daughter cell viability. In contrast, the mitochondrial division machinery is external to the organelle, allowing cells to flexibly regulate fission. Initially, the division site is marked by a pre-constriction defined by contact with ER tubules 12 and deformed by targeted actin polymerization [13][14][15] Live-cell Structured Illumination Microscopy (SIM) of Drp1-mediated mitochondrial constriction events enabled us to measure dynamic changes in membrane geometry leading up to and following division. We discovered that a fraction of mitochondria constrict dramatically before relaxing to an unconstricted state, termed 'reversals'. Using a custom-written image analysis package and classical elasticity theory, we calculated the energy differences between fission and reversal events, which al...