Super-resolution fluorescence imaging based on localization microscopy requires tuning the photoblinking properties of fluorescent dyes employed. Missing is a rapid way to analyze the blinking rates of the fluorophore probes. Herein we present an ensemble autocorrelation technique for rapidly and simultaneously measuring photoblinking and bleaching rate constants from a microscopy image time series of fluorescent probes that is significantly faster than individual single-molecule trajectory analysis approaches. Our method is accurate for probe densities typically encountered in single-molecule studies as well as for higher density systems which cannot be analyzed by standard single-molecule techniques. We also show that we can resolve characteristic blinking times that are faster than camera detector exposure times, which cannot be accessed by threshold-based single-molecule approaches due to aliasing. We confirm this through computer simulation and single-molecule imaging data of DNA-Cy5 complexes. Finally, we demonstrate that with sufficient sampling our technique can accurately recover rates from stochastic optical reconstruction microscopy super-resolution data.

The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.

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The human skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically-relevant mechanical forces lead to small, repairable membrane injuries in bone-forming osteoblasts, resulting in the release of ATP and stimulation of purinergic (P2) calcium responses in neighbouring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, extracellular diffusion and degradation, and purinergic responses in neighboring cells. The model was validated using experimental data obtained by measuring intracellular free calcium ([Ca 2+ ]i) elevations following mechanical stimulation of a single osteoblast. The validated single-cell injury model was then scaled to a tissue-level injury to investigate how purinergic responses communicate information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed the maximal distance from the injury at which purinergic responses were stimulated, as well as the overall number of responders. The peak ATP concentration reflected the severity of an individual cell injury and determined signal propagation velocity and temporal synchrony of responses. Peak ATP concentrations also discriminated between minor and severe injuries that led to the release of similar total amounts of ATP due to differences in injury repair dynamics. The third component was ADP-mediated signaling which became relevant only in larger tissue-level injuries, and it conveyed information about the distance to the injury site and its geometry. Taken together, this study identified specific features of extracellular ATP/ADP spatiotemporal signals that encode the severity of the mechanical stimulus, the distance from the stimulus, as well as the mechanoresilient status of the tissue.

Collagen alignment, also known as tumor associated collagen signature, has been used to predict worse prognosis in breast cancer. It has been shown in breast cancer studies that when collagen is aligned perpendicular to the tumor, it allows for cancer invasion. Cirrhosis is a risk factor for liver cancer