Fluorescence microscopy is the standard imaging technique to investigate the structures and dynamics of living cells. However, increasing the spatial resolution comes at the cost of temporal resolution and vice versa. In addition, the number of images that can be taken in sufficiently high quality is limited by fluorescence bleaching. Hence, super-resolved imaging at several Hertz of low fluorescent biological samples is still a big challenge and, especially in structured illumination microscopy (SIM), is often visible as imaging artifacts. In this paper, we present a TIRF-SIM system based on scan-mirrors and a Michelson interferometer, which generates images at 110 nm spatial resolution and up to 8 Hz temporal resolution. High resolution becomes possible by optimizing the illumination interference contrast, even for low fluorescent, moving samples. We provide a framework and guidelines on how the modulation contrast, which depends on laser coherence, polarization, beam displacement or sample movements, can be mapped over the entire field of view. In addition, we characterize the influence of the signal-to-noise ratio and the Wiener filtering on the quality of reconstructed SIM images, both in real and frequency space. Our results are supported by theoretical descriptions containing the parameters leading to image artifacts. This study aims to help microscopists to better understand and adjust optical parameters for structured illumination, thereby leading to more trustworthy measurements and analyses of biological dynamics.
The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis.
Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.
Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargos. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis and find that clathrin coats assemble first on flat membranes to ~50% of the coat area, before they become rapidly and continuously bent. We introduce a mathematical model that assumes a positive feedback for curvature generation of the clathrin coat. This Cooperative Curvature Model agrees excellently with experimental data in three cell lines, and likely describes a general pathway for clathrin coat remodeling during endocytosis.
Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.
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