A Fast Projection Imaging Method for the Quantification of the Dynamics of Endosome Maturation

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Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live-cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast ‘projections’ from the field of depth and shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocal and instant structured-illumination-microscopy (iSIM) microscopes. Since the initial publication of the method, we have made several improvements on system stablization and stepping speed, and have implemented the system onto two other imaging platforms other than our own microscope.

  Hu, X., Jalal, S., Sheetz, M., Bakke, O., Margadant, F., 2020. Micro-stepping Extended Focus reduces photobleaching and preserves structured illumination super-resolution features. J. Cell. Sci. https://doi.org/10.1242/jcs.240796