Full-field dual-color 100-nm super-resolution imaging reveals organization and dynamics of mitochondrial and ER networks.
Most structured illumination microscopes use a physical or synthetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. In this study, we designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination pat-terns of arbitrary orientation and period, limited only by the illumination aperture (NA = 1.45), the response time of a fast, piezo-driven tip-tilt mirror (10 ms) and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrate the performance of our instrument by imaging mitochondrial dynamics in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope provides a versatile, compact and cost-effective arrangement for super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles--important questions in both cell biology and neurophysiology.
Maia
M
Brunstein
Kai
K
Wicker
Karine
K
Hérault
Rainer
R
Heintzmann
Martin
M
Oheim
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000648:ultrastructure*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000648:ultrastructure*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation*; D007368:Interferometry / Q000295:instrumentation*; D008029:Lighting / Q000295:instrumentation*; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D008928:Mitochondria / Q000648:ultrastructure*