Full-field dual-color 100-nm super-resolution imaging reveals organization and dynamics of mitochondrial and ER networks

Opt Express. 2013 Nov 4;21(22):26162-73. doi: 10.1364/OE.21.026162.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Astrocytes / ultrastructure*
  • Cells, Cultured
  • Endoplasmic Reticulum / ultrastructure*
  • Equipment Design
  • Equipment Failure Analysis
  • Humans
  • Image Enhancement / instrumentation*
  • Interferometry / instrumentation*
  • Lighting / instrumentation*
  • Mice
  • Microscopy, Fluorescence, Multiphoton / instrumentation*
  • Mitochondria / ultrastructure*