Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Reto Fiolka, Lin Shao, E. Hesper Rego, Michael W. Davidson, Mats G L Gustafsson

Research output: Contribution to journalArticlepeer-review

250 Scopus citations


Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.

Original languageEnglish (US)
Pages (from-to)5311-5315
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number14
StatePublished - Apr 3 2012


  • Extended resolution
  • Frequency mixing
  • Multicolor
  • Patterned excitation

ASJC Scopus subject areas

  • General


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