Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Structured illumination microscopy (SIM) is a super-resolution technique.
• Existing SIM methods have limitations in imaging depth and speed for live multicellular organisms.

Purpose of the Study:

• To develop a 3D super-resolution imaging method for live multicellular organisms.
• To overcome limitations in imaging depth and speed of conventional SIM.

Main Methods:

• Utilized structured illumination microscopy (SIM) with sparse multifocal illumination patterns generated by a digital micromirror device (DMD).
• Physically rejected out-of-focus light to achieve 3D subdiffractive imaging.
• Achieved imaging speeds of one 2D image per second with resolutions of 145 nm laterally and 400 nm axially.

Main Results:

• Enabled 3D super-resolution imaging in samples eightfold thicker than previously possible with SIM.
• Successfully imaged GFP-labeled microtubules in live zebrafish embryos at depths greater than 45 micrometers.
• Captured dynamic cellular processes, including interactions between myosin IIA and F-actin, in 4D super-resolution without apparent phototoxicity.

Conclusions:

• The developed method provides a simpler, more accessible approach to 3D super-resolution microscopy.
• This technique facilitates advanced imaging of live biological systems, opening new avenues for research.
• The method is compatible with commercially available components and open-source software, promoting wider adoption.