Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm...

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Structured-illumination microscopy (SIM) doubles light microscope resolution.
• Achieving higher resolution requires nonlinear sample responses to illumination.
• Existing nonlinear SIM methods use high light intensities, damaging biological samples.

Purpose of the Study:

• To develop a biologically compatible superresolution imaging method.
• To achieve high resolution using ultralow light intensities.

Main Methods:

• Utilized nonlinear structured-illumination microscopy (NL-SIM).
• Employed reversible photoswitching of fluorescent proteins for nonlinearity.
• Applied ultralow light intensities, six orders of magnitude lower than saturation.

Main Results:

• Demonstrated approximately 40-nm resolution on purified microtubules using the photoswitchable protein Dronpa.
• Successfully visualized mammalian nuclear pore and actin cytoskeleton structures.
• Achieved superresolution imaging compatible with biological samples.

Conclusions:

• Nonlinear structured-illumination microscopy with photoswitchable proteins is a viable superresolution technique.
• This method overcomes the limitations of high light intensity in traditional nonlinear SIM.
• Enables high-resolution imaging of delicate biological structures.