Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

Area of Science:

• Biophysics
• Optical Microscopy
• Cell Biology

Background:

• Single-molecule super-resolution imaging requires optimal signal-to-background ratio (SBR) for precise 3D localization in thick mammalian cells.
• Existing methods often lack versatility in optimizing SBR for diverse cellular structures.

Purpose of the Study:

• To develop a versatile multimodal illumination platform for 3D single-molecule super-resolution imaging.
• To enable target-specific SBR optimization for enhanced nanoscale studies of cellular architecture.

Main Methods:

• Integration of light sheet (LS), epi-illumination, and total internal reflection fluorescence (TIRF) illumination schemes.
• Utilizing commercially available parts for a versatile and switchable optical platform.
• Point spread function (PSF) engineering for improved 3D localization.

Main Results:

• Demonstration of a multimodal platform enabling 3D single-molecule super-resolution imaging throughout mammalian cells.
• Flat-field TIRF illumination improved data quality for coverslip-adjacent structures with low background and uniform kinetics.
• LS illumination provided increased contrast for imaging structures throughout the cell.

Conclusions:

• The developed platform facilitates precise 3D nanoscale studies by optimizing SBR for different cellular targets.
• This versatile microscopy approach enhances super-resolution imaging capabilities for cellular architecture investigations.
• Validated for two-color imaging of focal adhesion complexes and actin in human osteosarcoma cells.