Combining 3D single molecule localization strategies for reproducible bioimaging

Area of Science:

• Biophysics
• Microscopy
• Optical Imaging

Background:

• Super-resolution microscopy is crucial for visualizing cellular structures.
• Existing methods often struggle with axial localization accuracy and stability.

Purpose of the Study:

• To develop a 3D super-resolution technique with high and stable axial localization precision.
• To enable robust multicolor 3D imaging in biological samples.

Main Methods:

• Utilized point spread function (PSF) shaping and supercritical angle fluorescence (SAF) for axial localization.
• Employed a dual-view imaging scheme to decouple and optimize axial and lateral detection.
• Demonstrated multicolor 3D super-resolution imaging of cellular and bacterial structures.

Main Results:

• Achieved a slowly varying localization precision down to 15nm over a 1μm range.
• Provided absolute axial information, yielding weakly anisotropic 3D resolution.
• Demonstrated drift-free, tilt-insensitive, and achromatic imaging performance.

Conclusions:

• The developed technique is readily implementable and robust against experimental biases.
• It is well-suited for multicolor 3D super-resolution microscopy of biological samples.
• The method facilitates imaging across multiple depths for comprehensive biological studies.