Homogeneous multifocal excitation for high-throughput super-resolution imaging

Area of Science:

• Biological imaging
• Microscopy techniques
• Quantitative biology

Background:

• Super-resolution microscopy is crucial for biological research.
• Low imaging throughput limits large dataset acquisition for quantitative biology.

Purpose of the Study:

• To enhance imaging throughput and field of view in super-resolution microscopy.
• To enable high-speed, multicolor, volumetric imaging for quantitative biological studies.

Main Methods:

• Integration of multifocal flat illumination for field-independent imaging (mfFIFI) into instant structured illumination microscopy (iSIM).
• Image stitching for extended field of view.
• Combination with expansion microscopy for ultrastructural imaging.

Main Results:

• Achieved >100 × 100 µm² field of view with iSIM, maintaining high-speed, multicolor, volumetric imaging at double the diffraction limit.
• Enabled fast live-cell super-resolution imaging of dozens of cells via image stitching.
• Acquired 3D images of thousands of centrioles per hour with ~35 nm resolution using iSIM and expansion microscopy.

Conclusions:

• Multifocal flat illumination for field-independent imaging (mfFIFI) significantly improves super-resolution microscopy throughput and field of view.
• The developed method allows for large-scale 3D mapping of centriolar structures and modifications.
• This advancement facilitates quantitative analysis of cellular components in large biological datasets.