Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective...

Area of Science:

• Cellular and Molecular Imaging
• Biophysics
• Microscopy Techniques

Background:

• Single-molecule super-resolution microscopy is crucial for nanoscale biological insights.
• Challenges include high background noise and slow imaging speeds, particularly for 3D multi-target imaging in mammalian cells.

Purpose of the Study:

• To develop novel methods for high-resolution, 3D, multi-target imaging of whole mammalian cells.
• To overcome limitations of fluorescence background and acquisition speed in super-resolution microscopy.

Main Methods:

• Developed a steerable, dithered, single-objective tilted light sheet for optical sectioning.
• Created a 3D nanoprinting microfluidic system for light sheet reflection.
• Integrated point spread function engineering, deep learning for emitter analysis, 3D stabilization, and Exchange-PAINT.

Main Results:

• Significantly reduced fluorescence background and increased imaging speed for 3D super-resolution.
• Achieved whole-cell, multi-target 3D imaging with enhanced precision.
• Demonstrated efficient solution exchange within microfluidic channels with integrated optics.

Conclusions:

• The novel microfluidic fabrication pipeline and optical innovations enable efficient, high-speed, 3D super-resolution imaging.
• This approach significantly advances the capability to study subcellular structures and their interactions at the nanoscale.
• The adaptable system offers a powerful tool for whole-cell biological investigations.