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

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Single-molecule super-resolution microscopy is crucial for nanoscale biological imaging.
• Challenges include high background noise and slow acquisition speeds in 3D whole-cell imaging.
• Existing methods struggle with multi-target imaging and maintaining precision.

Purpose of the Study:

• To develop an advanced microscopy platform for high-resolution, multi-target 3D imaging of whole mammalian cells.
• To overcome limitations of fluorescence background and slow imaging speeds.
• To enhance precision and efficiency in nanoscale subcellular structure analysis.

Main Methods:

• Developed a steerable, dithered, single-objective tilted light sheet (soTILT) for optical sectioning and background reduction.
• Engineered 3D nanoprinting microfluidic systems for light sheet reflection and automated solution exchange.
• Integrated point spread function engineering, deep learning for overlapping emitters, active 3D stabilization, and Exchange-PAINT for sequential multi-target imaging.

Main Results:

• The soTILT3D platform effectively reduces fluorescence background and increases imaging speed.
• Achieved high-precision nanoscale localization of individual molecules in 3D.
• Demonstrated successful whole-cell, multi-target 3D super-resolution imaging with improved performance.

Conclusions:

• soTILT3D enables efficient and precise whole-cell multi-target 3D super-resolution imaging.
• The developed microfluidic system and optical innovations address key limitations in the field.
• This platform advances the understanding of subcellular structures and their nanoscale interplay.