Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions

Area of Science:

• Microscopy and Imaging
• Bacterial Cell Biology
• Biophysics

Background:

• Understanding the spatial organization of proteins within bacteria is crucial for deciphering cellular functions.
• Existing imaging techniques often lack the resolution to visualize nanoscale structural arrangements in living cells.

Purpose of the Study:

• To demonstrate quantitative multicolor 3D subdiffraction imaging of fluorescent protein fusions in living Caulobacter crescentus.
• To achieve high-precision localization of multiple fluorescent labels within bacteria.

Main Methods:

• Utilized multicolor three-dimensional (3D) subdiffraction imaging.
• Employed a flexible locally weighted image registration algorithm for super-resolution data combination.
• Implemented surface-relief dielectric phase masks with a double-helix response for multicolor localization.

Main Results:

• Achieved single-molecule localization precisions of 20-40 nm.
• Combined super-resolution data with <10 nm error using the registration algorithm.
• Successfully distinguished and precisely localized two different fluorescent labels in 3D.

Conclusions:

• Quantitative multicolor 3D subdiffraction imaging is feasible for studying bacterial structural arrangements.
• The developed methods enable precise nanoscale localization of multiple targets in living bacteria.
• This technique offers a powerful tool for advancing the study of bacterial cell biology and molecular organization.