BeadBuddy: user-friendly, nanometer-scale registration of single-molecule imaging data

Area of Science:

• Microscopy
• Biophysics
• Optical Imaging

Background:

• Single-molecule localization microscopy (SMLM) provides nanoscale resolution in fluorescence imaging.
• Increasingly complex chromatic aberrations arise from advanced SMLM techniques like large-area cameras and multiplex imaging.
• These chromatic errors hinder accurate quantitative analysis in SMLM data.

Purpose of the Study:

• To develop an open-source software solution for modeling and correcting 3D, spatially varying chromatic errors in SMLM.
• To improve the precision and reliability of SMLM data analysis, particularly for challenging imaging conditions.

Main Methods:

• Utilized images of fluorescent beads to create a model for chromatic aberration correction.
• Developed BeadBuddy, a user-friendly software tool implementing the developed correction model.
• Applied the software to correct chromatic errors in 3D SMLM datasets.

Main Results:

• Successfully modeled and corrected 3D, spatially varying chromatic errors using fluorescent bead imaging.
• Demonstrated sub-voxel resolution in DNA Fluorescence In Situ Hybridization (FISH) experiments after correction.
• Validated the applicability of BeadBuddy across various SMLM modalities.

Conclusions:

• BeadBuddy effectively addresses complex chromatic aberrations in SMLM.
• The software enables more precise nanoscale imaging and analysis, particularly for DNA FISH.
• BeadBuddy is a versatile tool applicable to a wide range of SMLM techniques.