Highly accurate image registration for 3D multiplexed cyclic imaging using dense labeling in expandable tissue gels

Area of Science:

• Biotechnology
• Microscopy
• Molecular Imaging

Background:

• Multiplexed cyclic imaging in expandable tissue gels visualizes biomolecules at nanoscale resolution.
• Sparse labels (e.g., DAPI) have been used for registration but fail to capture full sample deformation.
• Inaccurate registration limits the precision of nanoscale imaging in 3D expandable tissues.

Purpose of the Study:

• To develop a dense labeling strategy for highly accurate image registration in 3D multiplexed cyclic imaging.
• To overcome limitations of sparse registration markers in expandable tissue gels.
• To enable nanoscale precision in imaging complex biological samples.

Main Methods:

• Tested NHS-ester functionalized fluorophores as fiducial markers for registration.
• Compared registration accuracy between sparse (DAPI) and dense (NHS-ester) labeling.
• Optimized experimental conditions to mitigate 3D nonlinear distortion during expansion/shrinking cycles.
• Investigated photobleaching and computational unmixing as alternatives to chemical de-staining.

Main Results:

• Dense label-based registration using NHS-ester staining demonstrated superior accuracy compared to DAPI.
• Identified suitable NHS-ester fluorophores for 3D multiplexed cyclic imaging.
• Showcased mitigation strategies for 3D nonlinear distortion, including re-embedding and alternative signal removal methods.
• Achieved nanoscale precision image registration in expanded specimens using dense labeling.

Conclusions:

• Dense labeling with NHS-ester provides a robust solution for accurate image registration in expandable tissue gels.
• Optimized experimental protocols effectively address sample distortion, enhancing imaging reliability.
• This dense labeling strategy is anticipated to advance multiplexed cyclic imaging applications in biological and medical research.