3D-Aligner: An advanced computational tool designed to correct image distortion in expansion microscopy for precise 3D reconstitution and quantitative analysis.

Expansion Microscopy (ExM) is an innovative and cost-effective super-resolution microscopy technique that has become popular in cell biology research. It achieves super-resolution by physically expanding specimens. Since its introduction, ExM has undergone continuous methodological developments to enhance its resolution and labeling capabilities. However, ExM imaging often encounters sample drift during image acquisition due to the physical movement of the expanded hydrogel, posing a significant challenge for accurate image reconstruction. Despite many proposed experimental solutions to mitigate sample drift, a universal solution has yet to be established. In response to this challenge, we developed 3D-Aligner, an advanced and user-friendly image analysis tool designed to computationally correct drift in ExM images for precise three-dimensional image reconstruction and downstream quantification. We demonstrate that 3D-Aligner effectively determines and corrects drift in ExM images with different expansion rates and various fluorescently labeled biological targets, showcasing its capabilities and robustness in drift correction. Additionally, we validate the precision of 3D-Aligner by comparing drift values across different labeled targets and highlight the importance of drift correction in quantification of biological structures.