List of imager DNAs for sequential imaging.

Multiplexed cyclic imaging in expandable tissue gels has been extensively studied to visualize numerous biomolecules at a nanoscale resolution in situ. Previous studies have employed sparse labels, such as DAPI or lectin staining, as registration markers. However, these sparse labels do not adequately capture the full extent of deformation across the entire region of interest. To overcome this challenge, we propose the use of dense labels, specifically fluorophore N-hydroxysuccinimide (NHS)-ester staining, as registration markers to achieve highly accurate image registration. We first tested several NHS-functionalized fluorophores as fiducial markers and identified the proper candidates for three-dimensional (3D) multiplexed cyclic imaging. We analyzed the registration accuracy between DAPI and NHS-ester staining and illustrated that dense label-based registration provides a more accurate registration performance. In the multiplexed imaging of expanded specimens, we observed that repetitive expansion/shrinking processes and chemical treatments for signal elimination can induce 3D nonlinear distortion. This sample distortion can be mitigated by re-embedding the tissue gel or replacing the chemical de-staining process with photobleaching-based signal removal or computational signal unmixing. With such an optimized experimental setup, we demonstrated 3D multiplexed cyclic imaging with nanoscale precision image registration. Finally, we prove that dense biological structures, such as actin, can be used as registration markers to achieve high registration accuracy. We anticipate that the proposed dense labeling strategy will overcome the technical limitations of multiplexed cyclic imaging in expandable tissue gels, offering high-precision registration. We expect it to be widely adopted by the biological and medical communities.