Visualizing primary cilia with 1-nm localization precision via expansion single-molecule localization microscopy

In search of a higher resolved image, a number of superresolution techniques surrounding the combination of single molecule localization microscopy (SMLM) and expansion strategies (ExM) developed rapidly. However, the effective resolution often restricts itself in relation to the expansion factor of the given sample. Resolving nano-scaled structures becomes a difficult task with the scarcity of information on this integration of superresolution techniques. To shed light on this, we propose a solution by combining a ten-fold robust expansion technique (TREx) with SMLM, termed TREx-SMLM, to achieve localization precision to an ∼1-nm regime. This solution compares and attentively selects different conditions in the re-embedding process prior to SMLM imaging, allowing TREx to be readily applied to SMLM with highly retained expansion factor. Moreover, we not only refine but lay out the foundation during the sample preparation process detailing each step in the procedure for simplicity and clarity. This overall workflow optimizes the gel rigidity, improves fluorescent signals, and promotes the feasible combination of expansion strategies to SMLM. To this, TREx-SMLM achieves an unprecedented imaging of cellular structures, specifically, axonemal microtubule, among other Ex-SMLM modalities.