Description
Expansion microscopy (ExM) provides a unique high-resolution solution for biological imaging that physically increases the dimension of biological samples to bypass the constraints of the light diffraction limit and avoid the requirement for sophisticated optical set-up. However, the expansion process may introduce physical distortions in the gel, compromising the accuracy for the 3D visualization of nanometer-scale cellular structures. Here, we present our efforts in applying vertically aligned nanopillar arrays in conjunction with ExM to achieve distortion calibration in 3D. Specifically, we fabricate ordered arrays of nanopillars with known coordinates along the x, y, and z axes. By referencing to the known coordinates of these nanopillars, we were able to accurately calibrate cellular protein positions in 3D. Furthermore, with nanopillar array-enabled 3D calibration, we located proteins involving in podosome rosettes—clusters of individual podosomes critical for cell migration and bone degradation, whose structural organization is lack of study. Using nanopillar array-enabled 3D calibration, we significantly improved the accuracy of podosome protein localization in 3D, enhancing our understanding of the podosome rosette's intricate organization. Our work provides a new solution to a robust 3D distortion calibration method for ExM, increasing the accuracy and reliability of nanoscale imaging for detailed cellular structures in 3D.
