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After data collection, post-localization 2D-to-3D transformation is applied to obtain 3D super-resolution structural and dynamic information. The complete protocol, including cell culture and sample preparation (6-7 d), SPEED imaging (4-5 h), data analysis and validation through simulation (5-13 h), takes ~9 d to complete.Microtissues with specific structures and integrated vessels play a key role in maintaining organ functions. To recapitulate the in vivo environment for tissue engineering and organ-on-a-chip purposes, it is essential to