Rapid Fabrication of Sub‐100 µm Microstructures In Situ via Femtosecond Laser for Correlative Imaging of Rare Cells

ABSTRACT Correlative light, electron, and ion microscopy provides a powerful multiscale framework for analyzing single cells, but integrating these methods remains challenging due to divergent specimen preparation requirements, especially for rare cell populations where low yield and sample loss limit success. Herein, we introduce a femtosecond (fs) laser ablation strategy that enables rapid material removal and targeted fabrication of 3D sub‐100‐µm microstructures on resin‐embedded biological substrates. The workflow is demonstrated using flow‐sorted mouse CD4 + T‐cell subsets: regulatory (Treg) and effector (Teff) cells. Following resin embedding, fs‐laser ablation was used to fabricate a half‐grid geometry featuring an array of micropillars (∼30 µm tip diameter), each containing single or multiple target cells while limiting laser‐induced damage to ∼3 µm. We validated the approach by preparing electron‐transparent membranes using a hybrid fs‑laser/focused ion beam scanning electron microscopy (FIB‐SEM) workflow and performing 3D FIB‐SEM tomography on single cells positioned on the fabricated micropillar tips. These analyses revealed well‐preserved subcellular architecture and enabled direct ultrastructural comparison between the T‐cell subsets. The grid geometry is also compatible with additional downstream analytical techniques, expanding opportunities for nanoscale chemical analysis. Overall, this work establishes a practical pathway that integrates precision micro/nanoengineering with high‑resolution correlative imaging for robust analysis of rare and limited cell populations.