Rapid Fabrication of Sub‐100 µm Microstructures In Situ via Femtosecond Laser for Correlative Imaging of Rare Cells
7 affiliations
- 1Department of Mechanical and Aerospace Engineering Monash University Clayton Victoria Australia
- 2Oujiang Laboratory Zhejiang Lab for Regenerative Medicine Vision and Brain Health Wenzhou Zhejiang China
- 3Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility Clayton Victoria Australia
- 4Monash Centre for Electron Microscopy Monash University Melbourne Victoria Australia
- 5Bioimaging Platform La Trobe University Melbourne Victoria Australia
- 6Frazer Institute, Faculty of Health, Medicine and Behaviour Sciences The University of Queensland Brisbane Queensland Australia
- 7Ian Frazer for Children's Immunotherapy Research Children's Health Research Centre Faculty of Health Medicine and Behaviour Sciences The University of Queensland Brisbane Queensland Australia
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.
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BibTeX
@article{djoulde2026rapid,
title = {{Rapid Fabrication of Sub‐100 µm Microstructures In Situ via Femtosecond Laser for Correlative Imaging of Rare Cells}},
author = {Aristide Djoulde and Yaping Chen and Vahid R. Adineh and Yang Liu and Boyin Liu and Di Yu and Jing Fu},
journal = {Small (Weinheim an Der Bergstrasse, Germany)},
year = {2026},
month = sep,
doi = {10.1002/smll.75551},
url = {https://doi.org/10.1002/smll.75551},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Rapid Fabrication of Sub‐100 µm Microstructures In Situ via Femtosecond Laser for Correlative Imaging of Rare Cells
AU - Aristide Djoulde
AU - Yaping Chen
AU - Vahid R. Adineh
AU - Yang Liu
AU - Boyin Liu
AU - Di Yu
AU - Jing Fu
PY - 2026
DA - 2026/09/08
JO - Small (Weinheim an Der Bergstrasse, Germany)
DO - 10.1002/smll.75551
UR - https://doi.org/10.1002/smll.75551
AB - 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.
SN - 1613-6810
SN - 1613-6829
ER -