Workflow and instrumentation for multiscale correlative imaging in voluminous cryogenic specimens
- 1European Molecular Biology Laboratory
Abstract
Cryo-electron tomography (cryo-ET) enables molecular-resolution imaging of intracellular environments, but is constrained by the need to generate electron-transparent sections of the biological specimen and thus a limited imaging volume. To alleviate such limitations, we present a multiscale correlative workflow that links cryo-confocal fluorescence microscopy (cryo-FLM), cryo-focused ion beam-scanning electron microscopy (cryo-FIB-SEM) volume imaging (cryo-vEM), and cryo-FIB serial lamellae lift-out for cryo-ET in high-pressure frozen voluminous specimens. An integral part of this workflow is a sample holder that preserves specimen orientation across instruments, reduces manual handling, and overcomes stage-tilt limitations in cryo-FIB-SEM hardware. Applied to vitrified colorectal cancer organoids, we demonstrate that this workflow enables fluorescence-guided site preparation for cryo-vEM, which provides biological context beyond the limited field of view of cryo-ET, and targeted lift-out for fabrication of multiple lamellae from defined regions of interest. The reconstructed tomograms reveal subcellular architecture and cell-cell interfaces. This workflow establishes a modular methodological framework for continuum-resolution cryogenic imaging of complex 3D biological specimens.
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BibTeX
@misc{dimprima2026workflow,
title = {{Workflow and instrumentation for multiscale correlative imaging in voluminous cryogenic specimens}},
author = {E. D'Imprima and E. Zagoriy and S. Mimietz-Oekler and R. Kirmse and A. Hallady and P. N. Mbonu and P. Ronchi and M. Jechlinger and J. Mahamid},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.14.759080},
url = {https://www.biorxiv.org/content/10.64898/2026.10.14.759080v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Workflow and instrumentation for multiscale correlative imaging in voluminous cryogenic specimens
AU - E. D'Imprima
AU - E. Zagoriy
AU - S. Mimietz-Oekler
AU - R. Kirmse
AU - A. Hallady
AU - P. N. Mbonu
AU - P. Ronchi
AU - M. Jechlinger
AU - J. Mahamid
PY - 2026
DA - 2026/10/14
PB - bioRxiv
DO - 10.64898/2026.10.14.759080
UR - https://www.biorxiv.org/content/10.64898/2026.10.14.759080v1
AB - Cryo-electron tomography (cryo-ET) enables molecular-resolution imaging of intracellular environments, but is constrained by the need to generate electron-transparent sections of the biological specimen and thus a limited imaging volume. To alleviate such limitations, we present a multiscale correlative workflow that links cryo-confocal fluorescence microscopy (cryo-FLM), cryo-focused ion beam-scanning electron microscopy (cryo-FIB-SEM) volume imaging (cryo-vEM), and cryo-FIB serial lamellae lift-out for cryo-ET in high-pressure frozen voluminous specimens. An integral part of this workflow is a sample holder that preserves specimen orientation across instruments, reduces manual handling, and overcomes stage-tilt limitations in cryo-FIB-SEM hardware. Applied to vitrified colorectal cancer organoids, we demonstrate that this workflow enables fluorescence-guided site preparation for cryo-vEM, which provides biological context beyond the limited field of view of cryo-ET, and targeted lift-out for fabrication of multiple lamellae from defined regions of interest. The reconstructed tomograms reveal subcellular architecture and cell-cell interfaces. This workflow establishes a modular methodological framework for continuum-resolution cryogenic imaging of complex 3D biological specimens.
ER -