Correlative Workflows
Cryo-Correlative, Multimodal
Imaging Workflows
Bringing Non-destructive, Whole-Cell Context to Cryo-CLEM Imaging
Correlative Light, Electron, and X-ray Microscopy (CLEXM)
Correlative Light, Electron, and X-ray Microscopy (CLEXM) integrates fluorescence microscopy, soft X-ray tomography, and electron microscopy into s single, non-destructive workflow on the same cryo-preserved sample. By linking molecular signals with whole-cell ultrastructure and nanometer-scale detail, CLEXM provides essential context across imaging scales. This content is critical for interpreting molecular function within the 3D architecture of intact cells, tissues and organoids. Because the workflow is non-destructive, samples can be re-imaged and correlated, enabling researchers to study disease mechanisms and therapeutic effects with greater confidence and depth.
Principles Behind CLEXM
Multimodal Data Acquisition from Same Sample
Cryo-CLEXM ensures that fluorescence, soft X-ray, and electron microscopy data are all collected from the same vitrified specimen. This eliminates sample-to-sample variation, strengthens correlations, and provides researchers with complementary molecular, structural and ultrastructural information within one coherent dataset.
Spatial Registration Across Imaging Modalities
Accurate spatial registration aligns fluorescence signals, 3D SXT reconstructions and electron microscopy detail. This principle ensures that features identified in one modality can be precisely located in another, providing confidence in interpreting molecular function in the context of ultrastructural organization.
Integration Across Spatial Scales
Cryo-CLEXM bridges the gap between molecular-level detail and larger tissue or organoid architecture. By integrating imaging across scales, it enables researchers to connect localized molecular events to broader cellular tissue and structures, advancing understanding of disease mechanisms and therapeutic responses.
Cryo Preservation of Native State
Maintaining samples in a vitrified state preserves ultrastructure and molecular distribution close to physiological conditions. By carefully managing the total soft X-ray photon flux, radiation damage is minimized and only becomes visible at low, single-digit Ångström resolutions, ensuring non-destructive imaging and reliable multimodal correlation.
Benefits of Cryo CLEXM Workflow
Cryo-CLEXM directly links molecular signals from fluorescence with ultrastructural context from SXT and EM. This enables researchers to correlate biological function with cellular architecture, providing deeper insights into disease mechanisms and therapeutic effects at multiple levels of organisation.
Combining multiple imaging modalities on the same sample streamlines workflows, reduces trial-and-error targeting, and maximizes data output while lowering sample preparation demands, making advanced multimodal imaging more accessible and practical for researchers.
Cryo-preservation and single-sample multimodal imaging greatly reduce artefacts associated with chemical fixation, sectioning or staining. Because correlation occurs on the same vitrified sample, the risk of damage or loss during transfers is minimized, ensuring higher data fidelity and reproducibility.
Example Imaging Workflows
In Situ Lamella
In the in-situ lamella workflow, cells grown on TEM grids are plunge-frozen and first screened with the SXT-100’s integrated fluorescence microscope to identify suitable targets. Non-destructive SXT then images the intact cell in 3D, providing essential whole-cell context and guiding the selection of a region of interest. A cryo-FIB-SEM thins this region into a ~150 nm lamella, registering its position within the whole-cell volume. The same grid is then transferred directly to a cryo-TEM where, without changing the holder, a high-resolution tomogram is produced and correlated with the SXT image to strengthen interpretation.
Cryo Lift-out
In this workflow, TEM grids containing high-pressure frozen tissue are screened by fluorescence microscopy to locate isolated fluorescent larvae within grid holes. A cryo-FIB-SEM then cuts successive 6-8 μm thick transversal slabs, which are lifted out and laid flat on a second grid. Each slab is imaged by SXT to identify and register regions of interest. These regions are subsequently thinned into lamella using cryo-FIB-SEM and analysed by cryo-TEM. This approach increases throughput and, by capturing the entire organisms volume, provides invaluable contextual information for interpreting high-resolution tomograms.
Thin Grid Waffle Freezing
Cells can be prepared on thin electron microscopy (EM) grids with approximately 10 µm bars and then high-pressure frozen using the waffle method. These samples can subsequently be imaged directly in the SXT-100 without requiring additional processing. This technique is especially advantageous for samples thicker than a few microns, as they can benefit from the enhanced vitrification quality provided by high-pressure freezing.
Driving Discoveries with
3D Cell Imaging
SXT provides the definitive native contrast unbiased insights of the cellular ultrastructure, uninhibited by complex and artifact prone sample preparation techniques and unreliant on fluorescent labels where biochemistry is unknown or signal is weak. This makes it ideal for assessing cellular morphology in whole frozen-hydrated cells as well as pairing with FM and EM techniques.
SXT visualises whole cell morphology, quantitatively capturing complex 3D networks, rare events and nanoparticle distribution to complement high resolution EM data.
The recent development of the SXT-100 microscope opens up the possibility of integrating this novel technique into light and electron imaging workflows. A typical volume reconstruction of 5000 – 10000 µm³ can be acquired in approximately one hour. FIB targeting and downstream electron microscopy to relate molecular behaviour to large scale structure.
Cryofixation is a technique used to rapidly preserve biological specimens by freezing them at extremely low temperatures. It is particularly valuable for cell and tissue imaging because it preserves cellular structures in a near-native state, minimising damage caused by the processes of fixation, dehydration, or embedding, which can occur with conventional fixation methods. Single cells are typically frozen by plunge freezing while tissue samples up to 200 μm can be high pressure frozen with 10 μm sections extracted for SXT.
The SXT-100 enables researchers to address central questions in cell biology, providing insights into how the entire organelle network spatially arranges within the cytoplasm of the cell. Analysis of SXT data reveals the statistical variation in morphological features across cell populations or conditions. Many key cellular processes can be investigated such as chromatin rearrangement, virus–host interactions, cell motility, parasite life cycle, and lymphocyte activation and function.
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