Understanding cellular behaviour at the subcellular level is essential for advancing both disease research and drug discovery. Imaging cells in their natural context within tissue or 3D models provides a more accurate representation of their architecture, interactions, and functionality compared to traditional 2D models. Unlike flat cultures, 3D models mimic the complexity of in vivo environments, preserving the spatial relationships and biochemical gradients that influence cellular behaviour. This enables researchers to observe phenomena such as cellular differentiation, drug responses, and disease progression in conditions that closely mirror reality, offering deeper insights and more predictive data for therapeutic development.

High-pressure freezing enables larger tissue samples or nano-biopsies, up to 200 microns thick, to be vitrified, preserving their native structure. Traditionally, to achieve high-resolution imaging (approx 1 nanometre) in specific regions of interest, cryo-focused ion beam (cryo-FIB) milling is used to extract “lift-outs” or “slabs” from the vitrified sample. These are then thinned to approximately 200 nanometers for imaging in a cryo-transmission electron microscope (cryo-TEM). However, this process has a significant drawback: a large portion of the original sample is destroyed during FIB milling, eliminating valuable structural context that could have provided insights into the surrounding tissue architecture and its relationship to the imaged lamella.

Integrating Soft X-ray Microscopy (SXM) into this workflow addresses this limitation by allowing an intermediate imaging step. Instead of directly thinning the sample to 200 nanometers, cryo-FIB is first used to produce 10-micron-thick slabs of tissue, which can then be imaged using SXM. This approach preserves much more of the sample’s structural context while achieving resolutions of approximately 40 nanometers. After SXM imaging, the same slabs can be further thinned to 200 nanometers for cryo-TEM imaging of the regions of interest.

Additional benefits of Integrating SXM into tissue imaging workflows:

• Improved Region-of-Interest Identification: SXM enables precise localization of regions of interest with sub-50-nanometer resolution, increasing the likelihood of capturing the desired structures in the final cryo-TEM lamella.
• Preservation of Structural Context: SXM imaging of the 10-micron slabs provides a broader structural overview, helping researchers interpret the subcellular features observed in the cryo-TEM within the context of the larger tissue environment.
• Reduced Risk of Sample Loss: By imaging thicker slabs before thinning, SXM reduces the chance of missing critical regions during the milling process, as the regions of interest are already mapped and identified.
• Complementary Modalities: The multimodal approach leverages the strengths of both SXM and cryo-TEM—SXM’s ability to image larger volumes at high resolution and cryo-TEM’s nanoscale detail—providing a more comprehensive understanding of the sample.
• Increased Efficiency: The SXM-guided workflow minimizes trial-and-error during cryo-FIB milling, potentially reducing the time and resources needed to produce high-quality lamella.
• Enhanced Quantitative Analysis: SXM’s ability to image larger tissue volumes enables researchers to perform quantitative analyses on cellular and subcellular structures across broader contexts, complementing the localized insights from cryo-TEM.

This integrated approach not only maximizes the value of each sample but also provides a richer dataset for understanding biological processes and disease mechanisms.

Click here to see a working example of cryo lift-out imaging on a C. elegans worm sample.