SXT-100 Standalone Imaging Workflow
(CLXM)

The SXT-100 supports a wide variety of sample-mounting formats to accommodate diverse research needs. Standard TEM grids and clipped grids are commonly used, while glass capillaries and half-grids provide alternatives for specific applications. For high-pressure frozen samples, custom holders enable full ±90° tilt rotation, ensuring complete tomographic data collection. This flexibility maximizes compatibility with existing cryo-workflows and broadens the range of biological specimens that can be imaged.

Cells are prepared on carbon-coated gold EM finder grids or in thin-walled glass capillaries. After incubation, grids are screened under a light microscope for confluency and protein expression, then plunge-frozen in liquid ethane after blotting excess fluid. For larger cells or tissue over 5μm thick, high-pressure freezing with the “waffle” technique produces samples under ~12μm, thin enough for SXT while preserving native ultrastructure.

The SXT-100 includes an integrated 5-channel widefield epifluorescence microscope with emission filters optimised for DAPI, GFP, RFP, Cy5 and Cy7 fluorophores. This microscope allows quick screening of the sample to identify individual, isolated cell candidates for SXT. The optical objective is conveniently located in the cryo sample chamber at 90 deg to the x-ray axis. Once FM overviews have been obtained the grid coordinates can be translated to the x-ray axis for imaging.
The user also has the option to perform cryo screening offline using one of the cryo microscopy stages currently on the market. This provides the opportunity to integrate higher resolution FM for correlation with SXT, such as cryo confocal or airyscan etc.

Rotation angles ranges are typically +/-60 deg (for grids) or +/-90 deg for glass capillaries or high pressure frozen pillar lift outs placed on pins. The latter is another key feature of the SXT-100, allowing for full rotation tomography and elimination of missing wedge artifacts associated with flat samples. Typical tomogram acquisition time varies from 30 min to 2 hours, depending on cell thickness.

Once prepared, grids are loaded into the transfer holder, which is picked up by a cooler transfer stick and kept inside a shroud under cold N2 gas during transfer to the SXT-100.

Samples are introduced vertically through a vacuum load lock and docked onto the cryo-stage for imaging. The entire transfer from bench to microscope takes less than one minute, maintaining vitrification and preventing ice contamination. After imaging, samples can be safely returned to storage.

After fluorescence microscopy images are acquired and the SXT tilt series is reconstructed into a 3D volume, the datasets are correlated to map molecular signals onto whole-cell ultrastructure.

This CLXM image links functional and structural information in a single view, enabling disease and therapeutic researchers to better interpret molecular processes within their native cellular context and gain deeper insights into disease mechanisms and treatment effects.