Applications for the CorrSight workflow fall generally in to three categories: prescreening, live-cell imaging, and cryo electron microscopy.
In prescreening applications the samples are already prepared for SEM before they are examined on the light microscope. Prescreening is most useful for finding very rare structures or events that require searching many samples. A flexible sample holder format compatible both with the light and the electron microscope can be configured to accommodate multiple samples in a variety of sizes and shapes. Images captured by the light microscope are seamlessly transferred to the SEM where they are automatically overlayed and aligned with the electron image (Figure 2).
The common software shared by the systems allows the operator to define the SEM imaging job while the sample is still on the LM, transferring the recipe and execution instructions along with the image and target coordinate data. Large sample areas can be designated for surveying in the SEM with multiple high-resolution images stitched together to cover the full area.
Live-cell imaging has been challenged by the need to view the live cells in physiologic conditions on the LM, and then fix and prepare them for the SEM, often at a specific moment in time defined by some triggering event—all while preserving the spatial correlation of the information in the LM and SEM observations. The CorrSight system addresses this challenge by providing an automated microfluidic sample-preparation system directly integrated with the LM platform.
The system can be programmed to execute complex, user-defined preparation protocols for resin-embedding and can be removed from the LM for steps that require a fume hood. Automation ensures the repeatability of the preparation. Fixing the sample is easily initiated upon observation of a triggering event, and precise control of timing permits correlation of timing information throughout an experiment. The sample can be observed on the LM throughout the preparation process to ensure optimal quality. The preparation results in an embedded sample ready for the SEM including the necessary information for relocation.
In Cryo electron microscopy the sample is fixed in amorphous ice to prepare it for the SEM. It has the advantage of preserving the sample in its most natural, fully hydrated state and native context. The greatest challenge is preventing the accumulation of ice contamination on the sample during LM observations and transfer to the vacuum environment of the SEM. CorrSight addresses this with a specialized cryo environment, allowing high-resolution light microscopy, while at the same time maintaining the vitrified state of the sample.
In addition, the CorrSight’s full enclosure supports the maintenance of a dry atmosphere to prevent the formation of ice on lenses or windows affecting the optical performance. The fixed position of the sample makes liquid nitrogen supply extremely easy and stable. The full workflow may include transfer from the CorrSight to a SEM/FIB (focused ion beam) system where a very thin sample is milled by an ion beam for subsequent examination in a transmission electron microscope.