Living organisms are complex systems with scales of organization spanning the range from Angstroms to meters. Even when consideration is limited to single cells and smaller, there is still a range of six orders of magnitude, from hundreds of micrometers to hundreds of picometers.
As a result, researchers in the life sciences often find themselves looking for the proverbial needle in a haystack, and it is a very small needle in a very big haystack. Once they find the needle, they need to analyze its detailed structure to try to understand how it functions. Unfortunately, the instruments and techniques that are good at finding the needle are not the same as those that are needed to investigate its fine-scale structure.
Light microscopists have developed a number of techniques that can identify and localize a specific structure or event. For example, they might use immuno-labelling to target a specific protein with specific antibodies and attach a fluorescent label to indicate its location with high sensitivity. Or they might engineer the genetic code of a cell so that it manufactures a fusion protein highlighting a protein of interest with a fluorescent protein. However, the resolution of light microscopes (LM) is fundamentally limited by the wavelength of the light, and they are unable to resolve structures smaller than one hundred nanometers.
Scanning electron microscopes (SEM) can resolve structures down to the nanometer scale, but they require specimens that have been specially prepared with fixing, staining, and embedding procedures. Correlative light and electron microscopy (CLEM) seeks to combine the strengths of each approach to allow scientists to understand the structure and function of biological entities at the nanoscale, within the larger context of the cells and tissues in which they naturally occur.
Recent advances in CLEM instrumentation have significantly enhanced the accuracy, speed, and ease of correlative microscopy. For example, FEI’s CorrSight system (Figure 1) provides a complete workflow from live-cell imaging on a versatile LM platform, through automatically preparing the sample for SEM, and then transferring it, along with image and navigation data, to an SEM for high-resolution imaging and analysis. By increasing the speed and repeatability of the CLEM workflow, CorrSight enables investigators to generate statistically significant results in a reasonable period of time.
CorrSight is built on a flexible, high-performance inverted light microscopy platform. The microscope is contained entirely within a sealed enclosure. A truly immobile sample stage allows unobstructed access to the sample for optimal support during imaging and for additional sample processing apparatus. Furthermore, the enclosure excludes ambient light, eliminating the need to locate the microscope in a dark room.
The light microscope can be configured with a broad range of optical techniques, ranging from a highly sensitive widefield system, a structured illumination set-up for high-contrast images without out-of-focus blur in thicker samples, to a spinning disk confocal, offering fast, optical sectioning in rapidly changing living specimens. The light microscope is controlled by the same software that controls the SEM, permitting seamless transfer of image and navigational data from one to the other, and most of all, making the use of the different systems as easy as possible.
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.
Correlative microscopy allows scientists to quickly and easily find specific structures within the vastly complex environment of a living organism, and analyze the relationships between these structures and their functions with nanometer-scale resolution. Purpose-built CLEM platforms such as CorrSight streamline and accelerate the workflow, removing much of the tedium and difficulty that have, until now, impeded adoption of the technique.