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MirrorCLEM simplifies correlative imaging of biological samples

31 Aug 2016

Hitachi and RIKEN platform allows observation using light and scanning electron microscopies.

Correlative light and electron microscopy (CLEM) is an methodology intended to combine the capabilities of two normally separate microscopy platforms.

Its goal involves the imaging of fluorescence-labelled proteins localized by light microscopy (LM) in combination with the visualization of subcellular structures possible by scanning electron microscopy (SEM), in as close to a complementary protocol as experimental constraints will allow.

The complexity and distinctly different data processing requirements of the two modalities, along with the need for multimodal labeling strategies and expensive equipment, has to date limited the expansion of the CLEM field. But a new platform designed by Japan's RIKEN research institute and Hitachi High-Technologies aims to allow a wider adoption of the technique.

The system, called MirrorCLEM, involved the joint development by Hitachi and RIKEN of a system for observing the ultrastructure of organelles labelled with green fluorescent proteins within living cells, while RIKEN designed the observation workflow and the preparation method of embedding a sample in resin to preserve both the GFP fluorescence and ultrastructure.

Hitachi High-Technologies in turn developed a dedicated jig for observing plastic sections mounted on cover slips under a field-emission SEM (FE-SEM) system, as well as the software for swiftly and accurately observing the same location with both modalities.

The new design of jig represents one of the significant differences between MirrorCLEM and other existing CLEM platforms, as the developers told optics.org. "Generally, other companies' CLEM systems are applicable only for their own electron microscope and a optical microscope," said Shota Sano of Hitachi High-Technologies. "Because the MirrorCLEM jig has been made applicable for many other brands of optical microscope, other customers can now introduce MirrorCLEM easily."

A faster, simpler procedure
Correlation of the two microscope modalities into a coordinated workflow involves first observing the mounted sections under a light microscope, from low magnifications up to sufficiently high power to observe the structures of interest. A field-emission SEM stage is coordinated to the target position defined by the low magnification LM system so that the FE-SEM observes the same field of view, from where it can move to any region of interest in the LM image.

MirrorCLEM can then display an overlay of the light microscope and FE-SEM images in real time, a significant advance over previous CLEM implementations according to the developers.

"The goal for MirrorCLEM is to allow observations to be made more easily and in a shorter time," commented Shota Sano. "In general, a CLEM analysis requires acquisition of images from both microscopes and the processing of those images for observation first, which takes time. Moreover, processing the images is an obstacle for CLEM analysis because the magnification and observation target is completely different between an electron microscope and optical microscope, making it hard to find the same area of the sample in both microscopes' images."

The MirrorCLEM system and its associated proprietary software allows an operator to proceed with the optical microscope observation and image processing steps in parallel. "Thanks to this, an overlay display of the optical microscope and electron microscope images in real time becomes available, shortening the time taken for the overall procedure and making the observation itself more straightforward," said Shota Sano.

The Hitachi SU8220 FE-SEM equipped with the MirrorCLEM system has already been used to clarify the ultrastructure of organelles in transgenic Arabidopsis thaliana plants, according to the company.

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