Correlative microscopy of samples of different topologies between light and electron microscopes

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Poster Session 1
Joerg Wissler (2), Martin Peschel (1), Kathrin Rudolph (2)
1. NIKON GmbH, Tiefenbroicher Weg 25, 40472 Düsseldorf, Germany
2. TESCAN GmbH, Zum Lonnenhohl 46, 44319 Dortmund, Germany

Correlative microscopy, Topographical sample, CLEM, SEM

Abstract text

A workflow for software-assisted correlative light and electron microscopy (CLEM) suitable for basic and sophisticated light microscopes is presented. Correlative microscopy is a permanent effort. The idea of correlative imaging and comparison sounds in the first moment easy but provides more challenges than often expected upon execution. Challenges include, for example, unexpected material contrasts, sample holder issues, lost, irretrievable region of interest positions to name only a few if correlative work between the different microscopic methods is performed. Often basic light microscopes do not provide any support for further correlated tasks although technically capable. CLEM additionally includes the challenge of different imaging preparation and environments. Light microscopy can be performed at normal atmospheric pressures. Electron microscopy (EM) needs vacuum conditions. In scanning electron microscopy (SEM), e.g., the sample cannot even be moved and viewed as easily as on most light microscopes. An additional limiting factor between the methods is often the limited field of view (FoV) of the light optics as well. The solutions for CLEM are often very specific for a certain task, complex engineered, and expensive due to the required precision. But for the most standard tasks in the lab, a simple correlation workflow would probably often suffice. Some SEMs provide the possibility of depicting large field of views of the samples. In those SEMs complete objects can be visualized in millimeter or centimeter scales and upon magnification also examined in detail up to the nanometer scale. The wide-field view is important and convenient for materials and life science samples. Orientation, the discovery of positions, and depictions of regions of interest are so enabled. On-stage cameras can help but not replace native large fields of view. The challenge is even more complex if topographic samples instead of flat samples are examined. The utilization of software image processing functions combined with a specific workflow enables to circumvent the limitations. Flat and topographical samples can be so examined in a similar manner. Tested on TESCAN and NIKON microscopes using for example the software packages CORAL and NIS, but not limited to, and basic equipment available, correlation of light and SEM sample images can be realized in lab real-time.