Correlative Fluorescence and Electron Microscopy

Correlative Fluorescence and Scanning Electron Microscopy

The SECOM system is a Fluorescence Microscope integrated with a Scanning Electron Microscope. The unique integrated design enables extremely fast correlative microscopy, with the highest optical quality and overlay accuracy.  With the automated alignment procedure, you are directly imaging the accurate location at a high resolution. The SECOM provide fluorescence imaging options for Super Resolution (stochastic techniques) and Confocal Microscopy.


Integrated Correlative Light and Electron Microscope

The SECOM platform is a fluorescence microscope made to be integrated with a scanning electron  icroscope. It enables you to do correlative microscopy extremely fast, with the highest optical quality and overlay accuracy. The integrated design provides, switching from fluorescence to electron imaging is seamless and instantaneous. And with the automated alignment procedure, you are directly imaging the right location at a high resolution. Imaging with the SECOM is just like using a fully equipped high-end optical wide-field microscope, without compromise on either optical or electron performance.
The system is installed as a retrofit by replacing the door to the vacuum chamber and can be
fitted to most scanning electron microscopes.

Imaging Thin Sections
Using the SECOM for thin sections allows you to use fluorescent markers for pinpointing regions of interest, locating rare events, screening large areas and identifying subcellular structures on a molecular basis. By using multicolour labelling you can have the enormous toolbox of fluorescent markers available in nanometer resolution electron images. Sectioning biological material that is resin embedded or cryo fixed is the ideal method to reveal subcellular details. This is why most electron microscopists are used to working with sectioned material. For fluorescence microscopy, the axial resolution is greatly enhanced as the sections can be as thin as 50 nm. Ultrathin sectioning can be applied to many different biological samples, such as cells in suspension or on a substrate, tissues and animal embryos.

Imaging Cultured Cells
The SECOM is the perfect tool to accurately inspect the morphology and surface topology of cultured cells. With the SECOM platform you have the unique opportunity to simultaneously image fluorescent markers together with all the different types of contrast available for SEM.  This provides a fast and straightforward method to study cell morphology in correlation with specific proteins of interest. Growing cells on a substrate is routine work in cell biology. With minor alterations in your sample preparation, you can take advantage of the high resolution and extra contrast provided by electron microscopy.

Integrated Workflow – Fast and Precise

Correlative imaging with an integrated system is very different from traditional correlative procedures. With the SECOM platform you acquire your fluorescence and electron images in one microscope, allowing you to quickly switch between both modalities. This drastically reduces the time from sample preparation to correlative imaging. After loading your sample, you immediately get your results: in one acquisition you get your fluorescence and high resolution electron images combined with an accurate overlay. Moreover, by easily switching between imaging modes you can quickly assess the quality of your sample preparation and adapt your imaging parameters accordingly.
Thanks to the automated alignment procedure of the SECOM, you can accurately correlate subcellular structures, without the risk of introducing a user-bias. The use of an integrated sample preparation protocol prevents unpredictable sample shrinkage and deformation. Since the sample is not moved
from ambient to vacuum conditions, it is guaranteed that your sample is in the same conformation for both light and electron microscopy.


HeLa cells expressing GFP (Image courtesy of C.J. Peddie                                                                 Pancreas (Image courtesy of Ben Giepmans)

and L.M. Collinson, CRUK LRI)  Pancreas (Image courtesy of Ben Giepmans)

Automated Overlay – High accuracy, independent of user and sample

Alignment of fluorescence and electron images is a crucial step in correlative microscopy. The alignment procedure of the SECOM is fully automated and achieves an accuracy of 50 nm or better, independent of the sample. This accuracy is achieved using a patented alignment procedure. With the SECOM platform you therefore always look at exactly the same location with both the fluorescence and the electron microscope. The key to this alignment procedure is the physical principle of cathodoluminescence. Light is generated at the position where the electron beam hits the glass. This light can be detected by the camera of the fluorescence microscope and acts as a temporary fiducial marker. By positioning the electron beam such that not one, but many spots are created, a grid of such temporary fiducial markers is generated.

                      Compatible with FEI, Zeiss, Tescan, Hitachi and JEOL

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