Integrated Microscopy – iLEM

The Integrated Laser and Electron Microscope: iLEM

Project of Dr. Sasha Agronskaia

Correlative light and electron microscopy (CLEM) is a technique that combines the powers of fluorescence microscopy and electron microscopy. Advantages of fluorescence microscopy are large field of view, high sensitivity and capability to find specific label/event. However, fluorescence microscopy has a limited spatial resolution (~0.3 μm). Electron microscopy has a superior spatial resolution (~0.5 Å) but a limited field of view. Normally, CLEM is carried out in two separate set-ups. Such an approach is slow and prone to errors. However, the CLEM can be simplified and speed up by integrating both microscopes into one. With this idea in mind an integrated correlative microscope is build in our group. This microscope consists of standard transmission electron microscope (Technai 12, FEI) wherein a homemade scanning fluorescence microscope is added to one of the ports. Imaging in both fluorescence and electron modes are done using the original TEM sample stage. Fluorescence microscope is perpendicular to the electron beam and therefore the sample needs to be 90° rotated before start of the fluorescence imaging. TEM imaging starts after completion of the fluorescence mapping of the sample. Inter-modal coordinate retrieval is fully automated and the switch between two modes of operation can be done within seconds. The integrated approach for the CLEM requires new sample preparation technique that can provide samples simultaneously suitable for both fluorescence and electron microscopy.

ILEM Principle of the set-up and an example of correlative images. (A): Mechanical drawing of the integrated laser and electron microscope (DM – Dichroic mirror). (B): The photograph inside TEM with iLEM installed (geometry of the TEM operation mode). (C): iLEM image of fluorescence beads of 0.2 μm diameter (obtained in fluorescence operation mode). (D): iLEM TEM image of the same groups of beads as in (C).


EM Staining and Cracking Particles

Project of Matthia Karreman (Supervision Prof. Gerritsen)

The integrated Laser and Electron Microscope (iLEM) combines a fluorescence microscope (FM) and a transmission electron microscope (TEM) in one set-up1. This novel form of correlative microscopy combines the best of both worlds: the large field of view of the FM with the superb resolution of the TEM. The regions of interest in the sample are identified with the FM based on localization of fluorescent probes, and subsequently these areas can effortlessly be retraced in the TEM and imaged at high resolution. The iLEM demands samples suitable for both FM and TEM imaging. Therefore, conventional methods for the preparation of biological material are not applicable (Figure 1). During my research we set out to develop and optimize sample preparation methods for iLEM2. Furthermore, the unique possibilities of the iLEM as a research tool were demonstrated in the field of both Life and Material Science3,4. The current focus of my work is on Facio Scapulo Humeral Dystrophy (FSHD), a form of muscle dystrophy. Here, we navigate to FSHD affected cells based on fluorescent labeling of a protein known to be involved in the development of the disease. Next, we can investigate these areas at high resolution. Another application of the iLEM, here in the field of heterogeneous catalysis, is the characterization of Fluid Catalytic Cracking (FCC) particles4. These particles are employed in the oil refinery industry, where they catalyze the breakdown of large molecules in crude oil fractions into products with lower molecular weight, like gasoline. The catalytic sites in the FCC particles were selectively stained with a fluorescent probe5, and next their structure was investigated in the TEM.

  1. A.V. Agronskaia et al, J. Struc. Biol. 2008
  2. M.A. Karreman, E.G. Van Donselaar et al, Traffic, 2011
  3. M.A. Karreman et al, Biol. of the Cell, 2009
  4. M.A. Karreman, I.L.C. Buurmans et al, Angew. Chem. Int. Ed., 2012.
  5. I.L.C. Buurmans et al., Nature Chemistry, 2011

EM staining
The effect of increasing amounts of heavy-metal stain on fluorescence intensity3. Thin sections of cells were labeled with Alexa 488 for a nuclear protein (A-C), contrasted with increasing amounts of heavy metal stain; little staining (A,D) increased staining (B,E) or heavy staining (C,F) and imaged with the iLEM. Top panels: fluorescence signal, bottom panels: high magnification TEM images. No cellular membranes could be seen in the TEM when the sections are only lightly stained (D). In contrast, membranes were clearly visible when more stain is used (E,F). Scale bar: 10 μm (A-C), 200 nm (D), 100 nm (E, F).

FCC particles
Imaging FCC particles with iLEM4. The overlay image (middle) shows an area within an FCC particle where both bright and low fluorescent signal can be observed; indicating high and low catalytic activity, respectively. The inset (right) shows that these sites are not only different in activity, but also in structure.

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