CLEM: Combining the Strengths of Light and Electron Microscopy

With the emergence of Green Fluorescent Protein (GFP) in the 1990s the interest of live cell imaging studies has been tremendous. This, coupled with faster and more sensitive detection systems on microscopes provides even greater opportunities. Life science research has learned such an incredible amount from these live cell imaging studies that it is hard to imagine what research would be like without these tools.

The field of electron microscopy has not fully locked onto the momentum of the light microscopy wave, but in recent years it has become evident that the resolution of the light microscope is in some cases limiting in resolving the scientific question. Therefore, there has been increasing interest in developing techniques that combine the live cell imaging aspect of GFP with the high resolution of EM (Figure 1). GFP is not directly visible in the EM (electron microscopy) but can be visualised using antibodies or by photo conversion. However, such methods are based on samples that are chemically fixed at room temperature. It is well documented that this can introduce artefacts. Such artefacts are not visualised at the LM (light microscopy) level since there is either no fixative present yet (live) or when fixed samples are studied the artefacts are below the resolution of the LM.

However, at the EM level they become apparent and that is exactly where we want to study structures at high resolution and in precise detail. There is an alternative fixation method that is based on physical fixation, namely, cryofixation. It fixes cells much faster and unlike chemical fixation is non-selective. High Pressure Freezing (HPF) is nowadays the most reliable method for cryofixation of cells and tissues. A sample is put under high pressure (2,000 bar) and milliseconds afterwards the sample is sprayed with liquid nitrogen. This prevents the formation and growth of ice crystals and fixes samples instantly with up to about 200–300 μm depth of well-preserved, vitreous sample.

In most instances this will involve freeze substitution, where the frozen water is removed from the sample with solvents such as acetone prior to resin infiltration and polymerisation at low temperature. Here too, Leica Microsystems has recently made it much easier for the scientist with the introduction of the Leica EM AFS2 with EM FSP that allows for automatic exchange of all the freeze substitution chemicals. Figure 3 shows an example of such an experiment. Epidermal Growth Factor (EGF) was coupled to quantum dots. Quantum dots are both fluorescent and electron dense and therefore excellent markers for CLEM studies. The EGF has been internalised for 30 minutes and is present in multi-vesicular bodies (MVB). The MVB appear as a dynamic structure with lots of extensions appearing and disappearing over time (Figure 3c).

When another extension was seen appearing the sample was taken and immediately frozen. The same cell is retraced and the structure of interest can now be visualised at high resolution, showing the connections. In conclusion, the Leica EM PACT2 plus EM RTS coupled with Leica Microsystems’ confocal microscopes offers life scientists an excellent solution that serves both to extend their toolbox and knowledge.