Correlative light and electron microscopy (CLEM) is the combination of fluorescence microscopy (FM) with high-resolution electron microscopy. The combination of the labelling power of fluorescence imaging and the high resolution structural information provided by electron microscopy makes correlative microscopy the perfect tool for studying the complex relation between form and function in biology.
In order to analyze the various aspects of the complex organization of cells, there is an increasing demand to study the same samples at different length scales. Ideally, the researcher would want to obtain a complete overview of a cell and thus require an image on a micrometer length scale, while at the same time analyze biomolecules in that same cell on the scale of a few nanometers. Correlative microscopy enables one to zoom in and out seamlessly on the same sample using an integrated fluorescence and electron microscope.
Figure 1: correlative microscopy image of projection neurons in songbird brain. Projection neurons in songbird brain. Imaging was performed using the SECOM platform (DELMIC) mounted on a Quanta 250 FEG SEM (FEI).
The great potential of CLEM lies in the combination of these two modalities: multi-color labelling together with high resolution contextual information. Traditionally CLEM is performed by correlating the results from these two different microscopy modalities, acquired using separate instruments, at separate locations, using potentially different sample preparation protocols. That approach results in procedures which are notoriously time-consuming and require high levels of expertise. Furthermore, creating an accurate unbiased overlay requires an independent set of features which can be used to align both modalities.
With the introduction of the SECOM platform, a completely integrated light and electron microscope, most of these difficulties can be overcome. The SECOM platform integrates light and electron microscopy in a single device by equipping the SEM with an inverted fluorescence microscope (see Figure 4).
Figure 4: A diagram showing the integrated CLEM system, the SECOM. The electron beam of the scanning electron microscope is shown in green. The added optical light path of the SECOM platform is shown in red.