Correlative Light and Electron Microscopy

Correlative light and electron microscopy

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.

Correlative microscopy image of projection neurons in songbird brain

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).

Fluorescence microscopy

Fluorescence microscopy owes its popularity to the immense variety of labelling strategies. Samples can be dyed, immuno-labelled, or labeled using genetically encoded fluorescent proteins. Furthermore, thanks to the spectral properties of these fluorescent tags, multiple labels can be identified simultaneously using light sources with different colors. In this way, it is very easy to pinpoint interesting parts and events with great accuracy.

 

Example of a Jablonski diagram of a fluorophore

Figure 2: Example of a Jablonski diagram of a fluorophore. This diagram depicts the processes that occur which cause an object to convert light from one wavelength to fluourescent light. Ground state energy states are shown in blue. Highest energy state is shown in orange and lowest singlet excited state is shown in red.


Scanning electron image of HeLa cell

Figure 3: Scanning electron image of HeLa cell.

Electron microscopy

Electron microscopy is the method of choice when one needs structural information at the nanometer scale. Because the wavelength of accelerated electrons is much shorter than that of visible light, the diffraction barrier can be overcome and smaller features can be visualized.

Abbe’s law of diffraction states that two points that are spaced less than d = λ0/(2NA), where λ0 is the free space wavelength and NA is the numerical aperture of the microscope, cannot be resolved by the microscope. This makes conventional optical microscopy unsuited for studies at the true nanoscale.

What distinguishes light from electron microscopy is not only the resolution; the type of contrast that is typically measured in EM is very distinct from FM. Whereas in FM one can only detect specific macromolecules that have been labelled, in EM one acquires primarily contextual information. Examples of useful applications of EM in the life sciences include membrane structures such as the endoplasmic reticulum, the Golgi apparatus, and vesicular structures.

Integrated CLEM

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).

Schematic_SECOM_text

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.

CLEM Webinar

For a straightforward overview of correlative light and electron microscopy, watch our CLEM webinar. In this webinar, we outline the relevance and the various applications of CLEM in the life sciences, as well as some of the challenges that one may encounter.

 
Watch Integrated CLEM Webinar

Applications


Application notes

White paper

Sample Preparation for Correlative Light and Electron Microscopy

One of the challenges associated with integrated CLEM is the preparation of samples suitable for both FM and EM. However, with the right methods one can easily prepare samples in such a way as to be able to benefit fully from correlative microscopy. If you would like to know more about the process of sample preparation for CLEM, you can download our white paper here.

Technical notes

Automated overlay technical note for correlative light and electron microscopy

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Further reading

Click here for an exhaustive list of resources related to correlative light and electron microscopy.