Correlative microscopy (CM)

About this technique

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Correlative microscopy (CM) helps us to understand how complex biological structures function, through the integration of knowledge of their dynamic behaviour and their molecular machinery. The combined use of light or laser microscopy and electron microscopy (EM) has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, often is referred to as correlative microscopy. It allows researchers to gain additional novel structure–function information and this provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or combined) microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches.

Among the more popular combinations to correlate are light plus either scanning or transmission electron microscopy (CLEM), and fluorescence plus either scanning or transmission electron microscopy.

The main issues that need to be considered in CM are those of:

• Positioning so that the same area can be located in both microscopes. Central to CM is the use of special sample devices that allow the relocation of the structures of interest over different instruments and length scales. Dedicated cover slips, culture dishes, EM supports and fiducial markers are readily available from commercial suppliers for this purpose. Some vendors offer total solutions by using sample transfer stages across different microscopy platforms for relocation purposes.
• Sample preparation will depend on the combination of techniques being carried out, and is crucial to success. Minimising any structural changes that occur during fixation or sectioning will help in the correlation process. Chemical or cryogenic fixation can be used for specimens. If sectioning is required on thicker samples, knife marks and crumpling of sections can introduce artifacts. It is therefore ideal to use wholemounts of cells to help reduce such problems if the nature of the sample allows it.
• Labeling techniques are available to allow visualisation of features in both techniques. For example, quantum dots are both luminescent and electron dense and come in different shapes, enabling different components to be labeled at the same time. Otherwise sequential labeling for fluorescence and then re-labeling for EM may be required.