Correlative Light Electron Microscopy (CLEM) methods and applications in cell biology

Session

Plenary Talk: Judith Klumperman - Correlative Light Electron Microscopy (CLEM) methods and applications in cell biology

Authors

Professor Judith Klumperman (1)

Affiliations

1. University Medical Center

Keywords

Correlative microscopy - immuno-EM -on-section CLEM - live cell CLEM - Life Sciences - Cell Biology - endo-lysosomal system

Abstract text

Fluorescence microscopy (FM) allows for quick navigation through a sample and comes with an extensive and sensitive toolbox for functional imaging and protein localization. Electron microscopy (EM) provides high-resolution information down to the nanoscale level and reveals essential structural information. The power of correlative light electron microscopy (CLEM) is to merge these two techniques, creating a novel imaging tool in which function and form are integrated at the single cell or organelle level. The application of CLEM is rapidly increasing and worldwide new methods are developed to increase the accuracy, sensitivity and throughput of CLEM methods. My lab develops sample preparation methods, and in collaboration with others, hardware and software tools for different types of CLEM. 

A main on-section CLEM approach applied in our lab uses ultrathin cryosections (the Tokuyasu technique). The sections are labeled with fluorescent and gold-tagged antibodies against specific antigens, and sequentially analyzed by FM and subsequently transmission EM. Cryosections - well-known for their use in immuno-EM – are great tools for CLEM since they generally preserve fluorescence signals. On-section CLEM is therefore highly suited to find the ‘needle in the haystack’ in complex biological samples, as well as to provide structural and context information to fluorescent ‘dots’. Some antigens are, however, not detectable by immuno-EM. By developing bimodal fiducial markers (visible in FM and EM) and dedicated software, we succeeded to improve the correlation between FM and EM images to such an extent (<20 nm accuracy), that we can use fluorescent label only to mark structures in EM. Since FM labels are generally more sensitive than EM labels, this opens up new possibilities to localize rare antigens. Moreover, we can now use FM to localize molecules for which no EM probes are available, such as lipids. Finally, we improved the throughput of CLEM to such an extent that we can correlate hundreds of spots from a single sample. Thus, we can perform quantitative, high-resolution localization studies of a range of molecules and probes for which no EM labels are available, just by using their fluorescent overlay on EM images. See for references: 1-4. 

In addition, we developed live cell CLEM. Since EM requires fixation of cells, the subcellular dynamics of live cells is traditionally studied by FM in separate samples than those used for EM analysis. Consequently, our understanding of organelles is based on averages of dynamic data (by FM) and averages of ultrastructural data (by EM), without direct correlation between the data sets. To address this gap, we developed a live-cell CLEM method that allows to trace individual compartments from live-cells to volume-EM methods, such as focused ion beam scanning EM (FIB.SEM). This allows to integrate dynamic, molecular, morphological and high-resolution context information from one single sample and at the level of a single organelle (Figure 1). Building on this, we developed in collaboration with the Hans Gerritsen lab (University Utrecht) an integrated fluorescence – transmission EM for 3D-CLEM with increased throughput and correlation efficiency. We can achieve a sub-100 nm correlation efficiency between 3D-FM and 3D-EM datasets, and correlate each organelle imaged in FM with high-precision to the volume-EM data. This greatly benefits correlative analyses, enables imaging of multiple organelles of interest in 3D, and obviates the need for post-correlation in big datasets. See for references: 5-7

The cell biology focus of the lab is the cell’s digestive system. We aim to understand autophagy and the endo-lysosomal system in health and diseases and in relation to other cell organelles. In this talk I will give an overview of the different CLEM methods we use and illustrate these with examples of our own work and in collaboration with others.

 

References

1. Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy. Fokkema J, Fermie J, Liv N, van den Heuvel DJ, Konings TOM, Blab GA, Meijerink A, Klumperman J, Gerritsen HC. Sci Rep. 2018 Sep 11;8(1):13625. doi: 10.1038/s41598-018-31836-1. PMID: 30206379 

2. High accuracy, fiducial marker-based image registration of correlative microscopy images. Mohammadian S, Fokkema J, Agronskaia AV, Liv N, de Heus C, van Donselaar E, Blab GA, Klumperman J, Gerritsen HC. Sci Rep. 2019 Mar 1;9(1):3211. doi: 10.1038/s41598-019-40098-4. PMID: 30824844 

3 Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins. van der Beek J, de Heus C, Liv N, Klumperman J. J Cell Biol. 2022. Jan 3;221(1):e202106044. doi: 10.1083/jcb.202106044. Epub 2021 Nov 24. PMID: 34817533 

4 Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy. Fermie J., de Jager L., Foster H., Veenendaal T., de Heus C., van Dijk S., ten Brink C., Oorschot VM., Yang L., Li W., Muller W., Howes S., Carter A., Forster F., Posthuma G., Klumperman J, Gerritsen HC, Liv N. Cell Rep Methods. 2022. 16;2(5):100220. doi: 10.1016/j.crmeth.2022.100220. eCollection 2022 May 23.

5 Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy. Fermie J, Liv N, Ten Brink C, van Donselaar EG, Müller WH, Schieber NL, Schwab Y, Gerritsen HC, Klumperman J. Traffic. 2018 May;19(5):354-369. doi: 10.1111/tra.12557. Epub 2018 Mar 25. PMID: 29451726

6. Integrated super resolution fluorescence microscopy and transmission electron microscopy. Mohammadian S, Agronskaia AV, Blab GA, van Donselaar EG, de Heus C, Liv N, Klumperman J, Gerritsen HC. Ultramicroscopy. 2020 Aug; 215:113007. doi: 10.1016/j.ultramic.2020.113007. Epub 2020 May 6. PMID: 32470633 

7. Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes. Loginov, S., Fermie, J., Fokkema, J., Agronskaia, A.V., de Heus, C., Blab, G.A., Klumperman, J., Gerritsen, H.C. and Liv, N. Front Cell Dev Biol. 2022 Apr 11;10:829545. doi: 10.3389/fcell.2022.829545. eCollection 2022.