Xavier HEILIGENSTEIN

Institut Curie (Paris)

Xavier HEILIGENSTEIN

Institut Curie (Paris)

Résumé

Live CLEM, toward fast and automated multi-scale imaging of membrane exocytotic/recycling processes

Xavier Heiligenstein1,2,*, Martin Belle3,*, Perrine Paul-Gilloteaux1,2,4, Frédéric Eyraud3, Jérôme Heiligenstein3, , Graça Raposo1,2, Jean Salamero1,2

1Cell and Tissue Imaging Facility CNRS Institut Curie, PSL Research University, F-75248 Paris, France
2CNRS UMR 144, Paris, France
3CryoCapCell, Paris, France
4SFR Santé F Bonamy Nantes CNRS INSERM, Université de Nantes, F-44000 Nantes, France
*authors contributed equally to this work

Organelle and compartment dynamics are largely studied through imaging techniques. Primarily described by electron microscopy (EM) approaches, successive revolutions in light microscopy (LM) have brought dynamic information to the structure understanding of cell compartmentation. Often used in parallel, light and electron microscopy should now be directly correlated, gaining maximum benefit from the so-called “Correlative Light and Electron Microscopy (CLEM) approaches”.

CLEM allow linking molecular function to ultrastructure. Although no longer considered as an emerging methodology, technological developments are required to turn the method into routine. From sample preparation to image registration, several gaps remain before it turns into a beginner’s approach.

For optimal sample preparation at electron microscopy level, vitrification is to date considered the best approach (over chemical fixation). We developed a workflow around our own sample support: the CryoCapsule [1], Figure 1.

Taking advantage of this miniaturized petri dish, we designed a vitrification machine, physically linked to a light microscope to vitrify a specimen in less than 2 seconds: The HPM Live µ (Figure 1).

In combination with our image registration software eC-CLEM [2], we can achieve high spatio-temporal resolution in CLEM approaches for cell biology.

We use this comprehensive approach to address cell compartment ultrastructure in relation to their dynamics.

 

References:
[1] X. Heiligenstein, J. Heiligenstein, C. Delevoye, I. Hurbain, S. Bardin, P. Paul-Gilloteaux, L. Sengmanivong, G. Régnier, J. Salamero, C. Antony, and G. Raposo, “The CryoCapsule: Simplifying Correlative Light to Electron Microscopy.,” Traffic, vol. 15, no. 6, pp. 700–16, Jun. 2014.
[2] X. Heiligenstein, P. Paul-Gilloteaux, M. Belle, M.-C. Domart, B. Larijani, L. Collinson, G. Raposo, and J. Salamero, “eC-CLEM: flexible multidimensional registration software for correlative microscopies,” Nat. Methods, vol. 14, no. 2, pp. 102–103, 2017.
[3] The authors thank the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, “Investments for the future”). X.H. was funded by the Labex grant No. ANR-10-LBX-0038 part of the IDEX Idex PSL No. ANR-10-IDEX-0001-02 PSL. We acknowledge the FRM grant No. DEQ20140329491. MB, FE and JH are members of the CryoCapCell company.

 

figure_salamero

Figure 1. HPM live µ: A new generation of High Pressure Freezer, associated to a light microscope. The sample is loaded in the CryoCapsule before the experiment. The CryoCapsule is then loaded on top of the microscope objective where live cell imaging is performed. When commanded by the user, the sample transfers automatically for vitrification in the HPM Live µ. Following image registration between LM and EM is done using eC-CLEM.

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