Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell. This method appears to be an unprecedentedly fast (<3 hours), straightforward, and reliable solution to study the finer details of pathogen-host cell interactions and provides important insights into the complex and dynamic relationship between a pathogen and a host.

Department of Virology Veterinary Research Institute Brno CZ 62100 Czech Republic

Faculty of Science Charles University Prague Viničná 1594 7 Praha CZ 12800 Czech Republic

Faculty of Science University of South Bohemia in České Budějovice Branišovská 1760 České Budějovice CZ 37005 Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branišovská 31 České Budějovice CZ 37005 Czech Republic

Zobrazit více v PubMed

Steere A. C. Lyme disease. N Engl J Med 345, 115–125 (2001). PubMed

Livengood J. A. & Gilmore R. D. Jr. Invasion of human neuronal and glial cells by an infectious strain of PubMed

Wu J., Weening E. H., Faske J. B., Höök M. & Skare J. T. Invasion of eukaryotic cells by PubMed DOI PMC

Abbe E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für Mikroskopische Anatomie 9, 413–418 (1873).

Polishchuk R. S., Polishchuk E. V. & Luini A. Visualizing live dynamics and ultrastructure of intracellular organelles with preembedding correlative light-electron microscopy. Methods Cell Biol 111, 21–35, doi: 10.1016/B978-0-12-416026-2.00002-9 (2012). PubMed DOI

Dubochet J. PubMed

Dubochet J. & Sartori Blanc N. The cell in absence of aggregation artifacts. Micron 32, 91–99 (2001). PubMed

Schwartz C. L., Sarbash V. I., Ataullakhanov F. I., McIntosh J. R. & Nicastro D. Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J Microsc 227, 98–109 (2007). PubMed

Sartori A. PubMed

Rigort A., Villa E., Bäuerlein F. J., Engel B. D. & Plitzko J. M. Integrative approaches for cellular cryo-electron tomography: correlative imaging and focused ion beam micromachining. Methods Cell Biol 111, 259–281, doi: 10.1016/B978-0-12-416026-2.00014-5 (2012). PubMed DOI

Briegel A. PubMed DOI PMC

Li W., Stein S. C., Gregor I. & Enderlein J. Ultra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer accuracy. Opt Express 23, 3770–3783, doi: 10.1364/OE.23.003770 (2015). PubMed DOI

Moerner W. E. & Orrit M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999). PubMed

Mao B. PubMed PMC

van Driel L. F., Valentijn J. A., Valentijn K. M., Koning R. I. & Koster A. J. Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells. Eur J Cell Biol 88, 669–684, doi: 10.1016/j.ejcb.2009.07.002 (2009). PubMed DOI

Dunham-Ems S. M. PubMed DOI PMC

Cinatl J.

Klebe R. J. & Ruddle F. H. Neuroblastoma: cell culture analysis of a differentiating stem cell system. J Cell Biol 43, 69a (1969).

Ma Y., Sturrock A. & Weis J. J. Intracellular localization of PubMed PMC

Sultan S. Z. PubMed DOI PMC

Lemgruber L. PubMed DOI

Loussert C., Forestier C. L. & Humbel B. M. Correlative light and electron microscopy in parasite research. Methods Cell Biol 111, 59–73, doi: 10.1016/B978-0-12-416026-2.00004-2 (2012). PubMed DOI

Swulius M. T. PubMed DOI PMC

Briegel A. PubMed DOI PMC

Chang Y. W. PubMed DOI PMC

Koning R. I. PubMed DOI

Lučič V., Rigort A. & Baumeister W. Cryo-electron tomography: the challenge of doing structural biology PubMed DOI PMC

Hoyt F. H., Hansen B. T. & Fischer E. R. Secondary sublimation removes ice contamination for improved visualization of structures by cryo-SEM. Microsc Microanal (Suppl. S2) 16, 976–977, doi: 10.1017/S1431927610057673 (2010). DOI

Agronskaia A. V. PubMed DOI

Radolf J. D., Caimano M. J., Stevenson B. & Hu L. T. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10, 87–99, doi: 10.1038/nrmicro2714 (2012). PubMed DOI PMC

Rosa P. A., Tilly K. & Stewart P. E. The burgeoning molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol 3, 129–143 (2005). PubMed

Dunham-Ems S. M., Caimano M. J., Eggers C. H. & Radolf J. D. PubMed DOI PMC

Moriarty T. J. PubMed DOI PMC

Coburn J., Leong J. & Chaconas G. Illuminating the roles of the PubMed DOI PMC

Chmielewski T. & Tylewska-Wierzbanowska S. Interactions between PubMed

Kaufmann R. PubMed DOI PMC

Targeted volume imaging reveals early vascular interactions of Lyme disease pathogen in skin

Navigating infection by pathogenic spirochetes: The host-bacteria interface at the atomic level

Pathogenicity and virulence of Borrelia burgdorferi

Cathodoluminescence imaging of cellular structures labeled with luminescent iridium or rhenium complexes at cryogenic temperatures

Novel targets and strategies to combat borreliosis

Pleomorphism and Viability of the Lyme Disease Pathogen Borrelia burgdorferi Exposed to Physiological Stress Conditions: A Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy Study