A comprehensive study of fenestrations in liver sinusoidal endothelial cells using spectroscopy-based imaging mode of atomic force microscopy and superresolution fluorescence nanoscopy

Abstract number
552
Event
Virtual Early Career European Microscopy Congress 2020
Presentation Form
Submitted Oral
DOI
10.22443/rms.emc2020.552
Corresponding Email
[email protected]l
Session
LSA.6 - Applications of correlative microscopy of biological systems
Authors
PhD Bartlomiej Zapotoczny (6), professor Filip Braet (5), PhD Edyta Kus (3), PhD Katarzyna Ginda-Mäkelä (4), PhD Beata Klejevskaja (4), PhD student Oskar Szelest (2), professor Stefan Chlopicki (7), professor Marek Szymonski (1), professor Malgorzata Lekka (6)
Affiliations
1. Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy, and Applied Computer Science, Jagiellonian University
2. IRTech Sp. z o.o.
3. Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University
4. ONI (Oxford Nanoimaging Ltd.)
5. School of Medical Sciences (Discipline of Anatomy and Histology) – Cellular Imaging Facility, Charles Perkins Centre – Australian Centre for Microscopy & Microanalysis, The University of Sydney
6. The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences
7. Chair of Pharmacology, Jagiellonian University Medical College
Keywords

Atomic force microscopy, superfluorescence nanoscopy, fenestrations, live-cell imaging, correlative microscopy

Abstract text

Over the years evidence accumulated on the unique morphology of liver sinusoidal endothelial cells (LSECs), characterized by lack of basement membrane and presence of fenestrations – transmembrane pores – in their membrane[1]. Fenestrations, clustered in sieve plates, are involved in the dynamic transport of solutes and particles between the vascular space and the space of Disse. Sensitive to a variety of chemical and physical factors, fenestrations in live cells alter their structure rapidly. Because of the size of fenestrations, which is in range of 50-300 nm, their visualization remained reserved for electron microscopy-based approaches for over 20 years[2]

We applied spectroscopy-based imaging methods of Atomic Force Microscopy (AFM), to visualize fenestrations in LSECs[3]–[5]. Our methodology allowed to not only quantify fenestrations in living LSECs for the first time but also tracking alterations in their structures with unprecedented time (up to 9 seconds per frame) and spatial (<40 nm in a living cell) resolutions[4]. As a result, tracking a variety of cytoskeletal-based fenestrae-associated cytoskeletal structures became possible allowing for real-time observation of drugs influencing the morphology of LSEC[3], [5]

Here, we present the further development in the spectroscopy-based AFM enabling tracking of both morphology and the nanomechanical properties of LSECs together. The individual living cells analyzed firstly using AFM were further fixed and investigated using correlative AFM and super-resolution optical nanoscopy (namely dSTORM and STED). Altogether, presented comprehensive microscopy studies of LSECs provided detailed information about the cytoskeletal processes dragging fenestrations opening versus closing. 

As the presence of open fenestrations is a mark of the proper condition of LSEC, we believe that the presented approach can be used for in-vitro pharmacology on fenestrations, allowing testing of future drugs. The proof-of-concept video showing the reversibility of drug treatment on the fenestrated morphology of LSEC will be presented.


References

[1]      E. Wisse,  J. Ultrastructure Res., vol. 31, no. 1–2, pp. 125–150, 1970.

[2]      F. Braet and E. Wisse, Comp. Hepatol., vol. 1, 2002.

[3]      B. Zapotoczny et al., Hepatology, vol. 69, no. 2, pp. 876–888, 2019.

[4]      B. Zapotoczny et al.,  Sci. Rep., vol. 7, no. 1, p. 7994, 2017.

[5]      B. Zapotoczny et al., Traffic, no. April, pp. 1–11, 2019.