Atomic Force Microscopy Reveals the Dynamic Morphology of Fenestrations in Live Liver Sinusoidal Endothelial Cells

B. Zapotoczny,K. Owczarczyk,M. Szymonski,E. Kus,S. Chlopicki,K. Szafranska

doi:10.1038/s41598-017-08555-0

B. Zapotoczny, K. Owczarczyk + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-08555-0

Copy DOI

Journal: Scientific Reports	Publication Date: Aug 11, 2017
Citations: 40	License type: open-access

Affiliation: Jagiellonian University

Abstract

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Highlights

We present a novel application of the atomic force microscopy (AFM) force imaging mode based on fast acquisition of the force versus distance (FD) curves in every pixel of the scanned area
Imaging based on force spectroscopy enabled us to construct images containing detailed information regarding both the topography and nanomechanical properties of whole live liver sinusoidal endothelial cells (LSECs) as well as s single sieve plate (Fig. 1)
We found that the ability to visualize the cytoskeleton structures[1, 25], or stress fibres was strongly related to the number of analysed FD curves per image area, the applied loading force, and the speed of FD curve acquisition (Supplementary Fig. 2)

Summary

Introduction

We present a novel application of the AFM force imaging mode based on fast acquisition of the force versus distance (FD) curves in every pixel of the scanned area. Reconstructing the topographic image from thousands of FD curves collected in milliseconds permits the high-resolution imaging of detailed topographical structures on LSECs and the observation of rapid alterations in fenestrations morphologies in live LSECs. Because of the flat, thin appearance of LSECs, short FD curves, i.e., below 200 nm could be used. When a relatively small area was scanned, high-resolution images could be acquired rapidly (time per frame up to 45 s for a single sieve plate)

Methods

Results

Conclusion