Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces

Julia Sajman,Kobi Hermon,Seon Kinrot,Eilon Sherman,Yair Razvag,Oren Yakovian,Shachar Schidorsky

doi:10.1038/s42003-021-01960-2

Abstract

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

Highlights

Cell-cell interfaces convey mechanical and chemical information in multicellular systems
We show the compatibility of our approach with multiple diffraction-limited and super-resolution microscopy (SRM) imaging modalities, including bright field, confocal and Stimulated Emission Depletion (STED) imaging, direct Stochastic Optical Reconstruction Microscopy (STORM), Super-resolution Optical Fluctuation Imaging (SOFI), and SuperResolution Radial Fluctuations (SRRF)
To minimize the background associated with cell-capturing layers, we decided to capture the cells on two opposing coverslips coated with reagents that promote cell adhesion (Fig. 1a)

Summary

Introduction

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Multiple approaches for super-resolution microscopy (SRM) have been introduced, including (but not limited to) single molecule localisation microscopy [SMLM; such as Photoactivated Localization Microscopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM)], Stimulated Emission Depletion (STED), Structured Illumination Microscopy (SIM) and their variants[10]. These methods break the diffraction limit of light, and can reach lateral resolutions of 20–30 nm. SRM methods are often highly sensitive to optical aberrations and mechanical vibrations They often require a very low background, especially for SMLM. Deconvolution of either diffraction-limited microscopy or SRM further increases these sensitivities, as the point-spread function (PSF)[14] is usually known and constant

Methods

Results

Conclusion