Spatially Selective Imaging of Cell–Matrix and Cell–Cell Junctions by Electrochemiluminescence

Abstract

Cell junctions are protein structures located at specific cell membrane domains that determine key processes in multicellular development. Here we report spatially selective imaging of cell junctions by electrochemiluminescence (ECL) microscopy. By regulating the concentrations of luminophore and/or co-reactant, the thickness of ECL layer can be controlled to match with the spatial location of different cell junctions. At a low concentration of luminophore, ECL generation is confined to the electrode surface, thus revealing only cell-matrix adhesions at the bottom of cells. While at a high concentration of luminophore, the ECL layer can be remarkably extended by decreasing the co-reactant concentration, thus allowing the sequential imaging of cell-matrix and cell-cell junctions at the bottom and near the apical surface of cells, respectively. This strategy not only provides new insights into the ECL mechanisms but also promises wide applications of ECL microscopy in bioimaging.Cell junctions are specific domains on the cell membrane that tether cells to the extracellular matrix or connect the lateral surfaces of adjacent cells. [1] The junctions located at the bottom of basal cell membrane are called cell-matrix adhesions, while those near the apical surface of cell are termed as cell-cell junctions. [2] They are not only responsible for the physical integration of individual cells to threedimensional tissues, [3] but also regulate a variety of biological processes in multicellular organisms, such as neuronal pathfinding, embryonic development, cancer invasion and metastasis. [4] Although cell junctions have distinct structures and functions, they are all involved in a continuous crosstalk. [5] Knowing precisely how cell-matrix and cell-cell junctions are distributed in the cellular structure is critical for understanding their functions and associated biological events. Surfacesensitive methods such as interference reflection, [6] total internal reflection fluorescence [7] and surface plasmon resonance microscopies [8] have been used in mapping cell-matrix adhesions, while electron microscopy and fluorescence microscopy (in particular confocal laser scanning microscopy, CLSM) are the most frequently used methods for imaging cell-cell junctions, [9] which however often require expensive facilities or specific immunofluorescent labelling.

Shadow Electrochemiluminescence Microscopy of Single Mitochondria

“…In this context, label‐free imaging is an appealing alternative and several groups have achieved the ECL visualization of single cells [19] . Particularly relevant to our work are findings recently reported by Su et al [19a, 20] . They imaged by ECL the cell‐matrix adhesions located on a silica nanochannel membrane and cell‐cell junctions.…”

Label‐Free Electrochemiluminescence Imaging of Single‐Cell Adhesions by Using Bipolar Nanoelectrode Array

“…Coupling with the reduction of highly concentrated oxygen confined underneath cells at the cathodic side, an ECL reaction occurring at the anodic side is used for sensitive imaging of single cells with positive contrast mode. This approach is different from the previous reports that used the diffusional hindrance of ECL reagents by single cells to achieve single‐cell ECL imaging with negative optical contrast [3–5,7] . Thus, our approach can achieve label‐free, fast and positive ECL imaging of single cells, enabling to visualize localized adhesion strength of single cells.…”

“…Electrochemiluminescence (ECL) microscopy as an emerging approach for single cell imaging has attracted an increasing number of interests because of its very low background, good temporal‐spatial resolution, and simplified instrumental setup [1] . Recently, various ECL based strategies have been proposed for imaging of cell membranes, [2] cell morphology, [3] cell‐matrix adhesions, [4] cell‐cell junctions, [5] hydrogen peroxide efflux [6] and single mitochondria [7] . For example, a remarkable breakthrough in single‐cell ECL imaging has been achieved by means of labeling cell membranes with ruthenium complex dye and permeabilization with Triton X‐100 for diffusion of co‐reactant through cell membranes [2b] .…”

A microscopy technique that images single reaction events in total darkness

“…By imaging the photon emissions at the surface of a nanoelectrode over time, Dong et al were able to visualize the surface with a spatial resolution down to 22 nanometres. They extended this approach to image cells attached to an electrode: the adhesive regions of the cells hinder the diffusion of ECL reactants to the electrode surface, blocking photon emission and thereby producing a negative image of those regions 14 (Fig. 1).…”