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.
A complex and heterogeneous cell microenvironment offers not only structural support for cells but also myriad biochemical and biophysical cues. These outside-in signals transmit into cells primarily through integrins, which are the important components of cell–matrix adhesions to direct and maintain cell behaviors and fate. In this work, we report a surface-sensitive imaging methodology for evaluating the difference in cell–matrix adhesions at the single cell level to dissect the impact of the chemical microenvironment on cell behaviors. Cells were cultured on silica nanochannel membrane (SNM) modified indium tin oxide (ITO) electrodes (SNM/ITO) with different terminal surfaces and imaged by electrochemiluminescence microscopy (ECLM). The results show that the surface tethered with Arg-Gly-Asp (RGD) groups can mediate robust cell–microenvironment interaction and those coated with silanol and (3-aminopropyl)triethoxysilane (APTES) groups transmit an intermediate adhesion, while oligo(ethylene glycol) (OEG) coated surface conveys the weakest cell–matrix adhesion. Specific recognition of integrins to different surfaces was further explored in conjunction with selective immunoblocking of different subunits. α6, α5, and α1 integrin subunits were found to recognize SNM, RGD/OEG, and APTES surfaces, respectively. The work provides not only insights into cell–microenvironment interaction but also guideline in the design and development of functional and biomimetic surface materials.
Main observation and conclusion Chemical analysis of single cells plays essential roles in both fundamental research and clinical applications. In this work, the electrochemiluminescence (ECL) generated at vertically aligned gold microtube electrode ensembles (MEEs) was applied for the local chemical sensing of single cells. Thanks to the structure of MEEs and the surface‐confined nature of ECL generation, the ECL is confined in single microtubes and the local variations of hydrogen peroxide concentration can be revealed by the changes of ECL signals at single MEEs. With this methodology, we studied the hydrogen peroxide efflux from single living cells, showing the subcellular heterogeneity in hydrogen peroxide release. With sufficient spatial resolution for the parallel single cell sensing as well as advantages including easy preparation and high throughput, the MEEs provide a promising platform for subcellular electroanalysis.
Cells tend to align and move by following anisotropic topographical cues, namely the phenomenon known as contact guidancean essential step in cell alignment, adhesion, and migration. The effect of topographical cues on individual cells has been investigated extensively, but that on cell aggregates still remains to be fully understood. Considering the high surface sensitivity of electrochemiluminescence (ECL) microscopy, it was used in this work to explore the impact of surface topography on cell behaviors. First, we studied the variations of cell–matrix adhesions of cells cultured on different topographical features. Both fibroblast-like and epithelial cells plated on microgrooved electrodes exhibited obvious contact guidance behavior. Then, the effect of surface topography on cellular collective migration was investigated. Topographic cues would be a barrier for cell migration if the orientation of microgrooves was perpendicular to the direction of migration; otherwise, it would be a helper. Finally, it was found that relaxation of cytoskeleton contractility or reduction in adhesion density could weaken the directed migration of leading cells, because the alteration of migration directionality was retarded. In contrast, such interactions were lost on the contact guidance response of follower cells, as they still aligned by following the topographic cues.
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.
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