In Situ Analysis of Membrane‐Protein Binding Kinetics and Cell–Surface Adhesion Using Plasmonic Scattering Microscopy

Abstract

Surface plasmon resonance microscopy (SPRM) is an excellent platform for in situ studying cellsubstrate interactions. However, SPRM suffers from poor spatial resolution and small field of view. Herein, we demonstrate plasmonic scattering microscopy (PSM) by adding a dry objective on a popular prism-coupled surface plasmon resonance (SPR) system. PSM not only retains SPRM's high sensitivity and real-time analysis capability, but also provides � 7 times higher spatial resolution and � 70 times larger field of view than the typical SPRM, thus providing more details about membrane protein response to ligand binding on over 100 cells simultaneously. In addition, PSM allows quantifying the target movements in the axial direction with a high spatial resolution, thus allowing mapping adhesion spring constants for quantitatively describing the mechanical properties of the cell-substrate contacts. This work may offer a powerful and cost-effective strategy for upgrading current SPR products.

Label-free imaging and biomarker analysis of exosomes with plasmonic scattering microscopy

“…Meanwhile, the PSM presents higher SNR than SPR because the PSM signal contains analyte scattering signals in addition to the SPR condition variation signals, which produces the ensemble SPR sensor output. 41 In addition, the PSM can also recognize the exosome binding sites owing to high spatial resolution and measure the signals resulting from WGA binding onto these areas to suppress the interference from the blank regions. Fitting of the curves with the first-order binding kinetics model determines the k on , k off , and K D .…”

“…For the SPR measurement of WGA binding to the exosomes captured from low concentration EV solution, the fluctuations will block the signals. Meanwhile, the PSM presents higher SNR than SPR because the PSM signal contains analyte scattering signals in addition to the SPR condition variation signals, which produces the ensemble SPR sensor output 41 . In addition, the PSM can also recognize the exosome binding sites owing to high spatial resolution and measure the signals resulting from WGA binding onto these areas to suppress the interference from the blank regions.…”

“…Herein, we report plasmonic scattering microscopy (PSM) technology for imaging exosomes, analysing multiple protein biomarkers, and quantifying their binding kinetics without labels. PSM was first developed to realize label-free singlemolecule imaging on SPR microscopy, where it has been demonstrated that PSM can provide Gaussian distributed point spread function for high spatial resolution and automatic image processing with conventional software such as ImageJ [36][37][38][39][40][41][42] . In this article, we demonstrated that the PSM constructed on the popular prism coupled SPR system can provide an SNR similar to SPR microscopy, millimetre-scale field of view as prism coupled SPR system, and Gaussian distributed point spread function, thus allowing exosome size distribution analysis automatically with a conventional open-source tool and capturing a large number of exosomes for biomarker analysis.…”

“…PSM was rst developed to realize label-free singlemolecule imaging on SPR microscopy, where it has been demonstrated that PSM can provide Gaussian distributed point spread function for high spatial resolution and automatic image processing with conventional soware such as ImageJ. [36][37][38][39][40][41][42] In this article, we demonstrated that the PSM constructed on the popular prism coupled SPR system can provide an SNR similar to SPR microscopy, millimetre-scale eld of view as a prism-coupled SPR system, and Gaussian distributed point spread function, thus allowing exosome size distribution analysis automatically with a conventional open-source tool and capturing a large number of exosomes for biomarker analysis. PSM also retains the high specicity and surface sensitivity of the SPR sensors and thus allows selecting exosomes from extracellular vesicles with antibody-modied sensor surfaces and in situ analysing binding kinetics between antibody and the surface protein biomarkers on the captured exosomes.…”

Label-Free Optical Imaging of the Electron Transfer in Single Live Microbial Cells

“…Advanced optical imaging techniques have revolutionized the studies of exploring single cells with high spatiotemporal resolution. , Methods such as fluorescent microscopy for membrane potential imaging have been used to detect microbial extracellular electron transfer . In addition, plasmonic imaging technique has also been successfully applied to the exploration of single-cell interfacial behaviors, such as biomolecular binding and signal transduction . Based on the sensitivity to the local refractive index, this technique allows in situ observation of single-cell biological functions, providing a new opportunity to image the extracellular electron transfer in live microbial cells.…”