Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics

Abstract

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell–cell interactions for infection, immunology, and cancer research.

The New Era of High-Throughput Nanoelectrochemistry

“…130 Leitao et al developed a high-speed SICM based on highbandwidth large-scale piezo actuator able to perform timeresolved, long-term topographical mapping of living eukaryotic cells. 131 They demonstrated continuous surface topography measurement of large areas (80 μm × 80 μm) with good spatial resolution (512 × 512 pixels) and temporal resolution (from 0.5 s/frame to 20 min/frame depending on scan area). In earlier work, similar large scan areas were achieved by Zhuang et al by developing a stitching algorithm able to "stitch" different scans in a postprocessing step.…”

The new era of high throughput nanoelectrochemistry

“…132 Leitao et al developed a high-speed SICM based on high-bandwidth large-scale piezo actuator able to perform time-resolved, long-term topographical mapping of living eukaryotic cells. 105 They used their system for continuous surface topography measurement of large areas (80 μm × 80 μm ) with good spatial resolution (512 × 512 pixels) and temporal resolution (from 0.5 s/frame to 20 min/frame depending on scan area). In earlier work, similar large scan areas were achieved by Zhuang et al by developing a stitching algorithm able to "stitch" different scans in a postprocessing step.…”

Start Me Up: How Can Surrounding Gangliosides Affect Sodium-Potassium ATPase Activity and Steer towards Pathological Ion Imbalance in Neurons?

“…Studying the physiological effect of lipids on membrane proteins and enzymes in actual “real” membranes, albeit challenging, is indispensable for thorough understanding of all membrane-related phenomena. The innovations in imaging techniques allow for detailed and accurate visualization of membrane dynamics in membrane systems and live cells [ 22 , 23 , 24 ]. For example, such visualisation showed that ganglioside GT1b together with synaptotagmin 1/2 serves as a receptor component for botulinum neurotoxin type B (BoNT/B) [ 25 ].…”