A team of researchers from the Swiss Federal Institute of Technology has developed a high-performance scanning ion conductance microscope (SICM) using the latest advances in nanopositioning, nanopore fabrication, microelectronics and control engineering .
Time-resolved scanning allows 3D visualization of dynamic structures in a eukaryotic cell membrane at nanometer resolutions.
Studying the functions of living cells and organelles at the nanoscale is essential to understanding the causes of disease. Traditional approaches, including electron microscopy, can unfortunately damage these cells.
Swiss researchers have developed a SCIM microscope that resolves spatio-temporally diverse three-dimensional processes on a eukaryotic cell membrane at an axial resolution of less than 5 nanometers. This may offer insight into intracellular interactions in fighting infection and disease.
The origins of scanning probe microscopy
Studying the complex functions of living cells at the nanoscale is a unique challenge. Researchers have developed a range of techniques to meet this challenge, including atomic force microscopy (AFM), scanning tunneling microscopy (STM) and scanning probe electrochemistry (SPE).
Scanning probe microscopy (SPM) forms images of surfaces using a probe that scans the sample. The technique made its first appearance in 1981 in the form of the scanning tunneling microscope, which produces atomic resolution images by scanning a specimen with a probe.
In scanning probe microscopes, piezoelectric actuators move the probe with electronically controlled atomic-level precision. The probe frame scans the sample. It captures discrete data points which are used to form an image. Its way of scanning is called a mode.
Scanning ion conductance microscopy (SICM) was developed by PK Hansma and colleagues at the University of California in 1989. An aqueous medium containing an electrolyte is a poor conductor.
A SCIM microscope scans a nanoprobe (micro-pipette with a 50-100 nm hole) near the surface of the sample. As the probe moves over the sample, ion currents pass through the pipette. The strengths of these currents vary with electrical resistance across the surface of the sample, revealing information about its composition.
In the jump mode described by the Swiss team, however, the nanoprobe is not scanned. It moves vertically up and down in a jumping motion.
The probe approaches the sample at a distance of 25-50 nm at specified points and retracts, providing discrete measurement points from which an image is formed. Above all, the probe never touches the sample, thus avoiding damage to the sample.
SCIM microscopy is therefore a powerful tool for capturing abrupt changes in a cell’s topography without affecting the sample.
Time-Resolved Scanning Ion Conductance Microscopy
Time-resolved SICM microscopes produce high-resolution profiles of cell shapes and surface features. However, these must be correlated with biochemical information and changes in the internal organization of cells.
The Swiss team integrated an inverted optical microscope with an SICM microscope, allowing them to combine recently developed super-resolution microscopy techniques in their approach.
The SICM setup consisted of a custom pipette Z-actuator (vertical actuator) embedded in a controlled-atmosphere device, essential for cell viability during imaging.
Imaging eukaryotic cells requires long-range (>10−20 μm) piezo actuators. This leads to a compromise between resonant frequency and actuator range. The team overcame this problem by adaptively slowing the speed of the pipette and applying a gain to the piezo motion based on the current interaction curve.
The Z actuator achieved a large mechanical displacement amplification of a scan range of 22 μm on the cell surface. It was driven by a bespoke piezo controller and integrated with a stepper motor stage to approach the sample.
The team used borosilicate and quartz nanopipettes to probe. They were made with a CO2 laser extractor with a radius of 20-60 nm. The quartz capillaries were shrunk to a radius of less than 10 nm using electron beam irradiation.
Many cellular processes occur on time scales of minutes or hours and are easily traceable with time-lapse SICM. Subcellular processes, such as endocytosis or infection, however, occur much faster. The Swiss team’s technique combines the ability to address large volumes of imaging (up to 220,000 μm3) relatively quickly with high-speed SICM imaging of small details in live cells.
The wide range of possible measurements (Scan sizes of 500 × 500 nm2 at 100 × 100 μm2, imaging speeds from 0.5 sec/frame to 20 min/frame; Number of pixels per image from 1 Kp to 1 Mp; A depth of view of 22 μm with an axial resolution of less than 10 nm) greatly enhances the range of biological studies facilitated by SICM microscopy.
References and further reading
Leitao, S., et al., (2021) Time-resolved scanning ion conductance microscopy for three-dimensional tracking of cell surface dynamics at the nanoscale. ACS Nano, [online] Available at: https://doi.org/10.1021/acsnano.1c05202
Liu, B., et al., (2013) Scanning Ion Conductance Microscopy: A Nanotechnology for Biological Studies in Living Cells. Frontiers in Physiology, [online] 3. Available at: https://doi.org/10.3389/fphys.2012.00483