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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 manufacturing, microelectronics and control engineering.
Image Credit: Shutterstock.com/Meletios Verras
Time-resolved scanning allows 3D visualization of dynamic structures in eukaryotic cell membranes at nanometer resolution.
Studying the functions of living cells and organelles on the nanoscale is essential to understand the causes of diseases. Unfortunately, traditional methods, including electron microscopy, can damage these cells.
Swiss researchers have developed a SCIM microscope that can analyze the diversified three-dimensional processes of time and space on eukaryotic cell membranes with a sub-5 nanometer axial resolution. This may help to gain insight into intracellular interactions in the fight against infection and disease.
Studying the complex functions of living cells on the nanoscale is a unique challenge. Researchers have developed a series of technologies to meet this challenge, including atomic force microscopy (AFM), scanning tunneling microscopy (STM), and scanning probe electrochemistry (SPE).
A scanning probe microscope (SPM) uses a probe that scans a sample to form a surface image. This technology first appeared in the form of a scanning tunneling microscope in 1981, which uses a probe to scan a sample to generate atomic resolution images.
In scanning probe microscopes, piezoelectric actuators move the probe with electronically controlled atomic precision. The probe raster scans the sample. It captures discrete data points used to form an image. The scanning method is called mode.
Scanning ion conductance microscope (SICM) was developed by PK Hansma and colleagues at the University of California in 1989. Aqueous media containing electrolytes are poor conductors.
The SCIM microscope scans the nanoprobe (micropipette with 50 to 100 nm holes) close to the surface of the sample. When the probe passes through the sample, the ionic current flows through the pipette. The intensity of these currents varies according to the resistance of the sample surface, revealing information about its composition.
However, in the jump mode described by the Swiss team, the nanoprobe is not raster-scanned. It moves vertically up and down in a jumping motion.
The probe approaches the sample and retracts at a distance of 25 to 50 nm at a designated point, thereby providing discrete measurement points that form an image. It is essential that the probe never touches the sample to prevent damage to the sample.
Therefore, the SCIM microscope is a powerful tool that can capture sharp changes in cell morphology without affecting the sample.
The time-resolved SICM microscope generates high-resolution contours of cell shapes and surface features. However, these needs are associated with changes in biochemical information and internal organization of cells.
The Swiss team integrated the inverted optical microscope into the SICM microscope, which allowed them to incorporate the recently developed super-resolution microscopy technology into their method.
The SICM setup includes a custom pipette Z actuator (vertical actuator) integrated into a controlled atmosphere device, which is essential for cell viability during imaging.
The imaging of eukaryotic cells requires long-distance (>10−20 μm) piezoelectric actuators. This leads to a trade-off between resonant frequency and actuator range. The team overcomes this problem by adaptively slowing down the pipette and applying gain to the piezoelectric motion as a function of the current interaction curve.
The Z actuator achieves a wide mechanical displacement magnification with a scanning range of 22 μm on the cell surface. It is driven by a custom piezo controller and integrated with a stepper motor platform used to approach the sample.
The team used borosilicate and quartz nanopipettes for detection. They are manufactured using a CO2 laser puller with a radius of 20 to 60 nm. The quartz capillary is irradiated with electron beam to shrink to a sub-10 nanometer radius.
Many cellular processes occur on time scales of minutes or hours, and can be easily tracked by delayed SICM. However, subcellular processes, such as endocytosis or infection, occur faster. The Swiss team's technology combines the ability to process large imaging volumes (up to 220,000 μm3) relatively quickly with high-speed SICM imaging of small details on living cells.
The possible measurement range is very wide (scan size from 500 × 500 nm2 to 100 × 100 μm2, imaging speed from 0.5 seconds/image to 20 minutes/image; the number of pixels per image is from 1 Kp to 1 Mp; depth of field is 22 μm (Axial resolution is less than 10 nm) Significantly expand the scope of biological research facilitated by SICM microscope.
Leitao, S., et al., (2021) Time-resolved scanning ion conductance microscope for three-dimensional tracking of nano-scale cell surface dynamics. ACS Nano, [online] available at: https://doi.org/10.1021/acsnano.1c05202
Liu, B., et al., (2013) Scanning ion conductance microscope: Nanotechnology for living cell biology research. Frontiers of Physiology, [Online] 3. Available at: https://doi.org/10.3389/fphys.2012.00483
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William Alldred is a freelance B2B writer with a bachelor's degree in physics from Imperial College London. William firmly believes that science and technology can change the power of society. He is dedicated to distilling complex ideas into compelling narratives. Williams' interests include particle and quantum physics, quantum computing, blockchain computing, digital transformation, and financial technology.
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