Fluidic force microscopy (FluidFM) combines atomic force microscopy (AFM) with micro-channeled probes connected to a pressure controller that enables force-sensitive nanopipette experiments under aqueous conditions.
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FluidFM offers unique advantages in simultaneous three-dimensional manipulation and mechanical measurements of a wide range of materials at the micro- and nanoscale, including biological specimens, semiconductors, polymers, and colloidal nanoparticles.
AFM is a widely used characterization technique in material science, electronics, biomedical research, and many other research fields. Since its invention in 1986 by Gerd Binnig, Calvin Quate, and Christoph Gerber, the AFM technique has undergone many improvements and became a widely-used surface imaging tool.
The technique evolved from scanning tunneling microscopy (STM), which is restricted to the characterization of electrically conductive materials only. In contrast, AFM allows obtaining atomic-resolved images of a wide variety of materials by scanning an ultra-sharp probe attached to a flexible cantilever over the sample surface.
The deflection of the cantilever is monitored by a laser beam reflected from the cantilever surface, thus enabling quantification of the variation of the interaction forces between the probe and the sample surface.
A topographic image of the sample surface with a sub-nanometer resolution is acquired by correlating the cantilever deflection versus the position of the scanning probe over the sample. At the same time, the technique allows obtaining quantitative information about the sample's mechanical properties.
Characterization Technique of Choice for Biological Applications
After becoming a surface-imaging tool of choice for semiconductors and materials science, AFM has increasingly been used in biological research for the characterization of cell organelles, quantification of protein-protein and DNA-protein interactions, cell adhesion forces, and electromechanical properties of live cells.
Owing to its compatibility with aqueous environments, AFM is considered one of the best non-invasive methods for studying biological samples in real-time under physiological conditions.
Over the past three decades, the AFM technique has undergone many improvements that broadened the scope of its application, including nanoscale lithography, along with electrical and magnetic characterization of specimens. One such advancement is the FluidFM, which combines conventional AFM with micro-channeled probes for local liquid dispensing via a nanofluidic circuit. The technology was initially developed in 2009 in the group of Prof. Tomaso Zambelli at the Swiss Federal Institute of Technology in Zürich (ETH Zürich, Switzerland) and later improved and commercialized by the spin-off company Cytosurge.
FluidFM technique relies on using a new type of cantilever with a hollow tip and integrated micro-channel in its interior, allowing to control femtoliter volumes of liquid with nanometer spatial precision and picoNewton force resolution. This approach enables isolation and injection of single cells, force-controlled patch clamping of live cells, and manipulation of micro- and nanoscale objects.
Biophysics and Manipulation of Live Cells
By positioning the FluidFM probe onto an individual cell and applying an underpressure in the fluidic channel, the cell can be tightly attached to the aperture of the probe's tip and picked up from the substrate. By reversing the pressure, the cell can be placed onto the desired spot.
With FluidFM-based single-cell manipulations, the researchers were able to transfer cells to targeted areas to study cell behavior or remove unwanted cells to facilitate the formation of cell colonies.
The ability to manipulate individual live cells proved crucial for single-cell force spectroscopy experiments (where cell-substrate or cell-cell interaction are characterized). In addition, the FluidFM technique enabled single-cell electrophysiology by simultaneously measuring the mechanical response of the cell and the ionic current recording in patch-clamp experiments.
Nano-Injection for CRISPR Gene Engineering
Since its discovery and development as a gene-editing technology, CRISPR has revolutionized biomedical research by offering a versatile gene engineering tool suitable for a broad range of organisms and applications, such as curing genetic disease, creating drought-resistant crops, and de-extinction projects.
The method requires the precise delivery of multiple guide RNA molecules into the target cells, which is far from trivial when using traditional transfection methods (where cell viability might be hindered by stress and toxicity).
Cytosurge developed a highly-automated genetic manipulation solution called FluidFM OMNIUM that can gently and precisely deliver the necessary compounds directly into the nucleus of any cell. This ensures that all the reagents have the optimum stoichiometry to maximize efficiency and eliminate cell stress.
Compared to conventional cell line development strategies, where obtaining stable monoclonal cell lines requires 12 to 14 weeks, the FluidFM technique can pick and nano-inject, and clone a single cell in less than three weeks from the transfection until the clones have been characterized.
The FluidFM OMNIUM system enables researchers to target the nuclei of a few dozen individual cells by a simple point-and-click approach, leading to an automatic injection into the selected cells at a rate of around five cells per minute. In parallel with all the different guide RNAs and protein complexes, a fluorescent marker was co-injected in the treated cells to monitor the injection process and identify the treated cells.
After 24 hours, the targeted cells were found and isolated by using the FluidFM micropipette probe and transferred into an empty well to guarantee the monoclonality of the resulting cell line.
Nanoscale 3D Printing
FluidFM technology also enables 3D printing of complex structures on a micrometer level, including difficult-to-print geometries such as overhangs. The Cytosurge team of specialists developed a proprietary micro 3D printing technology which, in 2019, was spun off into an independent company called Exaddon AG.
The latest generation of the company's CERES 3D printer combines positioning with nanometer accuracy, air pressure-driven liquid dispensing, electrochemical deposition, and optical force feedback. By employing the FluidFM nanopipette probes, the system deposits a metallic ion solution, which is then solidified via an electroplating process that takes place at room temperature.
The CERES micro 3D printer offers a printing volume of 200x200x200 µm, while the optical force feedback loop measures the forces acting on the printing tip and allows real-time monitoring of the printing process and ensuring completion of each voxel until the complete object is constructed.
Such in-situ control of the printing process leads to high-quality metal microstructures that are immediately ready for use without the need for any post-processing.
Continue reading: Determining the Viscosity of Nanofluids: Techniques and Applications
References and Further Reading
Li, M., et al. (2022) FluidFM for single-cell biophysics. Nano Res. 15, 773–786. Available at: https://doi.org/10.1007/s12274-021-3573-y
P. Monnier et al. (2021) FluidFM nano-injection overcomes delivery limitations of current CRISPR gene editing methods, accelerates cell line development cycles, and is poised to significantly broaden multiplexing capabilities. [Online] CRISPR Medicine News. Available at: https://crisprmedicinenews.com/news/fluidfm-nano-injection-overcomes-delivery-limitations-of-current-crispr-gene-editing-methods-accele (Accessed on 11 March 2022)
Saha, P., et al. (2020) Fundamentals and Applications of FluidFM Technology in Single-Cell Studies. Adv. Mater. Interfaces 7, 2001115. Available at: https://doi.org/10.1002/admi.202001115
C. Scott (2017) Cytosurge Develops Nanoscale FluidFM into Consumer-Friendly 3D Printing Process [Online] 3DPrint.com. Available at: https://3dprint.com/180243/cytosurge-eth-zurich-fluidfm (Accessed on 11 March 2022)
Meister, A., et al. (2009) FluidFM: Combining Atomic Force Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond. Nano Letters 9 (6), 2501-2507. Available at: https://doi.org/10.1021/nl901384x
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