An overview of single-cell manipulation methods and applications with FluidFM®
This short note aims to provide the reader with an overview of cell manipulation and more precisely, single-cell manipulation methods suited for a broad range of life sciences and biological applications.
Go straight to:
What is single cell manipulation?
First, what does cell manipulation mean?
The field of cell manipulation comprises a set of tools, instruments, methods, and technologies to accurately manipulate cells.  Numerous fields, such as life science, diagnostics, the pharmaceutical sector, and renewable energy are supported by cellular analysis.
Traditionally, the average response from a population of cells is measured using cell-based assays, which assume that an average response is characteristic of a typical cell within a population. This simplification, nevertheless, could lead to a false interpretation. That is why, with the fast development of microscopy techniques, various methods were introduced to visualize, study, and enumerate cellular and intracellular components at the scale of an individual cell.
Introduction to single-cell manipulation
The expression “single-cell manipulation” describes a range of methods and tools used to perform a set of actions on a single cell, from injecting, extracting, sorting, isolating, trapping, probing, picking, and placing. The manipulation of a single cell can be performed in the cell’s native environment but also in various biocompatible solutions while the cells are adhered to a surface or suspended in solution. Various tools and methods have been invented and developed to handle cells in their natural environments for a broad range of biological applications. Until today, single-cell study has been recognized as the most straightforward way to perform comprehensive heterogeneity studies on different topics from the aspects of cellular behavior to genetic expression. Thereby, a comprehensive single-cell study heavily relies on the use of high-throughput and efficient tools for manipulating and analyzing cells at the single-cell level.
Single-cell analysis is technically more difficult than bulk-cell analysis in terms of the sizes of cells and the concentrations of cellular components. Most cells, such as mammalian and bacteria cells, have sizes at the scale of microns. Therefore, manipulation of those cells at the single-cell level becomes difficult when using traditional biological tools, such as petri-dishes and well-plates. Additionally, most of the intracellular, extracellular components are presented in very small concentrations and have a wide range of concentrations, which demand highly sensitive and specific detection methods. The development of single-cell analysis led to the creation of different categories of single-cell manipulation.
Categories of single-cell manipulation
This first category of cellular manipulation represents the process of injecting a soluble compound repeatedly and semi-automatically, to the same single cell without destroying it. This function is of particular interest for many applications from genome engineering, disease modelling and drug discovery. The FluidFM technology offers this functionality with the combination of a FluidFM cantilever and a pressure controller to perform a precise and gentle single-cell nanoinjection. Unlike microinjection and other cell transfection methods, the direct intra-nuclear or cytoplasmic nanoinjection ensures the viability of the system and the reproducibility of the experiment.
The second category of cell manipulation illustrates the action of using a FluidFM nanosyringe to penetrate single living cells and extract their content for further investigation. This function complements the FluidFM spectrum of capabilities for single-cell analysis. The subsequent downstream analysis of the collected samples can then be carried out using well established procedures. The method has encountered a growing interest in various fields of research, from neurosciences, virology or Live-seq. The video on the right hand-side shows how the FluidFM technology can be employed to perform a gentle, accurate and direct extraction of cytoplasmic cellular content.
Measuring adhesion at the single cell level is key for the study of numerous biological systems from bacteria to mammalian cells. [2,3] With the FluidFM OMNIUM, live single-cell adhesion measurement are performed by reversibly immobilizing a cell onto the cantilever by applying and maintaining adequate negative pressure of the fluidic channel on the FluidFM probe. This allows the measurement to be realized in native conditions. The cellular adhesion can be quantified multiple times for high reproducibility, throughput and efficiency.
More categories of single-cell manipulation exist such as single-cell isolation, single-cell sorting or single-cell sequencing. Those are application dependent and won’t be detailed here. Nonetheless, the three categories of cellular manipulation described above can be performed with a range of techniques presented in the next section.
Existing methods to perform single cell manipulation
Over the years, various techniques have arisen to perform single cell manipulation from optical tweezers to atomic force microscopy (AFM).
Those tools give access to a broad range of crucial information about biological systems. A summary of some of the existing techniques is given below.
Optical Tweezers - Optical Trapping
The use of optical traps - laser light - for the manipulation of biological particles relies on the forces due to radiation pressure. The force causes the biological dielectric particles to be pushed into a highly concentrated, high-intensity light beam.  Over the years, to manipulate individual biological cells and carry out intricate physical and mechanical characterizations, optical tweezers have become a common tool. Optical Tweezers are an interesting choice for these characterizations due to their adaptability to liquid media settings, non-contact force for manipulating cells, and force resolution as accurate as 100 aN. Their numerous uses include adding foreign substances into individual cells, placing cells to precise regions, and sorting cells in microfluidic devices. 
In cell biology, electrical detection techniques have been successfully applied. Because various cells have varied dielectric characteristics, they respond to an electrical field in different ways. Dielectrophoresis (DEP), one of many electrokinetic detection techniques, has been extensively employed for cell sorting, cell separation, molecular analysis, single-cell mechanobiology, and cell deformation.  The DEP approach offers high throughput assessment, excellent accuracy, and compatibility with microfluidics. Nowadays, DEP is employed broadly in the complex spatial manipulation of viruses, bacteria, cells and sub-micron biological particles [7–9].
The mechanical and electrical characteristics of a single cell can be characterized using the micropipette aspiration technique.  Micropipette aspiration works by applying accurate and sensitive negative pressure from a pressure-based controller to the suction or aspiration of a cell through a micropipette. The cell is initially immobilized on the tip of a micropipette before being drawn into the tube by suction. The position of the cell can be followed using a microscope to calculate the length of the pipetted section of a cell through the micropipette tube.  However, this method is only applicable to the nonadherent cells and presents a relatively low throughput.
Long-established as the gold standard for single-cell analysis, flow cytometry or laser scanning cytometry quickly screens fluorescent-labeled cells in a flow.  Employed for a large spectrum of biological applications, flow cytometry is automated, capable of many detections, and effective at sorting single cells, but it is bulky, expensive, physically complex, and requires relatively large sample quantities. In addition, it is limited to cell analysis at a single time-point. As a result, flow cytometry cannot be used to continuously monitor cell dynamics.
Microfluidics has been developed as a platform-level and ever-evolving technology for single-cell manipulation and analysis for almost two decades, with the capacity to handle and manipulate fluids in the range of µL to pL. Many advantages over traditional methods exist for microfluidics. The microfluidic chip can, first and foremost, be flexibly built to meet the demands of various single-cell manipulation and analysis applications. Second, highly sensitive detections can be achieved by using microfluidic devices, which can operate with very small volumes of liquid (down to the pL level). Thirdly, microfluidics makes it possible to manipulate and analyze the sample in parallel at high throughput, which is advantageous for the statistically significant single-cell analysis. Fourthly, it is simple to combine many functions onto a single chip, enabling automation as well as preventing contamination and mistakes that come from human error. [13-17] Despite those advantages, microfluidics requires the access to specific equipment and can be time consuming due to the low flow rates and the recurrent experimental problems related to clogging or surface treatment.
Atomic Force Microscopy (AFM)
Among the listed techniques, the main advantage of the AFM is that it can simultaneously acquire the structures and properties of individual cells with a high spatiotemporal resolution in aqueous conditions. AFM is a reliable tool to investigate the structures and properties of native biological samples at the micro/nanoscale under near-physiological conditions.  Yet, the AFM remains only applicable to adherent cells and shows a relatively low throughput.
The FluidFM technology
Over the years, the range of cell-manipulation techniques increased as a result of new technological developments. AFM and microfluidics were combined to form a technology named "Fluidic force microscopy" (FluidFM) using microchanneled cantilevers with nanoscale apertures. The hollow cantilevers' connection to a pressure controller, which enables their operation in liquid as force-controlled nanopipettes under optical control, is a key component of the technology. In a variety of biological systems, proof-of-concept studies performed with the FluidFM, showed a wide range of single-cell manipulation, including extraction, deposition, adhesion, and injection. 
Distinguish between extracting contents directly from the nucleus of a cell or from the surrounding cytosol.
Direct volume quantification
The extracted volume can be directly quantified with extreme accuracy down to 0.1 picoliter!
Extract. Inject. Pick-up. Place. Isolate single-cell, accurately and reliably.
Gently extract from cytoplasm while keeping the cell alive and fully viable.
Save the physiological context
During extraction, keep the targeted cell in its context next to its neighboring cells and conserve established cell-cell interactions.
Semi-automated repetition of the gentle extraction several times on the same cell, e.g. before and after stimulation by a specific drug.
Applications of single-cell manipulation with FluidFM
An example of single-cell manipulation for temporal transcriptomics
Chen, Wanze, et al. (2022) used the single-cell manipulation capability of the FluidFM technology as a solid basis to transform single-cell RNA sequencing (scRNA-seq) from an end point to a temporal analysis platform with a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction. With this novel method, this study enabled the coupling of a cell’s ground-state transcriptome to its downstream molecular or phenotypic behavior. As a first approach, this work demonstrates that Live-seq can be used to directly map a cell’s trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide stimulation, and of adipose stromal cells pre- and post-differentiation. 
 Yun, Hoyoung, Kisoo Kim, and Won Gu Lee. "Cell manipulation in microfluidics." Biofabrication 5.2 (2013): 022001.
 Bar-Or, Y. “The Effect of Adhesion on Survival and Growth of Microorganisms.” Experientia 46, no. 8 (August 1990): 823–26. https://doi.org/10.1007/BF01935532.
 Khalili, Amelia, and Mohd Ahmad. “A Review of Cell Adhesion Studies for Biomedical and Biological Applications.” International Journal of Molecular Sciences 16, no. 8 (August 5, 2015): 18149–84. https://doi.org/10.3390/ijms160818149.
 Ashkin, Arthur, James M. Dziedzic, and T. Yamane. "Optical trapping and manipulation of single cells using infrared laser beams." Nature 330.6150 (1987): 769-771.
 Zhang, Hu, and Kuo-Kang Liu. "Optical tweezers for single cells." Journal of the Royal Society interface 5.24 (2008): 671-690.
 Hosseini, Imman I., et al. "Cell properties assessment using optimized dielectrophoresis-based cell stretching and lumped mechanical modeling." Scientific reports 11.1 (2021): 1-13.
 Pohl H A 1951 The motion and precipitation of suspensoids in divergent electric fields J. Appl. Phys. 22 869–71
 Pethig R and Markx G H 1997 Applications of dielectrophoresis in biotechnology Trends Biotechnol. 15 426–32
 Gascoyne P R C and Vykoukal J 2002 Particle separation by dielectrophoresis Electrophoresis 23 1973
 Pu, Huayan, et al. "Micropipette aspiration of single cells for both mechanical and electrical characterization." IEEE Transactions on Biomedical Engineering 66.11 (2019): 3185-3191.
 Davidson, P.M. et al. (2019) “High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells,” Lab on a Chip, 19(21), pp. 3652–3663. Available at: https://doi.org/10.1039/c9lc00444k.
 Chen, Jian, et al. "Microfluidic impedance flow cytometry enabling high-throughput single-cell electrical property characterization." International journal of molecular sciences 16.5 (2015): 9804-9830.
 Yin, Huabing, and Damian Marshall. "Microfluidics for single cell analysis." Current opinion in biotechnology 23.1 (2012): 110-119.
 Luo, Tao, et al. "Microfluidic single-cell manipulation and analysis: Methods and applications." Micromachines 10.2 (2019): 104.
 Gao, Dan, et al. "Recent advances in single cell manipulation and biochemical analysis on microfluidics." Analyst 144.3 (2019): 766-781.
 Reece, Amy, et al. "Microfluidic techniques for high throughput single cell analysis." Current opinion in biotechnology 40 (2016): 90-96.
 Kim, Hyun Soo, Timothy P. Devarenne, and Arum Han. "A high-throughput microfluidic single-cell screening platform capable of selective cell extraction." Lab on a Chip 15.11 (2015): 2467-2475.
 Li, Mi, et al. "Advances in atomic force microscopy for single-cell analysis." Nano Research 12.4 (2019): 703-718.
 Guillaume-Gentil, Orane, et al. "Force-controlled manipulation of single cells: from AFM to FluidFM." Trends in biotechnology 32.7 (2014): 381-388.