An overview of single-cell manipulation methods and applications with FluidFM®
This short note aims to provide the reader with an overview of tools employed for accurate cell manipulation and more specifically single-cell manipulation methods, suited for a broad range of life sciences and biological applications.
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About the importance of an accurate cellular manipulation
In many biological research fields, tools are needed to accurately manipulate cells.  In neurosciences, cell manipulation can be employed to study the development of a subpopulation of neurons that produce a specific neurotransmitter. Isolating the subpopulation of interest allows to perform a neuron-by-neuron analysis. In drug discovery, cell manipulation is omnipresent from the design of the engineered cell line to the injection of the soluble compound to be tested into each individual cell. In cancer research, the study of gene expression, the investigation of cellular heterogeneity in a tumor or again, the exploration of the immune cell populations related to the tumor microenvironment – all strongly rely on cell manipulation and subsequent analysis. Overall, an accurate manipulation of cells is at the basis of each and every thorough cell analysis and resulting key findings in biology.
Traditionally in cell analysis, 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, can lead to a false interpretation. That is why, with the fast development of microscopy techniques, various methods were introduced to visualize, enumerate and manipulate cellular and intracellular components at the scale of an individual cell. The need for single-cell analysis pushed the creation of tools to manipulate accurately and non-destructively, individual cells. The expression “single-cell manipulation” was then introduced to describe 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.
At first, single-cell analysis was 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 was difficult when using traditional biological tools, such as petri-dishes and well-plates. Additionally, most of the intracellular, extracellular components are present in very small concentrations and have a wide range of concentrations, which demand highly sensitive and specific detection methods. Lastly, cellular heterogeneity implies a broad variety of cell sensitivity and experimental conditions that need to be matched to ensure cell viability and a sufficient throughput and reliable tracking. Hence, with those numerous challenges, different categories of single-cell manipulation were developed, supported by various versatile techniques and technologies.
Consequently, with those challenges, different categories of single-cell manipulation were developed, supported by various techniques and technologies.
Categories of single-cell manipulation
A first category of cellular manipulation - single-cell injection - 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.
A second category - single-cell extraction - illustrates the action of using a tool to penetrate single living cells and extract their intracellular content for further investigation. This function has encountered a growing interest in various fields of research, from neurosciences, virology or single-cell RNA sequencing (scRNA-seq).
A third type of cellular manipulation can be performed by approaching a cell with a probe to measure the interaction between the probe and the cell during both approach and retraction of the probe from the cell surface. This process is reported in the literature as single-cell adhesion measurement. Indeed, measuring adhesion at the single cell level is key for the study of numerous biological systems, from bacteria to mammalian cells. [2,3]
More categories of single-cell manipulation exist such as single-cell isolation, single-cell sorting or single-cell patterning. 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
Until today, single-cell analysis 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 single-cell study requires the use of high-throughput and efficient tools for manipulating and analyzing cells at the individual cell level. 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 (OT)
For Sorting - Patterning - Cell Adhesion - Injection
Over the years, to manipulate individual biological cells and carry out intricate biophysical/biomechanical characterizations, optical tweezers have become a common tool. Optical tweezers are based on strongly focused laser beams that can trap and manipulate micro- to nanosized dielectric particles. The use of optical traps – laser light, on the cells, considered as dielectric particles – causes the cells to be pushed into a highly concentrated, high-intensity light beam.  From this process, various actions can be performed on the cell. Optical Tweezers are an interesting choice for cellular manipulation due to their adaptability to liquid media settings, non-contact force for manipulating cells, and force resolution as accurate as 100 aN. However, as the power of the OTs is in the range of a few hundred milliwatts in the sample plane, it is not sufficient to counteract the internal pressure of the cell or break the cell membrane to force external particles or internal organelles to move in or out of the cell, respectively. Therefore, OT has been used in conjunction with other micromanipulation tools to perform cell injection or extraction.  Among others, their numerous uses include adding foreign substances into individual cells, placing cells to precise regions, and sorting cells in microfluidic devices. 
For Sorting - Separation - Cell Adhesion
Dielectrophoresis (DEP) has been extensively employed for cell sorting, cell separation, molecular analysis, single-cell mechanobiology, and cell deformation.  Because various cells have varied dielectric characteristics, they respond differently and specifically to an electrical field. Thus, the DEP approach offers high throughput assessment and excellent accuracy. Nowadays, DEP is employed broadly in the complex spatial manipulation of viruses, bacteria, cells and sub-micron biological particles [8–10] but remains limited to cell sorting, separation and force measurement.
For Injection - Extraction - Isolation
The mechanical and electrical characteristics of a single cell can be characterized using the micropipette aspiration technique.  Micropipette aspiration or pulling works by applying accurate and sensitive negative pressure from a pressure-based controller to the suction or aspiration of a cell through a glass micropipette or microneedle. 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.  Despite this method appearing as straightforward and user-friendly, the technique is only applicable to nonadherent cells and presents a relative low throughput with a risk of killing the cell during the process of micro-injection or -extraction.
For Cell Sorting
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.
For Cell Sorting – Isolation – Trapping - Extraction - Injection
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 with a microfluidic chip.
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. [14-18] 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. Lastly, several on-chip microtools were also developed to perform extraction from or injection into cells but those microfluidic methods implied cell lysis. 
Atomic Force Microscopy (AFM)
For Cell Adhesion - Visualization
An Atomic Force Microscope is an instrument that allows to gain both quantitative and qualitative information on various surface physical properties from size, morphology, roughness but also on the interaction forces between a tip and a surface, down to the nanometer scale. An AFM is composed of a nanometer-sized tip attached to cantilever. The tip moves based on the tip-surface interactions while approaching to and retracting from the surface. Those changes are tracked by focusing a laser beam on the tip with a photodiode. 
One of the main advantages of the AFM is that it can simultaneously acquire the structure and properties of individual cells with a high spatiotemporal resolution in aqueous conditions. In addition, an AFM can operate in air and in liquid with a very gentle and controlled approach. It is therefore a reliable tool to characterize 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
For Injection - Extraction - Patterning - Cell Adhesion
AFM and microfluidics were combined to form a technology named "Fluidic force microscopy" (FluidFM) using micro-channeled 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 injection, extraction, and adhesion measurement detailed in the following section.
Single-cell injection with the FluidFM
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 cell and the reproducibility of the experiment.
Single-cell extraction with FluidFM
The FluidFM nanosyringe can be employed to penetrate single living cells and collect cytoplasmic extracts on the very same cell while keeping the cell alive, and perform further downstream 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.
Single-cell adhesion with FluidFM
With the FluidFM OMNIUM, semi-automated single-cell adhesion measurements 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.
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. 
An example of Organelle cellular manipulation – extraction and injection
Gäbelein, Christoph G., et al. (2022) offered a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. Upon the extraction of a set number of mitochondria, a morphological transformation was observed. A pearls-on-a-string phenotype was obtained due to locally applied fluidic forces. mitochondria. With this work, the transplantation of healthy and drug-impaired mitochondria into primary keratinocytes became possible and enabled the monitoring of mitochondrial subpopulation rescue. 
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