Single-cell injection with FluidFM®
How do you inject a cell with FluidFM?
FluidFM | A non-invasive single-cell injection technology
The field of single-cell analysis has always found a strong interest among the research community because it allows for the quantification of a number of important factors in biology and life sciences such as the study and modulation of cellular function and the response to external stimuli. The simple fact of cellular heterogeneity suffices in demonstrating the need to develop versatile tools and methods to study specifically, non-invasively and reproducibly, the content and the reaction of a single cell to controlled environmental and experimental conditions. Up to now, the field of single-cell analysis was dominated by the combination of cell micromanipulation, sorting or isolation followed by cell lysis. A new, groundbreaking method for single-cell analysis based on the FluidFM® technology allows scientist to investigate the behavior of individual cells in their natural environment.
The new method based on the FluidFM OMNIUM, was developed at ETH Zurich and dramatically broadened the future horizon of single-cell analysis. The procedure makes it possible to inject into and collect from individual cells, reproducibly and reliably, directly in their native environment while preserving the entire cellular context. Consequently, single-cell injection can be applied repeatedly and semi-automatically to the same cell without destroying it in the process. These novel capabilities are game changer for a number of applications from genome engineering, disease modelling and drug discovery. Yet, many years of research and development have been required to obtain such an effective method for single-cell injection. In fact, originally, microinjection and other cell transfection methods were initially employed to transfect a single cell. The development of nanoinjection has defined the starting point of a new era of direct intra-nuclear or cytoplasmic injection.
Example of direct intranuclear nanoinjection into mouse primary hepatocyte injected with CRISPR-Cas9 RNP complexes.
The evolution of single-cell injection techniques
What are the differences between microinjection and nanoinjection?
Microinjection is part of the current tools available to realize physical delivery into cells alongside with alternative physical transfection methods. Microinjection showed a certain number of advantages compared to other techniques due to the precision of delivery dosage and timing, the high efficiency of transfection and the low cytotoxicity. Nevertheless, some important drawbacks were found, such as the laborious and slow processes which caused a lower throughput of transfected cells. 
Despite those limitations, microinjection has been employed extensively in research to inject various types of biological samples. In the 1980s, J.E. Celis (1984) investigated the process of microinjection into cells with micropipettes and compared it with other transfer techniques.  Over the years, several microinjection protocols and setup have been investigated. More than twenty years later, Yan Zhang (2007) proposed a single-cell microinjection technique to both attached and suspended cells, comprising primary cells, cell lines and protozoan.  Despite several efforts made to decrease cellular stress and improve the delivery efficiency, the microinjection presented still important drawbacks. But the fast development of fields such as microfluidics, improved the process of microinjection into a single cell.
A. Andamo and K. Jensen (2008) went beyond the limit of traditional methods by reporting a single-cell microinjection in which fluid streams direct into a cell onto a fixed microneedle. This proposal simplified the process and developed the potential of flow through automated microinjection of cells.  The precision and control over the injected volumes improved over time. Y. Chow et al., (2016) pushed further the technique by developing the concept of precise microinjection with a quantitatively controlled injection volume based on injection pressure and time.  Nowadays, microinjection is still employed to study cell division of mammalian cell culture. C. Day and E. Hinchcliffe (2022) described a microinjection setup to analyze mammalian cells in mitosis with same cell live and fixed imaging. 
Moving away from microinjection, the term “nanoinjection” was reported in the literature to describe a broad range of methods from a targeted multiphoton optoporation of vital cells , a silicon microchip “nanoinjector” composed of a microelectromechanical system with an electrically conductive lance , also termed Lance Array Nanoinjection (LAN) , to a hybrid microfluidic chip with a true 3-dimensional nanoinjection structure for precise direct delivery of biomolecule into single cells . With the evolution of the instrumentation, the nanoinjection method became more and more accurate and controlled, to reach a peak with the development of the FluidFM technology. The nanoinjection process with FluidFM combines the precise control of microfluidics with the accuracy and gentleness of an AFM probe. Compared to existing methods, the FluidFM nanoinjection presents a number of benefits. With the FluidFM technology, researchers can sample individual cells within a tissue culture directly within their native environment. The method can thereby be repeatedly applied to the same cell. The FluidFM-based single-cell injection does not require the cells under investigation to be separated and physically removed from their native environment. Furthermore, the cells are not destroyed in the process and can thereby be repeatedly sampled to study them over longer time periods.
Benefits and features of the FluidFM single-cell injection
Distinguish between injected contents directly from the nucleus of a cell or from the surrounding cytosol.
Direct volume quantification
The injected volume can be directly quantified with extreme accuracy down to 0.1 picoliter!
Inject. Pick-up. Place. Isolate single-cell, accurately and reliably.
Gently inject into the cytoplasm or nucleus while keeping the cell alive and fully viable.
Save the physiological context
During injection, keep the targeted cell in its context next to its neighboring cells and conserve established cell-cell interactions.
Semi-automated repetition of the gentle injection several times on the same cell.
Applications of single-cell injection with the FluidFM
In the following, the single-cell nano-injection demonstrates its potential in a broad of biological applications.
Nanoinjection into HeLa cells
Here, CRISPR-Cas9 ribonucleoproteic (RNP) complexes were injected in HeLa cells. The cells were monitored over 5 hours, prior to and after the nanoinjection of the complex.
Example of nanoinjection into adherent cells. HeLa cells, throughput up to 200 cells injected per hour could be achieved.
Examples of nano-injection into adherent cells. CRISPR-Cas9 ribonucleoproteic (RNP) complexes injected in HeLa cells. Top left:30min post injection. Top right:1h post injection. Bottom left: 2h post injection. Bottom right: 4h post injection. In the case of HeLa cells, throughput up to 200 cells injected per hour could be achieved.
Nanoinjection of proteins into cells
Many types of compounds can be injected into cells using the FluidFM technology. Find below an example of a nanoinjection of Cas9-GFP protein in Human iPS cell nucleus. A full experimental protocol was prepared, and the experiment performed in-house with the FluidFM OMNIUM. For the full experimental protocol, click here.
Example of nanoinjection into adherent cells. Injection of Cas9-GFP protein in Human iPS cell nucleus.
Cytoplasmic and nuclear nanoinjection into Mouse Primary Hepatocyte
Two types of nanoinjections into a mouse primary hepatocyte with CRISPR-Cas9 RNP complexes could be achieved with the FluidFM technology. Both nuclear and cytoplasmic direct nanoinjection could be performed.
Example of cytoplasmic nanoinjection into Mouse primary hepatocyte injected with CRISPR-Cas9 RNP complexes.
Nanoinjection into single fungal cells
Fungal cells represent a challenge for intracellular injection and extraction due to their cell wall. Up to now, the most popular techniques for intracellular delivery into fungi relied on the first breakdown of the cell wall to produce protoplasts, a process that is extremely time-consuming, inefficient, inconsistent, and detrimental to cell survival.  Guillaume-Gentil and Orane, et al. (2022) employed the FluidFM technology to inject various solutions into and extract cytoplasmic fluid from individual fungal cells, including unicellular model yeasts and multicellular filamentous fungi. The FluidFM technology offered a strain-free and cargo-independent approach for manipulating and analyzing fungi. 
Nanoinjection into organelles
Very recently, Gäbelein, Christoph G., et al. (2022) proposed 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. 
 Zhang, Yan, and Long-Chuan Yu. "Microinjection as a tool of mechanical delivery." Current opinion in biotechnology 19.5 (2008): 506-510.
 Celis, Julio E. "Microinjection of somatic cells with micropipettes: comparison with other transfer techniques." Biochemical Journal 223.2 (1984): 281.
 Zhang, Yan. "Microinjection technique and protocol to single cells." (2007).
 Adamo, Andrea, and Klavs F. Jensen. "Microfluidic based single cell microinjection." Lab on a Chip 8.8 (2008): 1258-1261.
 Chow, Yu Ting, et al. "Single cell transfection through precise microinjection with quantitatively controlled injection volumes." Scientific reports 6.1 (2016): 1-9.
 Day, Charles, Alyssa Langfald, and Edward H. Hinchcliffe. "Using Microinjection of Mammalian Cultured Cells to Study Cell Division." Mitosis. Humana, New York, NY, 2022. 105-122.
 Stracke, Frank, Iris Rieman, and Karsten König. "Optical nanoinjection of macromolecules into vital cells." Journal of Photochemistry and Photobiology B: Biology 81.3 (2005): 136-142.
 Aten, Quentin T., et al. "Nanoinjection: pronuclear DNA delivery using a charged lance." Transgenic research 21.6 (2012): 1279-1290.
 Sessions, John W., et al. "CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection." SpringerPlus 5.1 (2016): 1-11.
 Yun, Chang-Koo, et al. "Nanoinjection system for precise direct delivery of biomolecules into single cells." Lab on a Chip 19.4 (2019): 580-588.
 Guillaume-Gentil, Orane, et al. "Injection into and extraction from single fungal cells." Communications biology 5.1 (2022): 1-10.
 Gäbelein, Christoph G., et al. "Mitochondria transplantation between living cells." PLoS biology 20.3 (2022): e3001576.