An overview of single-cell extraction with FluidFM®
This short note provides the reader with an overview of a novel and non-invasive single-cell extraction method suited for a broad range of life sciences and biological applications - the single-cell biopsy.
What is single-cell extraction?
The field of single-cell analysis has always found a strong interest among the research community because it allows for the quantification of several 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 analytical methods to examine specifically, non-invasively and reproducibly, the content of a single cell.
Up to now, the field of single-cell analysis was dominated by the combination of cell micromanipulation, sorting or isolation followed by cell lysis. With the development of the techniques, a non-destructive single-cell extraction method has arisen: the single-cell biopsy. Unlike methods like micromanipulation or laser cutting, the single-cell biopsy allows to extract sub-cellular content from the cytoplasm or the nuclear without compromising cellular viability.
Single-cell biopsy | A non-destructive single-cell extraction method
Nowadays, researchers are more and more interested in studying the behavior of individual cells within a population. Studying the properties and behavior of individual cell rather than the conduct of an entire cell population can lead to a much deeper understanding of the underlying biological processes.
The single-cell biopsy method could revolutionize biological research as it opens a completely new dimension for the study of individual cells.
The procedure makes it possible to sample the content of individual cells for various analyses directly in their native environment while preserving the entire cellular context.
Consequently, single-cell extraction can be applied repeatedly to the same cell without killing it in the sampling process. Thereby, single-cell biopsy appears as a promising alternative to achieving non-destructive sampling and cell-context preservation. The method has encountered a growing interest in various fields of research, from neurosciences, virology or transcriptomics. The video on the right hand-side shows how the technology can perform a gentle, accurate and direct extraction of nuclear or cytoplasmic cellular content.
Gently extract from cytoplasm or nucleus 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 extraction
In the following, single-cell extraction demonstrates its potential in a broad of biological applications from single-cell molecular analysis to temporal transcriptomics.
Single-cell extraction for molecular analyses
Guillaume-Gentil, Orane, et al (2016) demonstrated the direct application of the FluidFM technology to perform quantitative and spatiotemporal single-cell analysis of cytoplasmic and nucleus soluble molecules. The novel non-invasive single-cell extraction method allowed researchers to detect enzymatic activities and transcript abundances. Furthermore, this study proved the ability of cells to withstand extraction of up to several picoliters.  Later, the approach was employed for single-cell mass spectrometry by adding a step of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This work showed the ability of the method to detect and identify twenty metabolites recovered from the cytoplasm of individual HeLa cells. 
Injection into and extraction from 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. 
Live-seq enables temporal transcriptomic recording of single cells
Chen, Wanze, et al. (2022) employed the FluidFM technology as a solid basis to establish Live-seq - a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction. With this groundbreaking approach, 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.  This publication proves that Live-seq can address a number of biological problematics by transforming scRNA-seq from an endpoint to a temporal analysis approach.
Organelle extraction and injection
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. 
 Guillaume-Gentil, Orane, et al. "Tunable single-cell extraction for molecular analyses." Cell 166.2 (2016): 506-516.
 Guillaume-Gentil, Orane, et al. "Single-cell mass spectrometry of metabolites extracted from live cells by fluidic force microscopy." Analytical chemistry 89.9 (2017): 5017-5023.
 Guillaume-Gentil, Orane, et al. "Injection into and extraction from single fungal cells." Communications biology 5.1 (2022): 1-10.
 Chen, Wanze, et al. "Live-seq enables temporal transcriptomic recording of single cells." Nature 608.7924 (2022): 733-740.
 Gäbelein, Christoph G., et al. "Mitochondria transplantation between living cells." PLoS biology 20.3 (2022): e3001576.