Single-cell extraction - Single-cell biopsy with FluidFM®  

This short note aims to provide 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.

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Single-cell biopsy | A non-destructive single-cell extraction method

Single-cell biopsy - The story

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 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. 

Single-cell extraction with the FluidFM by Cytosurge

The story behind single-cell biopsy by Cytosurge

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 was developed at ETH Zurich and 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 destroying it in the sampling process. 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. Thereby, single-cell biopsy appears as a promising alternative to achieving non-destructive sampling and cell-context preservation.

Single-cell biopsy - The method behind the single-cell extraction function

Single-cell biopsies represent a new method to perform non-invasively, continuously and in a controlled manner, single-cell analysis via either single-cell extraction or injection. The newly developed technique relies on the usage of a FluidFM nanosyringe to penetrate single living cells and extract their content for further investigation. The subsequent downstream analysis of the collected samples can then be carried out using well established procedures. This way scientists can discover subtle differences between individual cells within a population at a molecular level. The method can therefore also be applied to discover and investigate very rare cell types.  The method has encountered a growing interest in various fields of research, from neurosciencesvirology or transcriptomics


The video on the right hand-side shows how the FluidFM technology can be employed to perform a gentle, accurate and direct extraction of nuclear or cytoplasmic cellular content. Originally, the single-cell biopsy method relies on the approach developed initially to perform nanoinjection. Thus, the story behind the single-cell biopsy comes from the evolution of single-cell injection techniques from microinjection to nanoinjection.

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Microinjection vs. Nanoinjection

At the origin of the single-cell extraction method is the evolution of single-cell injection techniques, notably from microinjection to nanoinjection. 

Microinjection is part of the current tools available to realize mechanical delivery into cells alongside with other physical transfection methods. Microinjection is advantageous compared to other technique due to the precision of delivery dosage and timing, the high efficiency of transfection and the low cytotoxicity. Nevertheless, microinjection presents also important drawbacks such as labor intensive and time-consuming processes which reduce the number of cells that can be transfected or transduced in a sample. [1] 

Single-cell extraction with the FluidFM by Cytosurge - Microinjection Illustration

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. [2] Over the years, several microinjection protocols and setup have been tested. 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. [3] Despite numerous efforts to reduce cellular stress and optimize the delivery efficiency, the microinjection showed still several limitations. But the fast development of fields such as microfluidics, improved the process of microinjection into a single cell. 

A. Andamo and K. Jensen (2008) pushed the limit of conventional methods by reporting a single-cell microinjection in which fluid streams direct into a cell onto a fixed microneedle in contrast to moving a microneedle towards an immobilized cell. This proposal simplified microinjection and developed the potential of flow through automated microinjection of cells. [4] 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. [5] 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. [6] 

Single-cell extraction with the FluidFM by Cytosurge - Eukaryotic cell Illustration

Moving away from microinjection, the term “nanoinjection” was employed in the literature to describe a broad range of techniques from a targeted multiphoton optoporation of vital cells [7], a silicon microchip “nanoinjector” composed of a microelectromechanical system with an electrically conductive lance [8], also termed Lance Array Nanoinjection (LAN) [9], to  a hybrid microfluidic chip with a true 3-dimensional nanoinjection structure for precise direct delivery of biomolecule into single cells [10]. With the evolution of the protocols and setups, 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 has numerous advantages. Thanks to the unique properties of the underlying 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. In contrast to established methods for single-cell analysis, FluidFM-based single-cell extraction 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. 

Features and benefits of nanoinjection for single-cell extraction

Unprecedented Precision

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!

All-in-one platform

Extract. Inject. Pick-up. Place. Isolate single-cell, accurately and reliably.

Non-invasive extraction

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.

Continuous analysis

Semi-automated repetition of the gentle extraction several times on the same cell, e.g. before and after stimulation by a specific drug.

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Applications of single-cell extraction

In the following, nanoinjection for single-cell extraction has demonstrated 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. [11] 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. [12]

Injection into and extraction from single fungal cells

Single-cell extraction with the FluidFM by Cytosurge

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. [13] 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. [13]

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. [14] 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.

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Organelle extraction and injection using FluidFM

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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. [15]

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[1] Zhang, Yan, and Long-Chuan Yu. "Microinjection as a tool of mechanical delivery." Current opinion in biotechnology 19.5 (2008): 506-510.

[2] Celis, Julio E. "Microinjection of somatic cells with micropipettes: comparison with other transfer techniques." Biochemical Journal 223.2 (1984): 281.

[3] Zhang, Yan. "Microinjection technique and protocol to single cells." (2007).

[4] Adamo, Andrea, and Klavs F. Jensen. "Microfluidic based single cell microinjection." Lab on a Chip 8.8 (2008): 1258-1261.

[5] Chow, Yu Ting, et al. "Single cell transfection through precise microinjection with quantitatively controlled injection volumes." Scientific reports 6.1 (2016): 1-9.

[6] 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.

[7] 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.

[8] Aten, Quentin T., et al. "Nanoinjection: pronuclear DNA delivery using a charged lance." Transgenic research 21.6 (2012): 1279-1290.

[9] 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.

[10] 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.

[11] Guillaume-Gentil, Orane, et al. "Tunable single-cell extraction for molecular analyses." Cell 166.2 (2016): 506-516.

[12] 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.

[13] Guillaume-Gentil, Orane, et al. "Injection into and extraction from single fungal cells." Communications biology 5.1 (2022): 1-10.

[14] Chen, Wanze, et al. "Live-seq enables temporal transcriptomic recording of single cells." Nature 608.7924 (2022): 733-740.

[15] Gäbelein, Christoph G., et al. "Mitochondria transplantation between living cells." PLoS biology 20.3 (2022): e3001576.