Is single-cell analysis always destructive?

Single-cell omics has transformed modern cell biology. Technologies such as single-cell RNA-seq or spatial transcriptomics allow researchers to profile thousands of cells at once and uncover cellular heterogeneity at unprecedented resolution.

But they share the same limitation: the cell does not survive the measurement. What we obtain is a highly detailed snapshot of a single moment in time.

For many biological processes, however, snapshots are not enough. Differentiation, infection, drug response or immune activation are dynamic processes that unfold over time. Ideally, we would like to observe how specific cells change during that process.

So the question becomes:
Can we interact with a cell, measure something from it and still keep it alive?

The recent Nature Methods Primer "Fluidic Force Microscopy" on FluidFM highlights an experimental approach designed exactly for this type of question.

In this blog post, we take the publication as a starting point and look at the underlying technology and some of the applications that have emerged from it in recent years.

Figure 1 & 2- FluidFM Omnium platform for single-cell manipulation. The system integrates atomic force microscopy with microfluidic pressure control, allowing researchers to interact with individual living cells using a hollow cantilever probe. The probe functions as a force-controlled nanopipette and can inject molecules, extract cytoplasm, apply compounds locally or measure mechanical properties of single cells.

AFM meets microfluidics

The FluidFM Omnium combines atomic force microscopy with microfluidics in a single microscopy platform. At the heart of the system is a hollow AFM cantilever connected to a pressure controller. Instead of a solid AFM tip, the probe contains a tiny microchannel and a microscopic aperture at its end. This effectively turns the cantilever into a force-controlled nanopipette.

With accurate positioning and picoliter pressure control, the same system can be used to:

• inject molecules into individual cells
• extract tiny amounts of cytoplasm
• locally apply compounds or drugs to a single cell
• measure adhesion forces and mechanical properties
• pick up, move or isolate individual cells

Rather than representing a single assay, FluidFM behaves more like a toolbox for targeted single-cell experiments.

Taking molecular snapshots from living cells

One particularly interesting application is taking cytoplasmic biopsies. Here, the probe gently penetrates the cell membrane and aspirates a tiny amount of cytoplasm. The extracted material can then be analyzed with downstream molecular techniques.

This approach became especially exciting with the development of Live-seq, a method that enables transcriptomic analysis of living cells by repeatedly sampling small cytoplasmic biopsies over time.

Chen et al. described this approach in Nature in 2022: Live-seq enables temporal transcriptomic recording of single cells. Instead of sequencing a cell once and destroying it, Live-seq allows researchers to follow transcriptional changes in the same cell across multiple time points.

A key technological driver behind this development is the progress in ultra-low input RNA sequencing kits. To support such workflows, Cytosurge partnered with Lexogen to combine FluidFM sampling with the LUTHOR HD library preparation kit, which can detect thousands of genes from extremely small RNA quantities obtained from only a few picoliter of cytoplasm. Together, these technologies make it possible to reconstruct transcriptional trajectories from living cells.

Delivering molecules into individual cells

Another widely used application is force-controlled nano-injection. This addresses a problem many labs encounter: How do you deliver controlled amounts of molecules into individual cells, especially into cells that are otherwise difficult to edit or to transfect?

With FluidFM, molecules, proteins, RNA or also whole CRISPR complexes can be injected directly into the cytoplasm or into the nucleus of a selected cell. This principle is also used by the CellEDIT service and enables genome editing even in otherwise difficult-to-modify cell types.

Mechanics, adhesion and cell manipulation

FluidFM probes can also be used in a completely different way: by gently attaching a cell to the cantilever via suction. Researchers can measure adhesion forces between cells and substrates or study how mechanical properties change under different conditions. Because cells can be repeatedly picked up and released, adhesion measurements can be performed across many cells with relatively high throughput.

The same pick-and-place principle can also be used to isolate individual cells from cultures or to reposition cells on the surface.

Where the field is heading

Single-cell biology has largely been shaped by high-throughput omics technologies. At the same time, there is growing interest in experiments that allow direct manipulation of individual cells.

As these different applications mature, experiments become possible that would have been extremely difficult only a few years ago, for example repeatedly sampling RNA from a cell after perturbing it with injected molecules or even transferring cellular components or organelles between cells.

Technologies like FluidFM therefore do not compete with high-throughput single-cell omics. Instead, they complement them by enabling mechanistic experiments at the level of individual cells.

REFERENCES:

Chen, W., Guillaume-Gentil, O., Rainer, P.Y. et al. Live-seq enables temporal transcriptomic recording of single cells. Nature 608, 733–740 (2022). https://doi.org/10.1038/s41586-022-05046-9

Zambelli, T., Guillaume-Gentil, O., Sarajlic, E. et al. Fluidic force microscopy. Nat Rev Methods Primers 6, 15 (2026). https://doi.org/10.1038/s43586-025-00463-2