# Emerging mechanobiology techniques to probe intracellular mechanics

Kelly Molnar, Jean-Baptiste Manneville

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Kelly Molnar, Jean-Baptiste Manneville. Emerging mechanobiology techniques to probe intracellular mechanics. npj Biological Physics and Mechanics, 2025, 2 (1), pp.12. 10.1038/s44341-025-00016-4. hal-05411995

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https://doi.org/10.1038/s44341-025-00016-4

# Emerging mechanobiology techniques to probe intracellular mechanics

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Kelly Molnar & Jean-Baptiste Manneville

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Studying the physical properties of sub-cellular components is increasingly important in understanding cell mechanics. This review focuses on the most advanced techniques available for investigating intracellular mechanics. We distinguish methods that act as force generators and those that act as force sensors. We highlight six state-of-the-art techniques, with increased spatial and temporal resolutions: optogenetics, Brillouin microscopy, bacterial cells and nanorobots, optical tweezers, membrane tension probes, and magnetic particles.

It is now widely accepted that most cellular functions depend on the mechanical properties of both the environment and the cell itself. Mechanobiology studies how cells sense and respond to physical cues and forces and how they transduce mechanical signals into biochemical responses. While a large amount of work has been performed in the past forty years to understand cell mechanics at the whole cell level, technical advances have recently made possible the study of intracellular mechanotransduction, i.e. how forces are transmitted within the cell at the subcellular level. Applying and measuring forces inside cells allows to address key questions, for instance in the fields of cell migration and division. These processes involve the deformation and rearrangement of cellular components, which can be studied by either applying or monitoring forces in real-time. For instance, cell migration often involves the deformation of the nucleus as the cell passes through confined spaces¹,². Similarly, in the field of cellular energetics and metabolism, the central role of cell mechanics is increasingly recognized. Intracellular forces are required for the transport, the fission, and fusion of mitochondria, which govern mitochondrial activity and hence energy production³⁻⁵. Besides mitochondria and the nucleus, most intracellular compartments in the secretory pathway (the endoplasmic reticulum (ER), the Golgi apparatus, endosomes) are also able to sense and respond to forces⁴,⁶,⁷. Because intracellular transport regulates lipid and protein production, sorting, export and import and, as such, is involved in most cell functions, including polarity, migration, division, the mechanosensitivity of endomembranes is critical for mechano responses.

The physical properties of sub-cellular components, such as organelles and the cytoplasm, are thus currently an active topic of research⁴,⁶⁻⁹. Although many reviews have explored the techniques related to cells and tissues¹⁰,¹¹, less has been written about the most current methods adapted to investigating the cell interior at a high spatio-temporal resolution, which is the focus of this review. Increased spatial and temporal sensitivity allows for the observation of minute or transient processes. In cancer for instance, changes in physical properties, such as cell viscosity, are correlated with increased metastatic potential¹². Furthermore, improvements in the

precision of measurements are essential for enhancing computer models of biological processes.

Two important distinctions have to be made at this point. The first concerns techniques that act as a force generator vs. techniques that act as a force sensor. To fully study the mechanical properties of an organelle, it is important to be able to exert forces on it and also to quantify its rheological properties. Some of the experimental techniques detailed in this review are one, or the other, or both. The second distinction concerns techniques that act exclusively at the intracellular level vs. techniques that act at the transcellular or extracellular levels. In the case of a force generator for instance, exclusively intracellular means that the method is able to exert a force that is localized in space, as in the case of optical tweezers as described below, which can cause a local deformation of an organelle on the order of a few micrometres. The transcellular or extracellular techniques are more global and less spatially resolved. In this review, we will focus only on the former kinds of spatially localized methods. It should also be noted that all these methods have specific temporal characteristics so that a force can be exerted and/or measured for different amounts of time, at different frequencies and in a reversible or irreversible fashion.

In this review, we highlight six different state-of-the-art experimental techniques that are specially adapted to the study of the mechanical properties of sub-cellular components. They are (ordered from force generators to mechanical sensors): optogenetics, bacterial cells and nanorobots, optical tweezers (OT), Brillouin microscopy, tension probes, and magnetic particles (Table 1, Fig. 1). We emphasize these techniques because, unlike their forerunners, they allow for a higher degree of precision in space and time, and can thus provide information about the organelle of interest without simultaneously altering other cell components. For example, common ways of deforming a cell and its components are by using a cell stretcher¹³⁻¹⁵, a parallel microplate rheometer¹⁶, a microfluidic device¹⁷, or an atomic force microscope (AFM) cantilever¹⁸. Another way is to put cells on different surfaces with textured adhesive micropatterns; this causes them to stretch to different degrees, and thus their internal organelles will stretch as well¹⁹,²⁰. Similarly, plating cells on stiff or soft substrates of controlled rigidity allows

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<table><caption>**Table 1 | Physical quantities and orders of magnitude**</caption><thead><tr><td>Technique</td><td>Description</td><td>Order of magnitude and unit</td><td>Source</td></tr></thead><tbody><tr><td>Optogenetics</td><td>Force of individual myosin motor<sup>a</sup></td><td>pN</td><td>138</td></tr><tr><td>Bacterial cells/ nanorobots</td><td>Force of bacterial cell upon collision</td><td>unknown</td><td>none</td></tr><tr><td>Optical tweezers</td><td>Force exerted by optical trap</td><td>pN to nN</td><td>139,140</td></tr><tr><td>Brillouin microscopy</td><td>Elastic moduli</td><td>kPa to GPa</td><td>85</td></tr><tr><td>Tension probes</td><td>Tension</td><td>0.01 to 1 mN/m</td><td>115</td></tr><tr><td>Magnetic particles</td><td>Force acting on particles</td><td>fN to nN</td><td>141,142<sup>b</sup></td></tr></tbody></table>

G giga 10⁵, n nano 10⁻⁵, p pico 10⁻¹², f femto 10⁻¹⁵.

ªOptogenetic constructs exist for the control of molecular motors such as myosin. Forces exerted by other motors or cytoskeletal elements are of the same order of magnitude.

ᵇLarger force values are not well-established intracellularly.

**Fig. 1 | Emerging techniques for intracellular mechanobiology.** a Optogenetics. An anchor protein (green) is inserted into the plasma membrane or the membrane of a specific organelle. Blue light activation from a laser causes the recruitment of the genetically modified target protein, often a molecular motor, to the anchor. The yellow star indicates the conformational change of the anchor protein upon photoactivation. b Bacterial cells and nanorobots. A single bacterial cell (e.g. *Shigella flexneri*, in green) moves in an uncontrolled manner through the cytoplasm, propelled by its actin comet tail. Collisions with intracellular organelles generate intracellular forces. For instance, collision with mitochondria causes mitochondrial fission. Figure inspired by images from Helle et al.⁴⁸. c Optical tweezers. A µm-diameter bead (green) trapped by optical tweezers (OT) can push against and deform the nucleus (purple). The infra-red (IR) laser generating the optical trap can be moved to displace the bead within the cytoplasm or, if the position of the trap is fixed, the nucleus can be moved towards the bead by displacing the microscope stage. d Brillouin microscopy. Photons from the laser interact with phonons (yellow) in an intracellular structure and cause Brillouin scattering to occur. Interaction between light and phonons induces a shift in the frequency of scattered light which can be related to the rheological moduli of intracellular compartments. e Tension probes. Mechanical planarization of the twisted Flipper molecule increases the conjugation and thus connects the donor and acceptor regions. This difference is at the origin of the difference in the lifetimes. The stars (*) refer to different functional groups, which target specific organelle membranes. f Magnetic particles. In Magnetic Rotational Spectroscopy (MRS), magnetic rods of a few micrometres in length will oscillate back and forth in the cell cytoplasm under a rotating magnetic field or switch to a rotating regime depending on the frequency of the rotating field. The magnetic field is generated by an electromagnet positioned near the sample.

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to modify cell contractility²¹ and osmotic shocks can be used to enlarge or shrink cells. On the sensing side, the gold standard for measuring mechanical properties has been AFM, although this technique is limited to the cell exterior and surface topologies²²,²³. Similarly, micropipette aspiration of living cells can allow for the measurement of their overall viscous and elastic properties, as well as those of their organelles²⁴,²⁵. Furthermore, the generation of intracellular forces can include photoablation, the use of a laser

to cut cellular components, usually cell-cell junctions. This technique was also used to cut intracellular cytoskeletal fibres, for instance, actin bundles²⁶, inside cells. Chromophore-assisted laser inactivation (CALI) is another technique that allows the specific inactivation of proteins in a spatio-temporal controlled manner and, when applied to cytoskeletal proteins or molecular motors, can modulate intracellular forces²⁷,²⁸. However, because these techniques induce a relaxation in intercellular or intracellular tension
rather than apply controlled forces and because inferring forces from these experiments requires complex modelling, we do not develop them further and refer the interested reader to the following recent reviews²⁹⁻³⁴.

Here, we argue that the near future of intracellular mechanics should revolve around the six experimental techniques outlined in this review. This is not intended to be a comprehensive review of all available techniques, and our mention of particular organelles is incidental. We illustrate each technique with a small number of chosen examples.

# Force generators: methods to apply forces at the intracellular level

## Optogenetics

In this section, we examine the role of optogenetics, a cutting-edge experimental technique, in establishing the mechanosensitivity of intracellular organelles. We give an overview of how it works at the molecular level, detail a recent application to the ER in living cells, and describe the many advantages of this technique.

Optogenetics is a technique that uses light to control the expression and/or localization of specific proteins in living cells, enabling precise spatial and temporal regulation of cellular functions. This is achieved using two key components: a light-sensitive protein that changes conformation upon illumination, and a second protein, which is typically the protein of interest, that interacts with the light-sensitive protein³⁵,³⁶. Two such couples are the Cry2/CIB1 and the iLID/SspB constructs³⁷,³⁸. The light-sensitive protein can be engineered to be organelle-specific, acting as an anchor that localizes to a particular cellular compartment (Fig. 1a). By restricting the illumination pattern, highly specific activation of the target protein can be achieved in a precise, sub-cellular spatial manner³⁹. The physical quantities involved are the area of the illumination region of interest (ROI), the duration and intensity of the illumination, and the concentration of the photosensitive proteins in the ROI. The activation is rapid, usually on the sub-second timescale, and persists for up to several minutes after the excitation beam is removed³⁸.

A recent application of this technique established the mechanosensitivity of the ER⁴⁰. The first element was the use of LIMER (light-inducible ER-specific mechanostimulator) to deliver the force to the ER by shining light on the organelle, which causes the recruitment of molecular motors (a modified kinesin-1). This caused the ER to contract upon the localized application of blue light (470 nm). The researchers hypothesized that this contraction would cause the opening of mechanosensitive ion channels in the ER membrane, and likely cause the release of calcium ions ($Ca^{2+}$), as transfer of $Ca^{2+}$ across the ER membrane is known to be essential for various cellular activities⁴¹,⁴². GCaMP6, a genetically encoded intensiometric cytosolic $Ca^{2+}$ indicator was used for its stable expression, reduced toxicity, and sub-cellular specificity⁴⁰,⁴³. To identify the ion channels, TRPV1 and PKD2⁴⁴,⁴⁵, researchers applied the TRPV1 antagonist, SB-366791⁴⁶, and used a PKD2 knockdown, observing a significant reduction in ion flux.

Many advantages of optogenetics are illustrated in this study. It allows for a protein-specific control in space using a ROI for the activation photons, and in time, the activation being on the order of seconds³⁸. It can be done in vivo, with no known damage to cells, and the activation is orthogonal to biological processes (meaning other proteins will not be unintentionally recruited). The disadvantages are the relative complexity of the molecular biology needed to establish a stable cell line expressing the optogenetic constructs. Light penetration can be an issue on complex media such as the human brain, with many lipid-water interfaces which cause refraction, thus limiting the spatial resolution of the activation photons as the medium becomes thicker⁴⁷. Overall, in terms of non-invasiveness and specificity, it is one of the leading techniques available to deliver forces to the organelles of living cells.

## Bacterial cells and nanorobots

Although typically abiotic, the force delivery mechanism in one recent study was a living bacterial cell, freely moving inside the eukaryotic cytoplasm and colliding with the mitochondrial network and the observed effect was

mitochondrial fission⁴⁸. This unusual approach was used to explore the question of how organelles avoid the catastrophic consequences of co-existing, intertwined with one another in a densely packed cellular environment⁴⁹. The *Shigella flexneri* bacterium uses the cytoskeletal actin of its host to stimulate rapid movement via polymerisation⁵⁰, reaching cytoplasmic speeds of up to 0.5 µm/s⁵¹. In sum, forces are applied to organelles in vivo by allowing a pathogenic bacterial cell to move freely and collide with the obstacles in its path with a force that is not precisely known (Fig. 1b).

Cellular mitochondria form dynamic cell-wide networks that undergo fission and fusion as a result of metabolic and mechanical cues³⁻⁵ and are highly regulated under normal conditions, the biochemistry of which is well-known⁵²,⁵³. For reviews on the links between mitochondrial morphology and biomechanics, see refs. 3,4. In ref. 48, mitochondrial fission events act as a readout of force sensing.

Unlike the other techniques described in this review, the use of freely moving, living bacteria to deliver intracellular forces in vivo is not a well-established technique, and although very intriguing, suffers from many obvious limitations: the direction of bacterial motion is random, and the magnitude of the force induced by each impulse is not necessarily the same. This limits how reproducible and quantitative experiments could ever be. It is however possible to improve this approach by combining it with other methods highlighted in this review. For instance, the direction and speed of the bacteria could be controlled using optogenetics by modulating the actin polymerization machinery, which causes propulsion⁵⁴,⁵⁵. One could also attempt to set the initial position of the bacteria using optical tweezers.

A similar technique is based on nanorobots, which are common in the biomedical literature, mainly as drug delivery systems or biosensors, but surprisingly have been less explored in the context of intracellular mechanics, with the notable exception of certain magnetic probes discussed below. These nanorobots are outfitted with either a magnetic field, chemical, light, or ultrasound-based propulsion system⁵⁶. One example is the Magnetically Propelled Nanomotor (MPN), which has a helical shape, is composed of biocompatible materials, and can be guided with a rotating magnetic field³⁷. Although intended as a delivery system (for instance in cell transfection), nanorobots like the MPN could in principle be used to probe intracellular mechanical properties, possibly calibrating these devices by pressing them against a bead held in optical tweezers (OT). In addition, photothermal materials convert near-infra red (IR) light into thermal energy on asymmetric nanorobots, thus causing propulsion⁵⁶. These devices range in morphologies from spherical, to urchin-like or flower-like⁵⁸⁻⁶⁰. These often suffer from the same flaws as magnetic particles, discussed below, which is that they either go towards or away from some external field and cannot easily engage in more complex trajectories. Notice here the partial similarity to optogenetics, but in this case, light is used to control an abiotic system. For a comprehensive review of this topic and the wide variety of nanorobots, see ref.⁵⁶.

## Optical tweezers

Optical tweezers (OT) have been used in a wide range of biological systems from individual proteins in vitro⁶¹ to reconstituted or isolated organelles⁶²,⁶³, intracellular organelles, cells⁶⁴⁻⁶⁶, multicellular aggregates⁶⁷, or Drosophila⁶⁸,⁶⁹, and zebrafish embryos⁷⁰. Here we focus on applications at the intermediate scale: the cytoplasm and organelles in living cells.

OT allows for the controlled displacement of a small object using a focused laser beam. In the case of intracellular mechanics, this object is usually a chemically inert, dielectric bead with a diameter of roughly 1 µm. Endogenous dense granules, such as lipid droplets, have also been used⁷¹. The incident light is scattered by the bead. If the bead is off-centre, there will be an increase in the scattered light on leaving that same side of the bead, thus pushing it back towards the middle of the trap. This is achieved using an IR laser which neither damages living cells nor interferes with fluorescent imaging. In the linear regime, i.e. at low bead displacement from the trap centre, OT behaves as a Hookean spring of stiffness $k$ (in N/m). The force $F$ (in N) exerted by the trap on the bead is given by $F = k\Delta x$ where $\Delta x$ is the displacement of the bead away from the centre of the trap (Fig. 1c).
Because of this spring-like behaviour, optical tweezers have a dual use, first as force generators inside living cells, and second as stiffness sensors (see below). They can both deliver and measure forces in the hundreds of pico- Newtons, with sub-micrometre accuracy.

# Force sensors: methods to measure forces and rheology at the intracellular level

## Optical tweezers

OT has been widely used to study cellular and intracellular rheology⁶. By moving the trap toward an organelle or through the densely crowded cytoplasm, rheological properties such as elasticity and viscosity can be measured⁹. This is done by tracking the location of the bead relative to that of the trap centre. These will differ when an obstruction is encountered, and the difference will increase with the rigidity of the obstacle. OT has been used in this way to study the visco-elasticity of the cytoplasm⁹,¹⁹,⁷²,⁷³. OT also allowed to study the effects of different cytoskeletal inhibitors on the stiffness of the Golgi apparatus in cellulo⁷⁴. In another study, a trapped bead was pressed against the ER (in this case from outside of the cell) in order to trigger the release of Ca²⁺ through mechanosensitive ion channels in the ER membrane, which was visualised using a genetically encoded ion reporter⁷⁵. In contrast with AFM experiments, which do not allow a direct measurement of nuclear mechanics, indentation of the nucleus by OT has been recently developed in living cells to quantify the local viscoelastic properties of the nucleus in living mammalian cells⁷⁶,⁷⁷, and in zebrafish cells⁷⁸⁻⁸⁰.

The advantages of this recent approach to intracellular mechanics have to do mainly with the minimal invasiveness of the technique. The beads will be spontaneously internalized by many cell types. These beads are chemically inert and can be moved about with no obvious damage to the cell. When combined with live cell fluorescence imaging, OT allows to either apply a force or measure rheological properties of a specific intracellular organelle at the local scale, typically in regions of the size of the bead (1 µm). However, despite order-of-magnitude level improvement compared to external techniques such as cell stretching, osmotic shocks, or AFM, which operate at the scale of the cell (10 µm), OT does not apply or measure forces at the sub-micrometre level. Further, when measuring the stiffness of an organelle, one has to also consider the role of cytoplasm crowding, which hinders bead motion and makes absolute measurements of rheological quantities difficult. More detailed reviews discussing intracellular OT are available⁶,⁹,⁸¹.

## Brillouin microscopy

A well-known technique in materials science that has become well-established over the past decade in biological systems is confocal Brillouin microscopy. It is based on the principle of Brillouin scattering⁸². Briefly, Brillouin scattering is caused by the interaction between light and phonons. Phonons result from spontaneous acoustic waves propagating inside a material due to thermal vibrations of molecules (Fig. 1d). The interaction between light and phonons induces a shift in the frequency of scattered light which can be used to measure the so-called longitudinal modulus (often denoted by M) of cell components. M is similar to the Young modulus, except it also accounts for the contraction that occurs in the direction orthogonal to stretching or the bulging orthogonal to a compression. The frequency shift is related to the velocity of the phonons, which can be used to calculate M.

Equation (1), taken from ref.⁸³, illustrates the relationship between the shift in the frequency of the light Δν and the longitudinal modulus M (with n and ρ the refractive index and the density of the material respectively, λ the incident wavelength, and θ the collection angle of the scattered light):

$$ \Delta\nu = \frac{2n}{\lambda} \sqrt{\frac{M}{\rho}} \sin\left(\frac{\theta}{2}\right) \quad (1) $$

Unfortunately, M cannot easily be related to the Young modulus E in every case⁸⁴. Equation (2) shows a simple relationship with K and G, the bulk

and shear moduli, respectively⁸⁵:

$$ M = K + \frac{4}{3} G \quad (2) $$

Although the precise absolute values of M, K, G, and E can be challenging to calculate, mainly because of the difficulty in obtaining reliable values for n and ρ, changes in stiffness can be detected and correlated with different cellular events⁸⁶. The confocal element of the system (the use of a pinhole to block out-of-focus light) is essential for intracellular experiments which require a sub-cellular resolution and enable superior 3D imaging⁸⁷, which is ideal for the complex environment of the cell interior.

Although Brillouin microscopy was initially used in materials science for the measurement of mechanical properties of materials ranging from rubber to ceramics⁸⁸,⁸⁹, this technique has recently been successfully applied to a wide range of biological systems such as the corneal lens⁹⁰, plaque stiffness in atherosclerosis⁹¹, bacterial meningitis⁹², the cytoskeleton⁹³, spider's silk⁹⁴, dentin⁹⁵, and generally for cells and tissues⁹⁶.

This promising method is label-free, non-destructive, and non-invasive. The sub-micron resolution allows studying subcellular structures, such as the nuclear envelope, nucleoli, and the cytoplasm⁸⁴,⁹⁷. However, it has some limitations. In biological samples, the refractive index as well as the density can be non-uniform across the zone of interest, but this has only a weak effect on the longitudinal modulus M⁹³,⁹⁸. Brillouin microscopy requires a high-resolution spectrometer to detect the small shift in frequency, a precise alignment of optical components, and advanced computational tools for the analysis needed to interpret the data. For additional technical information, a detailed review can be found in ref.⁸⁵.

## Tension probes

Förster Resonant Energy Transfer (FRET) sensors have been developed to investigate intramolecular tension at mechanosensitive modules within the cell, such as cell-substrate adhesions, cell-cell adhesions, or the nucleus⁹⁹,¹⁰⁰. For instance, it has been widely used to investigate the properties of focal adhesions¹⁰¹,¹⁰². FRET is an imaging technique involving two fluorophores: one donor which is excited by the laser from the imaging system, and one acceptor which is excited by the donor via energy transfer and which is also responsible for the emission¹⁰³. The proximity of the two fluorophores is correlated with emission intensity, making it ideal for tension sensing in the cell⁹⁹,¹⁰⁴.

In the following, we focus on recent advances in the field of membrane-embedded tension probes. For instance, lipid vesicles containing FRET sensors and the Laurdan probe have been employed to measure membrane tension¹⁰⁵. A FRET system called MSS imbedded parallel to the plasma membrane has been used to measure shear stress acting on a cell¹⁰⁶. FRET sensors have also been applied to nesprin, a constituent of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, to study mechanosotransduction at the nucleus¹⁰⁷,¹⁰⁸.

A relatively recent technique, developed just over a decade ago¹⁰⁹, and shown to be cell compatible¹¹⁰, is called Flipper. Flipper probes are long organic molecules containing cyclic groups that are inserted into the membranes of living cells and organelles. They provide information regarding lipid packing, which depends on both lipid composition and membrane tension (Fig. 1e). There are currently several commercially available organelle-specific Flipper probes for the ER, lysosomes, and mitochondria¹¹¹,¹¹², and this technique will be the focus of the remainder of this section.

The technique used to image Flipper probes is Fluorescence Lifetime Imaging Microscopy (FLIM). This method measures the time a fluorescent molecule remains in its excited state (the so-called fluorescence lifetime, typically of the order of 0.1–100 ns) by first illuminating the sample with an excitation pulse, and then capturing the photon emitted by the excited molecule when it returns to its ground state¹¹³. Flipper probes have broad absorption and emission spectra and are commonly excited at 488 nm, while emission is collected at around 600 nm. The typical values of Flipper
probe's lifetimes are 2–7 ns. Flipper molecules bear a donor fluorophore and an acceptor fluorophore at the two ends of the molecule, which twist as the result of changes in lipid packing, the increased fluidity of the membrane allowing for more flexibility¹¹⁴. The change of relative angle between the two fluorophores has an impact on the fluorescence lifetime and can be measured using FLIM. For the organic synthesis, as well as the detailed properties of different Flipper probes, see ref.¹¹¹.

In the case of Flippers, membrane tension consistently increases the lifetime of the probe¹¹⁵. These probes thus offer a way of collecting real-time, quantitative data in vivo in a non-invasive manner. The application to cells is usually easy, requiring only incubation of the probe at micromolar concentrations on the timescale of hours¹¹⁵. Chemical alterations to the probes using the HaloTag technology¹¹⁶, also allow for organelle-level specificity, although establishing a stable cell line expressing a Halo-tagged protein localized at the organelle of interest is required to avoid variations in expression levels typical of transient transfections. There is another major drawback of Flipper probes, beyond the obvious expertise demanded in the hardware and experimental FLIM set-up. The main issue with living systems is that their membranes have a highly dynamic composition¹¹⁷⁻¹¹⁹, and changes in lipid composition also impact the lifetime of the probes. One solution is to take advantage of the different time scales. As mentioned above, Flipper probes respond to changes in membrane tension in real-time, whereas changes in lipid composition usually occur on time scales of several minutes¹²⁰,¹²¹. The interpretation of results from these probes therefore requires caution when dealing with longer timescales.

## Magnetic particles

Magnetic particles are widely used in biology, often as detectors¹²²,¹²³ or even to isolate cells¹²⁴. Magnetic particles internalized inside cells can be controlled using an external magnetic field. The attractive or repulsive force can be varied by modifying the direction or magnitude of the magnetic field. Like OT, intracellular particles can be used as force generators¹²⁵, or as force sensors¹²⁶ and small micrometre-sized probes can report the rheological properties of gels and liquids in volumes of the order of 1 picolitre. In the latter study, reported effective viscosities ranged from the orders of 0.1 to 1 Pa.s depending on cell type. Magnetic particles can spontaneously internalize in cells¹²,¹²⁷, be endocytosed to form magnetic endosomes¹²⁸,¹²⁹, or be injected into the cytoplasm of living cells, as in *D. melanogaster* embryos¹³⁰ or into the nucleus¹³¹. Here we look at the role of magnetic particles as mechano-sensors rather than force generators.

The use of anisotropic paramagnetic probes interacting with an external magnetic field is referred to as Magnetic Rotational Spectroscopy (MRS)¹³²,¹³³. This technique has been used for the micro-rheology of complex fluids¹³⁴ and is increasingly applied inside cells. Micrometre size magnetic wires internalized inside living cells were used to measure the rheological properties of the cytoplasm in real time¹²,¹²⁷ (Fig. 1f). The wires appear to cross the plasma membrane without compromising cell viability and end up dispersed in the cytoplasm¹²⁶,¹²⁷. Rotating the wires by an external magnetic field at increasing speed induces a transition from a steady to a hindered motion which typifies viscoelastic materials¹³⁵,¹³⁶. As for OT, magnetic particles allow for the study of not only the mechanics of the cytoplasm but also of intracellular organelles. One recent example is given by ref.¹³¹, in which the mechanics of chromatin were probed using ferritin magnetic nanoparticles.

Magnetic particles are chemically inert, with no impact on cell viability, and are often internalized spontaneously. In the case of tissues, smaller ferrofluids can also be injected directly into living organisms¹³⁰, or be endocytosed by cells¹²⁸,¹²⁹. The external magnetic field does not interact with endogenous cell components, and the force vector of the field can easily be modulated to suit different experimental needs. In general, the spatial precision is lower than that of techniques such as optogenetics or OT, because the particles can only go towards or away from the magnet, or be made to spin. However, the development of magnetic nanoparticles of size of a few

tens of nanometres opens the possibility to apply force at smaller scales, in particular, to study chromatin mechanics¹³¹.

## Conclusions and outlook

In this final section, we briefly compare and contrast the different techniques described above. Compared to more conventional methods, such as cell stretching, AFM, osmotic shock, cell stretchers, and microfluidic devices, these newer intracellular techniques are much more precise in their ability to apply local forces and measure rheological properties. But, amongst themselves, there are important differences. Each technique requires specific expertise to be properly implemented, from molecular biology needed to obtain stable optogenetic cell lines, to chemistry and synthesis to build nanorobots, to optical components and data analysis for Brillouin microscopy. Furthermore, each of these techniques is minimally invasive, generally allowing for normal cell growth; and bio-orthogonal, i.e. not interfering with other physiological processes. We focus below on one key factor: the spatial control of force application and force measurement.

Optical techniques such as optogenetics and Flipper probes allow for organelle-level precision because each involves a functional group that is specific to the membrane of a particular organelle. However, this high degree of specificity and potentially high throughput does lack flexibility. For instance, to perform the same experiments on a different organelle, an entirely new construct is required. This is not the case for particle-based techniques, such as magnetic particles, bacteria/nanorobots, or for OT. After internalization into the cell, the particles will be found at different locations within the cytoplasm. The downside of this approach is that in order to study a particular organelle, a large number of cells must be scanned in order to use only those in which the probe is in the vicinity of the sub-cellular component of interest. These techniques are intrinsically low throughput since even a powerful magnet or laser can only typically move a single probe a few micrometres within the dense and crowded cytoplasm. OT offers the widest range of movement in 3D, whereas magnetic particles can either be moved towards or away from a given starting point. In a class of its own is Brillouin microscopy, which combines both of these strong points, sub-organelle level spatial precision, and the possibility to simultaneously study different types of organelles with a high throughput. Furthermore, magnetic particles and OT can have a dual use as both force generators and rheological sensors. This provides a tremendous practical advantage when choosing a technique to probe intracellular mechanics. In contrast, optogenetics, bacteria, and nanorobots are only force generators, while Flipper probes and Brillouin microscopy are only rheological sensors.

In conclusion, the six techniques described here, used either individually or in combination¹³⁷, should significantly improve our current understanding of intracellular mechanics in the coming years.

## Data availability

No datasets were generated or analysed during the current study.

Received: 10 November 2024; Accepted: 20 February 2025;

Published online: 04 April 2025

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**Acknowledgements**

This work was supported by the French Agence Nationale de la
Recherche (ANR), grant number ANR-22-CE13-0044-01 ‘Mechangolgi’
project.

**Author contributions**

K.M. wrote the first draft of the text and designed the figure. J-B.M. corrected and finalized the review.

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Correspondence** and requests for materials should be addressed to
Jean-Baptiste Manneville.

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