Fluid flow reconstruction around a free-swimming sperm in 3D.

We investigate the dynamics and hydrodynamics of a human spermatozoa swimming freely in 3D. We simultaneously track the sperm flagellum and the sperm head orientation in the laboratory frame of reference via high-speed high-resolution 4D (3D+t) microscopy, and extract the flagellar waveform relative to the body frame of reference, as seen from a frame of reference that translates and rotates with the sperm in 3D. Numerical fluid flow reconstructions of sperm motility are performed utilizing the experimental 3D waveforms, with excellent accordance between predicted and observed 3D sperm kinematics. The reconstruction accuracy is validated by directly comparing the three linear and three angular sperm velocities with experimental measurements. Our microhydrodynamic analysis reveals a novel fluid flow pattern, characterized by a pair of vortices that circulate in opposition to each other along the sperm cell. Finally, we show that the observed sperm counter-vortices are not unique to the experimental beat, and can be reproduced by idealised waveform models, thus suggesting a fundamental flow structure for free-swimming sperm propelled by a 3D beating flagellum.

Snail-inspired water-enhanced soft sliding suction for climbing robots.

Snails can stably slide across a surface with only a single high-payload sucker, offering an efficient adhesive locomotion mechanism for next-generation climbing robots. The critical factor for snails' sliding suction behaviour is mucus secretion, which reduces friction and enhances suction. Inspired by this, we proposed an artificial sliding suction mechanism. The sliding suction utilizes water as an artificial mucus, which is widely available and evaporates with no residue. The sliding suction allows a lightweight robot (96 g) to slide vertically and upside down, achieving high speeds (rotation of 53°/s and translation of 19 mm/s) and high payload (1 kg as tested and 5.03 kg in theory), and does not require energy during adhesion. Here, we show that the sliding suction is a low-cost, energy-efficient, high-payload and clean adhesive locomotion strategy, which has high potential for use in climbing robots, outdoor inspection robots and robotic transportation.

Bioinspired multiscale adaptive suction on complex dry surfaces enhanced by regulated water secretion.

Suction is a highly evolved biological adhesion strategy for soft-body organisms to achieve strong grasping on various objects. Biological suckers can adaptively attach to dry complex surfaces such as rocks and shells, which are extremely challenging for current artificial suction cups. Although the adaptive suction of biological suckers is believed to be the result of their soft body's mechanical deformation, some studies imply that in-sucker mucus secretion may be another critical factor in helping attach to complex surfaces, thanks to its high viscosity. Inspired by the combined action of biological suckers' soft bodies and mucus secretion, we propose a multiscale suction mechanism which successfully achieves strong adaptive suction on dry complex surfaces which are both highly curved and rough, such as a stone. The proposed multiscale suction mechanism is an organic combination of mechanical conformation and regulated water seal. Multilayer soft materials first generate a rough mechanical conformation to the substrate, reducing leaking apertures to micrometres (~10 µm). The remaining micron-sized apertures are then sealed by regulated water secretion from an artificial fluidic system based on the physical model, thereby the suction cup achieves long suction longevity on complex surfaces but minimal overflow. We discuss its physical principles and demonstrate its practical application as a robotic gripper on a wide range of complex dry surfaces. We believe the presented multiscale adaptive suction mechanism is a powerful unique adaptive suction strategy which may be instrumental in the development of versatile soft adhesion.

Feature-based 3D+t descriptors of hyperactivated human sperm beat patterns.

The flagellar movement of the mammalian sperm plays a crucial role in fertilization. In the female reproductive tract, human spermatozoa undergo a process called capacitation which promotes changes in their motility. Only capacitated spermatozoa may be hyperactivated and only those that transition to hyperactivated motility are capable of fertilizing the egg. Hyperactivated motility is characterized by asymmetric flagellar bends of greater amplitude and lower frequency. Historically, clinical fertilization studies have used two-dimensional analysis to classify sperm motility, despite the inherently three-dimensional (3D) nature of sperm motion. Recent research has described several 3D beating features of sperm flagella. However, the 3D motility pattern of hyperactivated spermatozoa has not yet been characterized. One of the main challenges in classifying these patterns in 3D is the lack of a ground-truth reference, as it can be difficult to visually assess differences in flagellar beat patterns. Additionally, it is worth noting that only a relatively small proportion, approximately 10-20% of sperm incubated under capacitating conditions exhibit hyperactivated motility. In this work, we used a multifocal image acquisition system that can acquire, segment, and track sperm flagella in 3D+t. We developed a feature-based vector that describes the spatio-temporal flagellar sperm motility patterns by an envelope of ellipses. The classification results obtained using our 3D feature-based descriptors can serve as potential label for future work involving deep neural networks. By using the classification results as labels, it will be possible to train a deep neural network to automatically classify spermatozoa based on their 3D flagellar beating patterns. We demonstrated the effectiveness of the descriptors by applying them to a dataset of human sperm cells and showing that they can accurately differentiate between non-hyperactivated and hyperactivated 3D motility patterns of the sperm cells. This work contributes to the understanding of 3D flagellar hyperactive motility patterns and provides a framework for research in the fields of human and animal fertility.

Human sperm rotate with a conserved direction during free swimming in four dimensions.

Head rotation in human spermatozoa is essential for different swimming modes and fertilisation, as it links the molecular workings of the flagellar beat with sperm motion in three-dimensional (3D) space over time. Determining the direction of head rotation has been hindered by the symmetry and translucent nature of the sperm head, and by the fast 3D motion driven by the helical flagellar beat. Analysis has been mostly restricted to two-dimensional (2D) single focal plane image analysis, which enables tracking of head centre position but not tracking of head rotation. Despite the conserved helical beating of the human sperm flagellum, human sperm head rotation has been reported to be uni- or bi-directional, and even to intermittently change direction in a given cell. Here, we directly measure the head rotation of freely swimming human sperm using multi-plane 4D (3D+t) microscopy and show that: (1) 2D microscopy is unable to distinguish head rotation direction in human spermatozoa; (2) head rotation direction in non-capacitating and capacitating solutions, for both aqueous and viscous media, is counterclockwise (CCW), as seen from head to tail, in all rotating spermatozoa, regardless of the experimental conditions; and (3) head rotation is suppressed in 36% of spermatozoa swimming in non-capacitating viscous medium, although CCW rotation is recovered after incubation in capacitating conditions within the same viscous medium, possibly unveiling an unexplored aspect of the essential need of capacitation for fertilisation. Our observations show that the CCW head rotation in human sperm is conserved. It constitutes a robust and persistent helical driving mechanism that influences sperm navigation in 3D space over time, and thus is of critical importance in cell motility, propulsion of flagellated microorganisms, sperm motility assessments, human reproduction research, and self-organisation of flagellar beating patterns and swimming in 3D space.

The reaction-diffusion basis of animated patterns in eukaryotic flagella.

The flagellar beat of bull spermatozoa and C. Reinhardtii are modelled by a minimal, geometrically exact, reaction-diffusion system. Spatio-temporal animated patterns describe flagellar waves, analogous to chemical-patterns from classical reaction-diffusion systems, with sliding-controlled molecular motor reaction-kinetics. The reaction-diffusion system is derived from first principles as a consequence of the high-internal dissipation by the flagellum relative to the external hydrodynamic dissipation. Quantitative comparison with nonlinear, large-amplitude simulations shows that animated reaction-diffusion patterns account for the experimental beating of both bull sperm and C. Reinhardtii. Our results suggest that a unified mechanism may exist for motors controlled by sliding, without requiring curvature-sensing, and uninfluenced by hydrodynamics. High-internal dissipation instigates autonomous travelling waves independently of the external fluid, enabling progressive swimming, otherwise not possible, in low viscosity environments, potentially critical for external fertilizers and aquatic microorganisms. The reaction-diffusion system may prove a powerful tool for studying pattern formation of movement on animated structures.

Primary entry trocar design and entry-related complications at laparoscopy in obese patients: meta-analysis.

BACKGROUND: Safe primary entry at laparoscopy could present challenges in obese patients. Various techniques have been proposed in previous studies, however, the characteristics of the actual device utilized may be more influential than the technique in achieving successful abdominal entry in patients with increased BMI. METHODS: This systematic review and meta-analysis included both randomized and non-randomized studies gathered with no date filters from MEDLINE, Embase, Scopus, Web of Science and Clinicaltrials.gov. PRISMA guidelines underpinned the conduct and reporting of the review. The meta-analysis of proportions was conducted using a generalized linear mixed model and analyses included random-effects models. The primary outcome was the proportion of first access vascular and visceral injuries incurred in the process of laparoscopic abdominal surgery in patients with a BMI >30 kg/m2. Subgroup analysis was performed for optical versus non-optically enabled devices. RESULTS: In total, 5403 patients were analysed across 13 observational studies with a mean BMI of 45.93 kg/m2. In 216 patients from two randomized studies, the mean BMI was 39.92 kg/m2. The overall incidence using a random-effects model was 8.1 per 1000 events of visceral and vascular injuries (95 per cent c.i. 0.003 to 0.024). Heterogeneity was statistically significant at I2 = 80.5 per cent (69.6 per cent; 87.5 per cent, P< 0.0001). In a subgroup analysis, a tendency towards reduced injuries when optical devices were employed was observed with one per 100 injuries in these trocars (95 per cent c.i. 0.001 to 0.018) versus four per 100 (95 per cent c.i. -0.019 to -0.102) in non-optically enabled devices. CONCLUSION: Injuries during primary laparoscopic entry undertaken in obese patient groups are uncommon. Due to considerable heterogeneity in the small number of examined studies, evidence was insufficient and largely of low quality to ascribe differences in the incidence of injuries to the characteristics of the primary entry trocar utilized.

The three-dimensional coarse-graining formulation of interacting elastohydrodynamic filaments and multi-body microhydrodynamics.

Elastic filaments are vital to biological, physical and engineering systems, from cilia driving fluid in the lungs to artificial swimmers and micro-robotics. Simulating slender structures requires intricate balance of elastic, body, active and hydrodynamic moments, all in three dimensions. Here, we present a generalized three-dimensional (3D) coarse-graining formulation that is efficient, simple-to-implement, readily extendable and usable for a wide array of applications. Our method allows for simulation of collections of 3D elastic filaments, capable of full flexural and torsional deformations, coupled non-locally via hydrodynamic interactions, and including multi-body microhydrodynamics of structures with arbitrary geometry. The method exploits the exponential mapping of quaternions for tracking 3D rotations of each interacting element in the system, allowing for computation times up to 150 times faster than a direct quaternion implementation. Spheres are used as a 'building block' of both filaments and solid microstructures for straightforward and intuitive construction of arbitrary three-dimensional geometries present in the environment. We highlight the strengths of the method in a series of non-trivial applications including bi-flagellated swimming, sperm-egg scattering and particle transport by cilia arrays. Applications to lab-on-a-chip devices, multi-filaments, mono-to-multi flagellated microorganisms, Brownian polymers, and micro-robotics are straightforward. A Matlab code is provided for further customization and generalizations.

3D+t feature-based descriptor for unsupervised flagellar human sperm beat classification.

Human spermatozoa must swim through the female reproductive tract, where they undergo a series of biochemical and biophysical reactions called capacitation, a necessary step to fertilize the egg. Capacitation promotes changes in the motility pattern. Historically, a two-dimensional analysis has been used to classify sperm motility and clinical fertilization studies. Nevertheless, in a natural environment sperm motility is three-dimensional (3D). Imaging flagella of freely swimming sperm is a difficult task due to their high beating frequency of up to 25 Hz. Very recent studies have described several sperm flagellum 3D beating features (curvature, torsion, asymmetries, etc.). However, up to date, the 3D motility pattern of hyperactivated spermatozoa has not been characterized. The main difficulty in classifying these patterns in 3D is the lack of a ground truth reference since differences in flagellar beat patterns are very difficult to assess visually. Moreover, only around 10-20% of induced to capacitate spermatozoa are truly capacitated, i.e., hyperactivated. We used an image acquisition system that can acquire, segment, and track spermatozoa flagella in 3D+t. In this work, we propose an original three-dimensional feature vector formed by ellipses describing the envelope of the 3D+t spatio-temporal flagellar sperm motility patterns. These features allowed compressing an unlabeled 3D+t dataset to separate hyperactivated cells from others (capacitated from non-capacitated cells) using unsupervised hierarchical clustering. Preliminary results show three main clusters of flagellar motility patterns. The first principal component of these 3D flagella measurements correlated with 2D OpenCASA head determinations as a first approach to validate the unsupervised classification, showing a reasonable correlation coefficient near to 0.7. Clinical relevance- The novelty of this work is defining a 3D+t feature-based descriptor consisting of a set of ellipses enveloping the flagellar motion of human sperm for its unsu-pervised classification. This is a new promising tool to determine the viability of human sperm to fertilize the egg.

Does the Rainbow Trout Ovarian Fluid Promote the Spermatozoon on Its Way to the Egg?

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)-sperm interaction reflect the specific features of the spawning process in this species.

A dynamic basal complex modulates mammalian sperm movement.

Reproductive success depends on efficient sperm movement driven by axonemal dynein-mediated microtubule sliding. Models predict sliding at the base of the tail - the centriole - but such sliding has never been observed. Centrioles are ancient organelles with a conserved architecture; their rigidity is thought to restrict microtubule sliding. Here, we show that, in mammalian sperm, the atypical distal centriole (DC) and its surrounding atypical pericentriolar matrix form a dynamic basal complex (DBC) that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking. During asymmetric tail beating, the DC's right side and its surroundings slide ~300 nm rostrally relative to the left side. The deformation throughout the DBC is transmitted to the head-tail junction; thus, the head tilts to the left, generating a kinking motion. These findings suggest that the DBC evolved as a dynamic linker coupling sperm head and tail into a single self-coordinated system.

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

Long-term segmentation-free assessment of head-flagellum movement and intracellular calcium in swimming human sperm.

Human spermatozoa are the archetype of long-term self-organizing transport in nature and are critical for reproductive success. They utilize coordinated head and flagellar movements to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to date, long-term analysis of the sperm head-flagellar movements, or indeed those of other flagellated microorganisms, remains elusive due to limitations in microscopy and flagellar-tracking techniques. Here, we present a novel methodology based on local orientation and isotropy of bio-images to obtain long-term kinematic and physiological parameters of individual free-swimming spermatozoa without requiring image segmentation (thresholding). This computer-assisted segmentation-free method evaluates, for the first time, characteristics of the head movement and flagellar beating for up to 9.2 min. We demonstrate its powerful use by showing how releasing Ca2+ from internal stores significantly alters long-term sperm behavior. The method allows for straightforward generalization to other bio-imaging applications, such as studies of bull sperm and Trypanosoma, or indeed of other flagellated microorganisms - appealing to communities other than those investigating sperm biology.

Egg-sperm interaction in sturgeon: role of ovarian fluid.

Fertilization of freshwater fish occurs in the environment which negatively affects a lifespan of gametes mostly due to the osmotic shock; therefore, male gametes should reach the female gamete, as soon as possible. The existence of mechanisms controlling the encounter of gametes would be highly expedient in this case. By analogy with other species for which guidance was demonstrated, it is likely that this control may be performed by ovarian fluid or substances released by eggs. The aim was to study the effect of ovarian fluid and egg-released substances on spermatozoa behavior in sterlet. It was found that the presence of a particular concentration of ovarian fluid (30% solution in water) had an inhibiting effect on spermatozoa motility initiation. Lower concentrations of the ovarian fluid improved the longevity of spermatozoa and did not affect their trajectories. Test of chemotactic response (using a microcapillary injection of fluids into the suspension of motile spermatozoa) showed no effect of ovarian fluid on spermatozoa behavior, while at the same time, the attracting effect of the egg-conditioned medium was evident (i.e., due to some substances released from the eggs during their contact with freshwater). The results of the fertilization test showed that the presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during a longer period of time. Thus, the combined physicochemical action of "female factors" affects sterlet gametes during fertilization and may be involved in the guidance and selection mechanisms.

Expanding the Limits of Computer-Assisted Sperm Analysis through the Development of Open Software.

Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is the Chemotactic Sperm Accumulation Module, a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allows different aspects of sperm quality to be evaluated, with many potential applications for researchers.

Human sperm uses asymmetric and anisotropic flagellar controls to regulate swimming symmetry and cell steering.

Flagellar beating drives sperm through the female reproductive tract and is vital for reproduction. Flagellar waves are generated by thousands of asymmetric molecular components; yet, paradoxically, forward swimming arises via symmetric side-to-side flagellar movement. This led to the preponderance of symmetric flagellar control hypotheses. However, molecular asymmetries must still dictate the flagellum and be manifested in the beat. Here, we reconcile molecular and microscopic observations, reconnecting structure to function, by showing that human sperm uses asymmetric and anisotropic controls to swim. High-speed three-dimensional (3D) microscopy revealed two coactive transversal controls: An asymmetric traveling wave creates a one-sided stroke, and a pulsating standing wave rotates the sperm to move equally on all sides. Symmetry is thus achieved through asymmetry, creating the optical illusion of bilateral symmetry in 2D microscopy. This shows that the sperm flagellum is asymmetrically controlled and anisotropically regularized by fast-signal transduction. This enables the sperm to swim forward.

Physical Biomarkers of Disease Progression: On-Chip Monitoring of Changes in Mechanobiology of Colorectal Cancer Cells.

Disease can induce changes to subcellular components, altering cell phenotype and leading to measurable bulk-material mechanical properties. The mechanical phenotyping of single cells therefore offers many potential diagnostic applications. Cells are viscoelastic and their response to an applied stress is highly dependent on the magnitude and timescale of the actuation. Microfluidics can be used to measure cell deformability over a wide range of flow conditions, operating two distinct flow regimes (shear and inertial) which can expose subtle mechanical properties arising from subcellular components. Here, we investigate the deformability of three colorectal cancer (CRC) cell lines using a range of flow conditions. These cell lines offer a model for CRC metastatic progression; SW480 derived from primary adenocarcinoma, HT29 from a more advanced primary tumor and SW620 from lymph-node metastasis. HL60 (leukemia cells) were also studied as a model circulatory cell, offering a non-epithelial comparison. We demonstrate that microfluidic induced flow deformation can be used to robustly detect mechanical changes associated with CRC progression. We also show that single-cell multivariate analysis, utilising deformation and relaxation dynamics, offers potential to distinguish these different cell types. These results point to the benefit of multiparameter determination for improving detection and accuracy of disease stage diagnosis.

Multiparameter antibiotic resistance detection based on hydrodynamic trapping of individual E. coli.

There is an urgent need to develop novel methods for assessing the response of bacteria to antibiotics in a timely manner. Antibiotics are traditionally assessed via their effect on bacteria in a culture medium, which takes 24-48 h and exploits only a single parameter, i.e. growth. Here, we present a multiparameter approach at the single-cell level that takes approximately an hour from spiking the culture to correctly classify susceptible and resistant strains. By hydrodynamically trapping hundreds of bacteria, we simultaneously monitor the evolution of motility and morphology of individual bacteria upon drug administration. We show how this combined detection method provides insights into the activity of antimicrobials at the onset of their action which single parameter and traditional tests cannot offer. Our observations complement the current growth-based methods and highlight the need for future antimicrobial susceptibility tests to take multiple parameters into account.

Flagellar ultrastructure suppresses buckling instabilities and enables mammalian sperm navigation in high-viscosity media.

Eukaryotic flagellar swimming is driven by a slender motile unit, the axoneme, which possesses an internal structure that is essentially conserved in a tremendous diversity of sperm. Mammalian sperm, however, which are internal fertilizers, also exhibit distinctive accessory structures that further dress the axoneme and alter its mechanical response. This raises the following two fundamental questions. What is the functional significance of these structures? How do they affect the flagellar waveform and ultimately cell swimming? Hence we build on previous work to develop a mathematical mechanical model of a virtual human sperm to examine the impact of mammalian sperm accessory structures on flagellar dynamics and motility. Our findings demonstrate that the accessory structures reinforce the flagellum, preventing waveform compression and symmetry-breaking buckling instabilities when the viscosity of the surrounding medium is increased. This is in agreement with previous observations of internal and external fertilizers, such as human and sea urchin spermatozoa. In turn, possession of accessory structures entails that the progressive motion during a flagellar beat cycle can be enhanced as viscosity is increased within physiological bounds. Hence the flagella of internal fertilizers, complete with accessory structures, are predicted to be advantageous in viscous physiological media compared with watery media for the fundamental role of delivering a genetic payload to the egg.

Cells Under Stress: An Inertial-Shear Microfluidic Determination of Cell Behavior.

The deformability of a cell is the direct result of a complex interplay between the different constituent elements at the subcellular level, coupling a wide range of mechanical responses at different length scales. Changes to the structure of these components can also alter cell phenotype, which points to the critical importance of cell mechanoresponse for diagnostic applications. The response to mechanical stress depends strongly on the forces experienced by the cell. Here, we use cell deformability in both shear-dominant and inertia-dominant microfluidic flow regimes to probe different aspects of the cell structure. In the inertial regime, we follow cellular response from (visco-)elastic through plastic deformation to cell structural failure and show a significant drop in cell viability for shear stresses >11.8 kN/m2. Comparatively, a shear-dominant regime requires lower applied stresses to achieve higher cell strains. From this regime, deformation traces as a function of time contain a rich source of information including maximal strain, elastic modulus, and cell relaxation times and thus provide a number of markers for distinguishing cell types and potential disease progression. These results emphasize the benefit of multiple parameter determination for improving detection and will ultimately lead to improved accuracy for diagnosis. We present results for leukemia cells (HL60) as a model circulatory cell as well as for a colorectal cancer cell line, SW480, derived from primary adenocarcinoma (Dukes stage B). SW480 were also treated with the actin-disrupting drug latrunculin A to test the sensitivity of flow regimes to the cytoskeleton. We show that the shear regime is more sensitive to cytoskeletal changes and that large strains in the inertial regime cannot resolve changes to the actin cytoskeleton.