Andrew Ekpenyong

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Viscoelastic Properties of Differentiating Blood Cells Are Fate- and Function-Dependent

Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (<seconds). Intriguingly, only the two circulating myeloid cell types have increased short time scale compliance and flow better through microfluidic constrictions. Moreover, all three differentiated cell types reduce their steady-state viscosity by more than 50% and show over 140% relative increase in their ability to migrate through tissue-like pores at long time-scales (>minutes), compared to undifferentiated cells. These findings suggest that reduction in steady-state viscosity is a physiological adaptation for enhanced migration through tissues. Our results indicate that the material properties of cells define their function, can be used as a cell differentiation marker and could serve as target for novel therapies.

Chromatin decondensation and nuclear softening accompany Nanog downregulation in embryonic stem cells

The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself-even prior to differentiation-is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.

Quantifying cellular differentiation by physical phenotype using digital holographic microscopy

Although the biochemical changes that occur during cell differentiation are well-known, less known is that there are significant, cell-wide physical changes that also occur. Understanding and quantifying these changes can help to better understand the process of differentiation as well as ways to monitor it. Digital holographic microscopy (DHM) is a marker-free quantitative phase microscopy technique for measuring biological processes such as cellular differentiation, alleviating the need for introduction of foreign markers. We found significant changes in subcellular structure and refractive index of differentiating myeloid precursor cells within one day of differentiation induction, and significant differences depending on the type of lineage commitment. We augmented our results by showing significant changes in the softness of myeloid precursor cell differentiation within one day using optical stretching, a laser trap-based marker-free technique. DHM and optical stretching therefore provide consequential parameterization of cellular differentiation with sensitivity otherwise difficult to achieve. Therefore, we provide a way forward to quantify and understand cell differentiation with minimal perturbation using biophotonics.

Separation of blood cells with differing deformability using deterministic lateral displacement(

Determining cell mechanical properties is increasingly recognized as a marker-free way to characterize and separate biological cells. This emerging realization has led to the development of a plethora of appropriate measurement techniques. Here, we use a fairly novel approach, deterministic lateral displacement (DLD), to separate blood cells based on their mechanical phenotype with high throughput. Human red blood cells were treated chemically to alter their membrane deformability and the effect of this alteration on the hydrodynamic behaviour of the cells in a DLD device was investigated. Cells of defined stiffness (glutaraldehyde cross-linked erythrocytes) were used to test the performance of the DLD device across a range of cell stiffness and applied shear rates. Optical stretching was used as an independent method for quantifying the variation in stiffness of the cells. Lateral displacement of cells flowing within the device, and their subsequent exit position from the device were shown to correlate with cell stiffness. Data showing how the isolation of leucocytes from whole blood varies with applied shear rate are also presented. The ability to sort leucocyte sub-populations (T-lymphocytes and neutrophils), based on a combination of cell size and deformability, demonstrates the potential for using DLD devices to perform continuous fractionation and/or enrichment of leucocyte sub-populations from whole blood.

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

Significance Infectious diseases are responsible for one-third of all mortality worldwide. Innate immunity is critical for mounting host defenses that eliminate pathogens. Salmonella is a global food-borne pathogen that infects and replicates within macrophages. How inflammasomes—multimeric protein complexes that provide innate immune protection—function to restrict bacterial burden in macrophages remains unknown. We show that actin polymerization is critical for NLRC4 inflammasome activation in response to Salmonella infection. NLRC4 activation in Salmonella-infected cells prevents further uptake of bacteria by inducing cellular stiffness and antimicrobial responses, which prevent bacterial dissemination in the host. These results demonstrate a critical link between innate immunity and the actin cytoskeleton in the cellular defense against Salmonella infection. Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Myosin II Activity Softens Cells in Suspension

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

Diffraction imaging of spheres and melanoma cells with a microscope objective

Diffraction imaging of polystyrene spheres and B16F10 mouse melanoma cells embedded in gel has been investigated with a microscope objective. The diffraction images acquired with the objective from a sphere have been shown to be comparable to the Mie theory based projection images of the scattered light if the objective is translated to defocused positions towards the sphere. Using a confocal imaging based method to reconstruct and analyze the 3D structure, we demonstrated that genetic modifications in these cells can induce morphological changes and the modified cells can be used as an experimental model for study of the correlation between 3D morphology features and diffraction image data.

Bacterial infection of macrophages induces decrease in refractive index

Infection of cells by pathogens leads to both biochemical and structural modifications of the host cell. To study the structural modifications in a label-free manner, we use digital holographic microscopy, DHM, to obtain the integral refractive index distribution of cells. Primary murine bone marrow derived macrophages (BMDM) infected with Salmonella enterica serovar Typhimurium, undergo highly significant reduction in refractive index, RI, compared to uninfected cells. Infected BMDM cells from genetically modified mice lacking an inflammatory protein that causes cell death, caspase 1, also exhibit similar decrease in RI. These data suggest that any reduction in RI of Salmonella-infected BMDMs is pathogen induced and independent of caspase 1-induced inflammation or cell death. This finding suggests DHM may be useful for general real time monitoring of host cell interactions with infectious pathogens.

Determination of cell elasticity through hybrid ray optics and continuum mechanics modeling of cell deformation in the optical stretcher

The optical stretcher is a dual-beam trap capable of stretching individual cells. Previous studies have used either ray- or wave-optical models to compute the optical pressure on the surface of a spherical cell. We have extended the ray-optics model to account for focusing by the spherical interface and the effects of multiple internal reflections. Simulation results for red-blood cells (RBCs) show that internal reflections can lead to significant perturbation of the deformation, leading to a systematic error in the determination of cellular elasticity. Calibration studies show excellent agreement between the predicted and measured escape force, and RBC stiffness measurements are consistent with literature values. Measurements of the elasticity of murine osteogenic cells reveal that these cells are approximately 5.4 times stiffer than RBCs.