Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Transient nuclear deformation primes epigenetic state and promotes cell reprogramming.

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering.

Stress hormone signaling through ß-adrenergic receptors regulates macrophage mechanotype and function.

Critical functions of immune cells require them to rapidly change their shape and generate forces in response to cues from their surrounding environment. However, little is known about how soluble factors that may be present in the microenvironment modulate key aspects of cellular mechanobiology-such as immune cell deformability and force generation-to impact functions such as phagocytosis and migration. Here we show that signaling by soluble stress hormones through ß-adrenoceptors (ß-AR) reduces the deformability of macrophages; this is dependent on changes in the organization of the actin cytoskeleton and is associated with functional changes in phagocytosis and migration. Pharmacologic interventions reveal that the impact of ß-AR signaling on macrophage deformability is dependent on actin-related proteins 2/3, indicating that stress hormone signaling through ß-AR shifts actin organization to favor branched structures rather than linear unbranched actin filaments. These findings show that through remodeling of the actin cytoskeleton, ß-AR-mediated stress hormone signaling modulates macrophage mechanotype to impact functions that play a critical role in immune response.-Kim, T.-H., Ly, C., Christodoulides, A., Nowell, C. J., Gunning, P. W., Sloan, E. K., Rowat, A. C. Stress hormone signaling through ß-adrenergic receptors regulates macrophage mechanotype and function.

Activating p53 family member TAp63: A novel therapeutic strategy for targeting p53-altered tumors.

BACKGROUND: Over 96% of high-grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63, which shares many of the downstream targets and functions of p53, is rarely mutated in cancer. METHODS: A novel strategy is presented for circumventing alterations in p53 by inducing the tumor-suppressor isoform TAp63 (transactivation domain of tumor protein p63) through its direct downstream target, microRNA-130b (miR-130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer. RESULTS: Treatment with miR-130b resulted in: 1) decreased migration/invasion in HEYA8 cells (p53 wild-type) and disruption of multicellular spheroids in OVCAR8 cells (p53-mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B-cell lymphoma (Bcl)-inhibitor B-cell lymphoma 2-like protein 11 (BIM). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR-130b. In vivo, combined miR-130b plus CDDP exhibited greater therapeutic efficacy than miR-130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR-130b in 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP-treated mice, and 20% of DOPC-miR-130b plus CDDP-treated mice were living tumor free. Systemic injections of scL-miR-130b plus CDDP in a clinically tested, tumor-targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts. CONCLUSIONS: The miR-130b/TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad-spectrum therapeutic options for the majority of p53-altered cancers.

Microfluidic Mechanotyping of a Single Cell with Two Consecutive Constrictions of Different Sizes and an Electrical Detection System.

The mechanical properties of a cell, which include parameters such as elasticity, inner pressure, and tensile strength, are extremely important because changes in these properties are indicative of diseases ranging from diabetes to malignant transformation. Considering the heterogeneity within a population of cancer cells, a robust measurement system at the single cell level is required for research and in clinical purposes. In this study, a potential microfluidic device for high-throughput and practical mechanotyping were developed to investigate the deformability and sizes of cells through a single run. This mechanotyping device consisted of two different sizes of consecutive constrictions in a microchannel and measured the size of cells and related deformability during transit. Cell deformability was evaluated based on the transit and on the effects of cytoskeleton-affecting drugs, which were detected within 50 ms. The mechanotyping device was able to also measure a cell cycle without the use of fluorescent or protein tags.

Cancer cells become less deformable and more invasive with activation of ß-adrenergic signaling.

Invasion by cancer cells is a crucial step in metastasis. An oversimplified view in the literature is that cancer cells become more deformable as they become more invasive. ß-adrenergic receptor (ßAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known. Here, we show that activation of ß-adrenergic signaling by ßAR agonists reduces the deformability of highly metastatic human breast cancer cells, and that these stiffer cells are more invasive in vitro We find that ßAR activation also reduces the deformability of ovarian, prostate, melanoma and leukemia cells. Mechanistically, we show that ßAR-mediated cell stiffening depends on the actin cytoskeleton and myosin II activity. These changes in cell deformability can be prevented by pharmacological ß-blockade or genetic knockout of the ß2-adrenergic receptor. Our results identify a ß2-adrenergic-Ca2+-actin axis as a new regulator of cell deformability, and suggest that the relationship between cell mechanical properties and invasion might be dependent on context.

Quantitative Deformability Cytometry: Rapid, Calibrated Measurements of Cell Mechanical Properties.

Advances in methods that determine cell mechanical phenotype, or mechanotype, have demonstrated the utility of biophysical markers in clinical and research applications ranging from cancer diagnosis to stem cell enrichment. Here, we introduce quantitative deformability cytometry (q-DC), a method for rapid, calibrated, single-cell mechanotyping. We track changes in cell shape as cells deform into microfluidic constrictions, and we calibrate the mechanical stresses using gel beads. We observe that time-dependent strain follows power-law rheology, enabling single-cell measurements of apparent elastic modulus, Ea, and power-law exponent, ß. To validate our method, we mechanotype human promyelocytic leukemia (HL-60) cells and thereby confirm q-DC measurements of Ea = 0.53 ± 0.04 kPa. We also demonstrate that q-DC is sensitive to pharmacological perturbations of the cytoskeleton as well as differences in the mechanotype of human breast cancer cell lines (Ea = 2.1 ± 0.1 and 0.80 ± 0.19 kPa for MCF-7 and MDA-MB-231 cells). To establish an operational framework for q-DC, we investigate the effects of applied stress and cell/pore-size ratio on mechanotype measurements. We show that Ea increases with applied stress, which is consistent with stress stiffening behavior of cells. We also find that Ea increases for larger cell/pore-size ratios, even when the same applied stress is maintained; these results indicate strain stiffening and/or dependence of mechanotype on deformation depth. Taken together, the calibrated measurements enabled by q-DC should advance applications of cell mechanotype in basic research and clinical settings.

Wrinkling of milk skin is mediated by evaporation.

Wrinkling of thin films and membranes can occur due to various mechanisms such as growth and/or mismatch between the mechanical properties of the film and substrate. However, the physical origins of dynamic wrinkling in soft membranes are still not fully understood. Here we use milk skin as a tractable experimental system to investigate the physics of wrinkle formation in a thin, poroelastic film. Upon heating milk, a micron-thick hydrogel of denatured proteins and fat globules forms at the air-water interface. Over time, we observe an increase in the total length of wrinkles. By confocal imaging and profilometry, we determine that the composition and thickness of the milk skin appears to be homogeneous over the length scale of the wrinkles, excluding differences in milk skin composition as a major contributor to wrinkling. To explain the physical origins of wrinkle growth, we describe theory that considers the milk skin as a thin, poroelastic film where pressure is generated by the evaporative-driven flow of solvent across the film; this imparts in-plane stresses in the milk skin, which cause wrinkling. Viscous effects can explain the time-dependent growth of wrinkles. Our theoretical predictions of the effects of relative humidity on the total length of wrinkles over time are consistent with our experimental results. Our findings provide insight into the physics of the common phenomenon of milk skin wrinkling, and identify hydration gradients as another physical mechanism that can drive morphological instabilities in soft matter.

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness, Nuclear Volume, Contractility, and Cell Stiffness.

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 µm length, 3.7 µm height, and a decreasing width from 11.2 to 1.7 µm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 µm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 µm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.

Understanding diffusion theory and Fick's law through food and cooking.

Diffusion is critical to physiological processes ranging from gas exchange across alveoli to transport within individual cells. In the classroom, however, it can be challenging to convey the concept of diffusion on the microscopic scale. In this article, we present a series of three exercises that use food and cooking to illustrate diffusion theory and Fick's first law. These exercises are part of a 10-wk undergraduate course that uses food and cooking to teach fundamental concepts in physiology and biophysics to students, including nonscience majors. Consistent demonstration of practical applications in a classroom setting has the potential to fundamentally change how students view the role of science in their lives (15).

Nuclear envelope composition determines the ability of neutrophil-type cells to passage through micron-scale constrictions.

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

The cell as a material.

To elucidate the dynamic and functional role of a cell within the tissue it belongs to, it is essential to understand its material properties. The cell is a viscoelastic material with highly unusual properties. Measurements of the mechanical behavior of cells are beginning to probe the contribution of constituent components to cell mechanics. Reconstituted cytoskeletal protein networks have been shown to mimic many aspects of the mechanical properties of cells, providing new insight into the origin of cellular behavior. These networks are highly nonlinear, with an elastic modulus that depends sensitively on applied stress. Theories can account for some of the measured properties, but a complete model remains elusive.

Using Histologic Image Analysis to Understand Biophysical Regulations of Epithelial Cell Morphology.

Epithelial mechanics and mechanobiology have become 2 important research fields in life sciences and bioengineering. These fields investigate how physical factors induced by cell adhesion and collective behaviors can directly regulate biologic processes, such as organ development and disease progression. Cell mechanics and mechanobiology thus make exciting biophysics education topics to illustrate how fundamental physics principles play a role in regulating cell biology. However, the field currently lacks hands-on activities that engage students in learning science and outreach programs in these topics. One such area is the development of robust hands-on modules that allow students to observe features of cell shape and mechanics and connect them to fundamental physics principles. Here, we demonstrate a workflow that engages students in studying epithelial cell mechanics by using commercial histology slides of frog skin. We show that by using recently developed artificial intelligence-based image-segmentation tools, students can easily quantify different cell morphologic features in a high-throughput manner. Using our workflow, students can reproduce 2 essential findings in cell mechanics: the common gamma distribution of normalized cell aspect ratio in jammed epithelia and the constant ratio between the nuclear and cellular area. Importantly, because the only required instrument for this active learning module is a readily available light microscope and a computer, our module is relatively low cost, as well as portable. These features make the module scalable for students at various education levels and outreach programs. This highly accessible education module provides a fun and engaging way to introduce students to the world of epithelial tissue mechanics.

Scalable Processes for Culturing Meat Using Edible Scaffolds.

There is increasing consumer demand for alternative animal protein products that are delicious and sustainably produced to address concerns about the impacts of mass-produced meat on human and planetary health. Cultured meat has the potential to provide a source of nutritious dietary protein that both is palatable and has reduced environmental impact. However, strategies to support the production of cultured meats at the scale required for food consumption will be critical. In this review, we discuss the current challenges and opportunities of using edible scaffolds for scaling up the production of cultured meat. We provide an overview of different types of edible scaffolds, scaffold fabrication techniques, and common scaffold materials. Finally, we highlight potential advantages of using edible scaffolds to advance cultured meat production by accelerating cell growth and differentiation, providing structure to build complex 3D tissues, and enhancing the nutritional and sensory properties of cultured meat.

Histone H1.0 couples cellular mechanical behaviors to chromatin structure.

Tuning of genome structure and function is accomplished by chromatin-binding proteins, which determine the transcriptome and phenotype of the cell. Here we investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that histone H1.0, which compacts nucleosomes into higher-order chromatin fibers, controls genome organization and cellular stress response. We show that histone H1.0 has privileged expression in fibroblasts across tissue types and that its expression is necessary and sufficient to induce myofibroblast activation. Depletion of histone H1.0 prevents cytokine-induced fibroblast contraction, proliferation and migration via inhibition of a transcriptome comprising extracellular matrix, cytoskeletal and contractile genes, through a process that involves locus-specific H3K27 acetylation. Transient depletion of histone H1.0 in vivo prevents fibrosis in cardiac muscle. These findings identify an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling force generation, nuclear organization and gene transcription.

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Altered physical phenotypes of leukemia cells that survive chemotherapy treatment.

The recurrence of cancer following chemotherapy treatment is a major cause of death across solid and hematologic cancers. In B-cell acute lymphoblastic leukemia (B-ALL), relapse after initial chemotherapy treatment leads to poor patient outcomes. Here we test the hypothesis that chemotherapy-treated versus control B-ALL cells can be characterized based on cellular physical phenotypes. To quantify physical phenotypes of chemotherapy-treated leukemia cells, we use cells derived from B-ALL patients that are treated for 7 days with a standard multidrug chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VDL). We conduct physical phenotyping of VDL-treated versus control cells by tracking the sequential deformations of single cells as they flow through a series of micron-scale constrictions in a microfluidic device; we call this method Quantitative Cyclical Deformability Cytometry. Using automated image analysis, we extract time-dependent features of deforming cells including cell size and transit time (TT) with single-cell resolution. Our findings show that VDL-treated B-ALL cells have faster TTs and transit velocity than control cells, indicating that VDL-treated cells are more deformable. We then test how effectively physical phenotypes can predict the presence of VDL-treated cells in mixed populations of VDL-treated and control cells using machine learning approaches. We find that TT measurements across a series of sequential constrictions can enhance the classification accuracy of VDL-treated cells in mixed populations using a variety of classifiers. Our findings suggest the predictive power of cell physical phenotyping as a complementary prognostic tool to detect the presence of cells that survive chemotherapy treatment. Ultimately such complementary physical phenotyping approaches could guide treatment strategies and therapeutic interventions. Insight box Cancer cells that survive chemotherapy treatment are major contributors to patient relapse, but the ability to predict recurrence remains a challenge. Here we investigate the physical properties of leukemia cells that survive treatment with chemotherapy drugs by deforming individual cells through a series of micron-scale constrictions in a microfluidic channel. Our findings reveal that leukemia cells that survive chemotherapy treatment are more deformable than control cells. We further show that machine learning algorithms applied to physical phenotyping data can predict the presence of cells that survive chemotherapy treatment in a mixed population. Such an integrated approach using physical phenotyping and machine learning could be valuable to guide patient treatments.

Engineering multicomponent tissue by spontaneous adhesion of myogenic and adipogenic microtissues cultured with customized scaffolds.

The integration of intramuscular fat-or marbling-into cultured meat will be critical for meat texture, mouthfeel, flavor, and thus consumer appeal. However, culturing muscle tissue with marbling is challenging since myocytes and adipocytes have different media and scaffold requirements for optimal growth and differentiation. Here, we present an approach to engineer multicomponent tissue using myogenic and adipogenic microtissues. The key innovation in our approach is the engineering of myogenic and adipogenic microtissues using scaffolds with customized physical properties; we use these microtissues as building blocks that spontaneously adhere to produce multicomponent tissue, or marbled cultured meat. Myocytes are grown and differentiated on gelatin nanofiber scaffolds with aligned topology that mimic the aligned structure of skeletal muscle and promotes the formation of myotubes in both primary rabbit skeletal muscle and murine C2C12 cells. Pre-adipocytes are cultured and differentiated on edible gelatin microbead scaffolds, which are customized to have a physiologically-relevant stiffness, and promote lipid accumulation in both primary rabbit and murine 3T3-L1 pre-adipocytes. After harvesting and stacking the individual myogenic and adipogenic microtissues, we find that the resultant multicomponent tissues adhere into intact structures within 6-12 h in culture. The resultant multicomponent 3D tissue constructs show behavior of a solid material with a Young's modulus of âˆ¼ 2 ± 0.4 kPa and an ultimate tensile strength of âˆ¼ 23 ± 7 kPa without the use of additional crosslinkers. Using this approach, we generate marbled cultured meat with âˆ¼ mm to âˆ¼ cm thickness, which has a protein content of âˆ¼ 4 ± 2 g/100 g that is comparable to a conventionally produced Wagyu steak with a protein content of âˆ¼ 9 ± 4 g/100 g. We show the translatability of this layer-by-layer assembly approach for microtissues across primary rabbit cells, murine cell lines, as well as for gelatin and plant-based scaffolds, which demonstrates a strategy to generate edible marbled meats derived from different species and scaffold materials.

Reduction of Intracellular Tension and Cell Adhesion Promotes Open Chromatin Structure and Enhances Cell Reprogramming.

The role of transcription factors and biomolecules in cell type conversion has been widely studied. Yet, it remains unclear whether and how intracellular mechanotransduction through focal adhesions (FAs) and the cytoskeleton regulates the epigenetic state and cell reprogramming. Here, it is shown that cytoskeletal structures and the mechanical properties of cells are modulated during the early phase of induced neuronal (iN) reprogramming, with an increase in actin cytoskeleton assembly induced by Ascl1 transgene. The reduction of actin cytoskeletal tension or cell adhesion at the early phase of reprogramming suppresses the expression of mesenchymal genes, promotes a more open chromatin structure, and significantly enhances the efficiency of iN conversion. Specifically, reduction of intracellular tension or cell adhesion not only modulates global epigenetic marks, but also decreases DNA methylation and heterochromatin marks and increases euchromatin marks at the promoter of neuronal genes, thus enhancing the accessibility for gene activation. Finally, micro- and nano-topographic surfaces that reduce cell adhesions enhance iN reprogramming. These novel findings suggest that the actin cytoskeleton and FAs play an important role in epigenetic regulation for cell fate determination, which may lead to novel engineering approaches for cell reprogramming.

Tracking lineages of single cells in lines using a microfluidic device.

Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.