X-ray phase-contrast tomography of cells manipulated with an optical stretcher.

X-rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X-ray phase-contrast imaging and tomography is combined with an X-ray-compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X-ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations.

Recent advancements to measure membrane mechanical and transport properties.

The natural vesicles, microscopic spherical structures defined by a single or many lipid bilayer membranes, not only entrap but are also dispersed in the aqueous environment. The space division between inner and outer compartments is also the basic characteristics of cell membranes playing several essential functions in all living organisms. Thus, vesicles are a simple model system for studying various cellular properties. In the last few decades, synthetic vesicles (or liposomes) have gained substantial popularity from many academia as model membranes and from many pharmaceutical industries as targeted and controlled drug delivery systems. The manufacturing of vesicles with desired characteristics that can entrap and release the drugs as required is one of the major challenges in this research area. To this end, a better understanding of the mechanical and transport properties of vesicles is essential to gain deeper insight into the fundamental biological mechanisms of vesicle formation and cellular uptake. The requirement has brought the modifications in membrane composition (with cholesterol, charged lipid, proteins, peptides, polymers, etc.) and solution conditions (with salts, pH, buffers, etc.). This article mainly focuses on the different techniques developed for studying the mechanical and transport properties of natural/synthetic vesicles. In particular, I thoroughly review the properties such as bending and stretching elastic moduli, lysis tension, and permeability of vesicle membranes.

High-throughput linear optical stretcher for mechanical characterization of blood cells.

This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. © 2015 International Society for Advancement of Cytometry.

Keratins significantly contribute to cell stiffness and impact invasive behavior.

Cell motility and cell shape adaptations are crucial during wound healing, inflammation, and malignant progression. These processes require the remodeling of the keratin cytoskeleton to facilitate cell-cell and cell-matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells is only partially understood. In this study, we address this issue in murine keratinocytes lacking all keratins on genome engineering. In contrast to predictions, keratin-free cells show about 60% higher cell deformability even for small deformations. This response is compared with the less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and 3D growth assays. These experiments reveal higher invasiveness of keratin-free cells. Reexpression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data show a unique role of keratins as major players of cell stiffness, influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that down-regulation of keratins observed during epithelial-mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells.

Evaluation of single-cell biomechanics as potential marker for oral squamous cell carcinomas: a pilot study.

OBJECTIVES: Early detection of oral cancer is a major health issue. The objective of this pilot study was to analyze the deformability of healthy and cancer cells using a microfluidic optical stretcher (OS). MATERIAL AND METHODS: Different cancer cell lines, primary oral cancer cells, and their healthy counterparts were cultivated and characterized, respectively. A measurable deformation of the cells along the optical axis was detected, caused by surface stress, which is optically induced by the laser power. RESULTS: All cells revealed a viscoelastic extension behavior and showed a characteristic deformation response under laser light exposure. The CAL-27/-33 cells exhibited the highest relative deformation. All other cells achieved similar values, but on a lower level. The cytoskeleton reacts sensitively of changing environmental conditions, which may be influenced by growth behavior of the cancer specimens. Nevertheless, the statistical analysis showed significant differences between healthy and cancer cells. CONCLUSION: Generally, malignant and benign cells showed significantly different mechanical behavior. Cancer-related changes influence the composition of the cytoskeleton and thus affect the deformability, but this effect may be superimposed by cell cultivation conditions or cell doubling time. These influences had to be substituted by brush biopsies to minimize confounders in pursuing investigations.

The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients.

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells-possible CTCs-were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin-Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

A quantitative high-resolution computational mechanics cell model for growing and regenerating tissues.

Mathematical models are increasingly designed to guide experiments in biology, biotechnology, as well as to assist in medical decision making. They are in particular important to understand emergent collective cell behavior. For this purpose, the models, despite still abstractions of reality, need to be quantitative in all aspects relevant for the question of interest. This paper considers as showcase example the regeneration of liver after drug-induced depletion of hepatocytes, in which the surviving and dividing hepatocytes must squeeze in between the blood vessels of a network to refill the emerged lesions. Here, the cells' response to mechanical stress might significantly impact the regeneration process. We present a 3D high-resolution cell-based model integrating information from measurements in order to obtain a refined and quantitative understanding of the impact of cell-biomechanical effects on the closure of drug-induced lesions in liver. Our model represents each cell individually and is constructed by a discrete, physically scalable network of viscoelastic elements, capable of mimicking realistic cell deformation and supplying information at subcellular scales. The cells have the capability to migrate, grow, and divide, and the nature and parameters of their mechanical elements can be inferred from comparisons with optical stretcher experiments. Due to triangulation of the cell surface, interactions of cells with arbitrarily shaped (triangulated) structures such as blood vessels can be captured naturally. Comparing our simulations with those of so-called center-based models, in which cells have a largely rigid shape and forces are exerted between cell centers, we find that the migration forces a cell needs to exert on its environment to close a tissue lesion, is much smaller than predicted by center-based models. To stress generality of the approach, the liver simulations were complemented by monolayer and multicellular spheroid growth simulations. In summary, our model can give quantitative insight in many tissue organization processes, permits hypothesis testing in silico, and guide experiments in situations in which cell mechanics is considered important.

Influence of hyaluronic acid binding on the actin cortex measured by optical forces.

Melanoma cells are often surrounded by hyaluronic acid (HA) rich environments, which are considered to promote tumor progression and metastasis. Induced effects in compound materials consisting of cells embedded in an extracellular matrix have been studied, however, alterations of the single cells have never been addressed. Here, we explicitly addressed single cell properties and measured HA-induced biomechanical changes via deformations induced solely by optical forces. With the optical stretcher setup, cells were deformed after culturing them in either the presence or absence of HA revealing the crucial interplay of HA with the CD44 receptor. To assess the role of CD44 in transducing effects of HA, we compared a CD44 expressing variant of the melanoma cell line RPM-MC to its natural CD44-negative counterpart. Our measurements revealed a significant stiffness change, which we attribute to changes of the actin cytoskeleton.

Particle Manipulation by Optical Forces in Microfluidic Devices.

Since the pioneering work of Ashkin and coworkers, back in 1970, optical manipulation gained an increasing interest among the scientific community. Indeed, the advantages and the possibilities of this technique are unsubtle, allowing for the manipulation of small particles with a broad spectrum of dimensions (nanometers to micrometers size), with no physical contact and without affecting the sample viability. Thus, optical manipulation rapidly found a large set of applications in different fields, such as cell biology, biophysics, and genetics. Moreover, large benefits followed the combination of optical manipulation and microfluidic channels, adding to optical manipulation the advantages of microfluidics, such as a continuous sample replacement and therefore high throughput and automatic sample processing. In this work, we will discuss the state of the art of these optofluidic devices, where optical manipulation is used in combination with microfluidic devices. We will distinguish on the optical method implemented and three main categories will be presented and explored: (i) a single highly focused beam used to manipulate the sample, (ii) one or more diverging beams imping on the sample, or (iii) evanescent wave based manipulation.

Optomechanical measurement of the role of lamins in whole cell deformability.

There is mounting evidence that the nuclear envelope, and particularly the lamina, plays a critical role in the mechanical and regulation properties of the cell and changes to the lamina can have implications for the physical properties of the whole cell. In this study we demonstrate that the optical stretcher can measure changes in the time-dependent mechanical properties of living cells with different levels of A-type lamin expression. Results from the optical stretcher shows a decrease in the deformability of cells as the levels of lamin A increases, for cells which grow both adherently and in suspension. Further detail can be probed by combining the optical stretcher with fluorescence microscopy to investigate the nuclear mechanical properties which show a larger decrease in deformability than for the whole cell.

Mechanical deformation induces depolarization of neutrophils.

The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.

A Comprehensive Review of Optical Stretcher for Cell Mechanical Characterization at Single-Cell Level.

This paper presents a comprehensive review of the development of the optical stretcher, a powerful optofluidic device for single cell mechanical study by using optical force induced cell stretching. The different techniques and the different materials for the fabrication of the optical stretcher are first summarized. A short description of the optical-stretching mechanism is then given, highlighting the optical force calculation and the cell optical deformability characterization. Subsequently, the implementations of the optical stretcher in various cell-mechanics studies are shown on different types of cells. Afterwards, two new advancements on optical stretcher applications are also introduced: the active cell sorting based on cell mechanical characterization and the temperature effect on cell stretching measurement from laser-induced heating. Two examples of new functionalities developed with the optical stretcher are also included. Finally, the current major limitation and the future development possibilities are discussed.

Impact of heating on passive and active biomechanics of suspended cells.

A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.

Dissecting the contribution of actin and vimentin intermediate filaments to mechanical phenotype of suspended cells using high-throughput deformability measurements and computational modeling.

Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently observed overexpression of vimentin in diverse types of cancer and underline the role of vimentin targeting drugs, such as Withaferin-A, as a potent cancerostatic supplement.