The regulatory role of cell mechanics for migration of differentiating myeloid cells.

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytoskeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.

Characterizing single suspended cells by optorheology.

The measurement of the mechanical properties of individual cells has received much attention in recent years. In this paper we describe the application of optically induced forces with an optical stretcher to perform step-stress experiments on individual suspended fibroblasts. The conversion from creep-compliance to frequency-dependent complex shear modulus reveals characteristic viscoelastic signatures of the underlying cytoskeleton and its dynamic molecular properties. Both normal and cancerous fibroblasts display a single stress relaxation time in the observed time and frequency space that can be related to the transient binding of actin crosslinking proteins. In addition, shear modulus and steady-state viscosity of the shell-like actin cortex as the main module resisting small deformations are extracted. These values in combination with insight into the cells' architecture are used to explain their different deformability. This difference can then be exploited to distinguish normal from cancerous cells. The nature of the optical stretcher as an optical trap allows easy incorporation in a microfluidic system with automatic trapping and alignment of the cells, and thus a high measurement throughput. This carries the potential for using the microfluidic optical stretcher to investigate cellular processes involving the cytoskeleton and to diagnose diseases related to cytoskeletal alterations.

Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy.

For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.

High-throughput rheological measurements with an optical stretcher.

The cytoskeleton is a major determinant of the mechanical strength and morphology of most cells. The composition and assembly state of this intracellular polymer network evolve during the differentiation of cells, and the structure is involved in many cellular functions and is characteristically altered in many diseases, including cancer. Here we exploit the deformability of the cytoskeleton as a link between molecular structure and biological function, to distinguish between cells in different states by using a laser-based optical stretcher (OS) coupled with microfluidic handling of cells. An OS is a cell-sized, dual-beam laser trap designed to nondestructively test the deformability of single suspended cells. Combined with microfluidic delivery, many cells can be measured serially in a short amount of time. With this tool it could be shown that optical deformability is sensitive enough to monitor subtle changes during the progression of cells from normal to cancerous and even a metastatic state. Stem cells can also be distinguished from more differentiated cells. The surprisingly low number of cells required for this assay reflects the tight regulation of the cytoskeleton by the cell. This suggests the possibility of using optical deformability as an inherent cell marker for basic cell biological investigation, diagnosis of disease, and sorting of stem cells from heterogeneous populations, obviating the need for external markers or special preparation. Many additional biological assays can be easily adapted to utilize this innovative physical method. This chapter details the setup and use of the microfluidic OS, the analysis and interpretation of data, and the results of a typical experiment.

Reconfigurable microfluidic integration of a dual-beam laser trap with biomedical applications.

A dual-beam fiber laser trap, termed the optical stretcher when used to deform objects, has been combined with a capillary-based microfluidic system in order to serially trap and deform biological cells. The design allows for control over the size and position of the trap relative to the flow channel. Data is recorded using video phase contrast microscopy and is subsequently analyzed using a custom edge fitting routine. This setup has been regularly used with measuring rates of 50-100 cells/h. One such experiment is presented to compare the distribution of deformability found within a normal epithelial cell line to that of a cancerous one. In general, this microfluidic optical stretcher can be used for the characterization of cells by their viscoelastic signature. Possible applications include the cytological diagnosis of cancer and the gentle and marker-free sorting of stem cells from heterogeneous populations for therapeutic cell-based approaches in regenerative medicine.

Muller cells are living optical fibers in the vertebrate retina.

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.

Quantifying the contribution of actin networks to the elastic strength of fibroblasts.

The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., Käs, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of approximately 220 Pa for normal fibroblasts and approximately 185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response.

Optical rheology of biological cells.

A step stress deforming suspended cells causes a passive relaxation, due to a transiently cross-linked isotropic actin cortex underlying the cellular membrane. The fluid-to-solid transition occurs at a relaxation time coinciding with unbinding times of actin cross-linking proteins. Elastic contributions from slowly relaxing entangled filaments are negligible. The symmetric geometry of suspended cells ensures minimal statistical variability in their viscoelastic properties in contrast with adherent cells and thus is defining for different cell types. Mechanical stimuli on time scales of minutes trigger active structural responses.

Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence.

The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.

Deformability-based flow cytometry.

BACKGROUND: Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker. METHODS: The optical stretcher is a new laser tool capable of measuring cellular deformability. A unique feature of this deformation technique is its potential for high throughput, with the incorporation of a microfluidic delivery of cells. RESULTS: Rudimentary implementation of the microfluidic optical stretcher has been used to measure optical deformability of several normal and cancerous cell types. A drastic difference has been seen between the response of red blood cells and polymorphonuclear cells for a given optically induced stress. MCF-10, MCF-7, and modMCF-7 cells were also measured, showing that while cancer cells stretched significantly more (five times) than normal cells, optical deformability could even be used to distinguish metastatic cancer cells from nonmetastatic cancer cells. This trimodal distribution was apparent after measuring a mere 83 cells, which shows optical deformability to be a highly regulated cell marker. CONCLUSIONS: Preliminary work suggests a deformability-based cell sorter similar to current fluorescence-based flow cytometry without the need for specific labeling. This could be used for the diagnosis of all diseases, and the investigation of all cellular processes, that affect the cytoskeleton.