Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.     

Using Optics to Measure Biological Forces and Mechanics

Spanning all size levels, regulating biological forces and transport are fundamental life processes. Used by various investigators over the last dozen years, optical techniques offer unique advantages for studying biological forces. The most mature of these techniques, optical tweezers, or the single-beam optical trap, is commercially available and is used by numerous investigators. Although technical innovations have improved the versatility of optical tweezers, simple optical tweezers continue to provide insights into cell biology. Two new, promising optical technologies, laser-tracking microrheology and the optical stretcher, allow mechanical measurements that are not possible with optical tweezers. Here, I review these various optical technologies and their roles in understanding mechanical forces in cell biology.     

Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos

Cells are not directly accessible in vivo and therefore their mechanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vivo assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of thermally excited nanoparticles embedded within the cytoplasm of developing embryos reflect local viscoelastic properties. As a proof of principle, we probe local viscoelastic properties of the cytoplasm of developing Caenorhabditis elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior-posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of the mitotic spindle stems from an asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.     

Structural and micromechanical characterization of type I collagen gels

In this paper we report a study where we use a novel optical tweezers technique to measure the local viscoelastic properties of type I collagen solutions spanning the sol-to-gel transition. We use phase contrast optical microscopy to reveal dense and sparse regions of the rigid fibril networks, and find that the spatial variations in the mechanical properties of the collagen gels closely follow the structural properties. Within the dense phase of the connected network in the gel samples, there are regions that exhibit drastically different viscoelastic properties. Within the sparse regions of the gel samples, no evidence of elasticity is found. In type I collagen gels, we find a high degree of structural inhomogeneity. The inhomogeneity in the structural properties of collagen gels and the corresponding viscoelastic properties provide benchmark measurements for the behavior of desirable biological materials, or tissue equivalents.     

Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells

A single-cell assay of active and passive intracellular mechanical properties of mammalian cells could give significant insight into cellular processes. Force spectrum microscopy (FSM) is one such technique, which combines the spontaneous motion of probe particles and the mechanical properties of the cytoskeleton measured by active microrheology using optical tweezers to determine the force spectrum of the cytoskeleton. A simpler and noninvasive method to perform FSM would be very useful, enabling its widespread adoption. Here, we develop an alternative method of FSM using measurement of the fluctuating motion of mitochondria. Mitochondria of the C3H-10T1/2 cell line were labeled and tracked using confocal microscopy. Mitochondrial probes were selected based on morphological characteristics, and their mean-square displacement, creep compliance, and distributions of directional change were measured. We found that the creep compliance of mitochondria resembles that of particles in viscoelastic media. However, comparisons of creep compliance between controls and cells treated with pharmacological agents showed that perturbations to the actomysoin network had surprisingly small effects on mitochondrial fluctuations, whereas microtubule disruption and ATP depletion led to a significantly decreased creep compliance. We used properties of the distribution of directional change to identify a regime of thermally dominated fluctuations in ATP-depleted cells, allowing us to estimate the viscoelastic parameters for a range of timescales. We then determined the force spectrum by combining these viscoelastic properties with measurements of spontaneous fluctuations tracked in control cells. Comparisons with previous measurements made using FSM revealed an excellent match.     

Effect of length,topology, and concentration on the microviscosity and microheterogeneity of DNA solutions

The viscoelastic behavior of chromosomal DNA, which is heterogeneously distributed within the nucleus, may influence the diffusion of nuclear organelles and proteins. To identify some of the parameters that affect DNA viscoelasticity, we use the high-throughput method of multiple-particle nanotracking to measure the microviscosity and degree of heterogeneity of solutions of chromosomal DNA, linear DNA, and circular double-stranded DNA over a wide range of concentrations and lengths. The thermally excited displacements of multiple fluorescent microspheres imbedded in DNA solutions are monitored with 5nm spatial resolution and 30Hz temporal resolution, from which mean-squared displacement (MSD) and viscosity distributions are generated. For all probed DNA solutions but the most concentrated solution of the longest molecules, the ensemble-averaged MSD increases linearly with time at all probed time scales, a signature of viscous transport. The associated mean viscosity of the DNA solutions increases slowly with concentration for circular DNA and more rapidly for linear DNA, but more slowly than predicted by theory. The heterogeneity of the DNA solutions is assessed by computing the relative contributions of the 10%, 25%, and 50% highest values of MSD and viscosity to the ensemble-averaged MSD and viscosity. For both linear DNA and circular DNA, these contributions are much larger than observed in homogeneous liquids such as glycerol. The microheterogeneity of the linear DNA solutions increases with concentration more significantly for linear DNA than circular DNA. These in vitro results suggest that the topology, local concentration, and length of DNA influence the microrheology and microheterogeneity of the DNA within the nucleus.     

Effect of cholesterol on viscoelastic properties of dipalmitoylphosphatidylcholine multibilayers as measured by a laser-induced ultrasonic probe

Using a novel laser-induced ultrasonic probe, we have examined the bulk viscoelastic properties of fully hydrated dipalmitoylphosphatidylcholine (DPPC) aligned multibilayers in terms of the anisotropic in-plane elastic stiffness (C11) and viscosity (eta 11). Our measurements of C11 are in accord with those reported on Brillouin light scattering on a similar system. Our measurements on viscosity are the first of their kind and are, on the average, a factor of 10 lower than microviscosities estimated by spectroscopic techniques. We report the first comprehensive study of the effects of cholesterol on the bulk mechanical properties of DPPC multibilayers. At temperatures above the phase transition temperature of DPPC (Tc), an increase in both C11 and eta 11 is noticed when cholesterol is incorporated in the multibilayers. However, at temperatures below Tc, no measurable changes are detected in either C11 or eta 11. These results, reflecting changes in the bulk viscoelastic properties of the multibilayers, differ from the changes reported by local fluidity parameters in that the latter indicate a decrease in the bilayer fluidity in the presence of cholesterol above Tc and an increase below Tc ("dual effect" of cholesterol). Our data suggest that the "dual effect" of cholesterol is noticeable only on a molecular scale. Increasing cholesterol concentrations higher than 20 mol % cease to further affect C11 or eta 11 of the DPPC multibilayers. This agrees with various results reported in the literature, by techniques measuring the local effects of cholesterol, and supports the changes in molecular organization postulated to occur when cholesterol concentration reaches 20 mol % in the lipid bilayers.     

Mechanical properties of high-G.C content DNA with a-type base-stacking

The sequence of a DNA molecule is known to influence its secondary structure and flexibility. Using a combination of bulk and single-molecule techniques, we measure the structural and mechanical properties of two DNAs which differ in both sequence and base-stacking arrangement in aqueous buffer, as revealed by circular dichroism: one with 50% G·C content and B-form and the other with 70% G·C content and A-form. Atomic force microscopy measurements reveal that the local A-form structure of the high-G·C DNA does not lead to a global contour-length decrease with respect to that of the molecule in B-form although it affects its persistence length. In the presence of force, however, the stiffness of high-G·C content DNA is similar to that of balanced-G·C DNA as magnetic and optical tweezers measured typical values for the persistence length of both DNA substrates. This indicates that sequence-induced local distortions from the B-form are compromised under tension. Finally, high-G·C DNA is significantly harder to stretch than 50%-G·C DNA as manifested by a larger stretch modulus. Our results show that a local, basepair configuration of DNA induced by high-G·C content influences the stretching elasticity of the polymer but that it does not affect the global, double-helix arrangement.     

Mapping of Bacterial Biofilm Local Mechanics by Magnetic Microparticle Actuation

Most bacteria live in the form of adherent communities forming three-dimensional material anchored to artificial or biological surfaces, with profound impact on many human activities. Biofilms are recognized as complex systems but their physical properties have been mainly studied from a macroscopic perspective. To determine biofilm local mechanical properties, reveal their potential heterogeneity, and investigate their relation to molecular traits, we have developed a seemingly new microrheology approach based on magnetic particle infiltration in growing biofilms. Using magnetic tweezers, we achieved what was, to our knowledge, the first three-dimensional mapping of the viscoelastic parameters on biofilms formed by the bacterium Escherichia coli. We demonstrate that its mechanical profile may exhibit elastic compliance values spread over three orders of magnitude in a given biofilm. We also prove that heterogeneity strongly depends on external conditions such as growth shear stress. Using strains genetically engineered to produce well-characterized cell surface adhesins, we show that the mechanical profile of biofilm is exquisitely sensitive to the expression of different surface appendages such as F pilus or curli. These results provide a quantitative view of local mechanical properties within intact biofilms and open up an additional avenue for elucidating the emergence and fate of the different microenvironments within these living materials.     

Micro- and macrorheology of jellyfish extracellular matrix

Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish.     

High frequency viscoelastic behaviour of low molecular weight hyaluronic acid water solutions

Hyaluronic acid (HA) is a polysaccharide widely used in biomedical applications, due to its elevated biocompatibility and the peculiar viscoelastic properties of its solutions. Although the viscoelastic behaviour of HA solutions has been extensively studied in the literature it has been often reported in the range of low frequency (1-100 Hz) and high salt concentration, whereas the main rheological peculiarities of this molecule are expected at high frequency (>100 Hz) and low salt concentration. In this work we studied the viscoelastic properties of low molecular weight HA (155 kDa) in wide range of concentrations (0.01-20 mg/ml) at low ionic strength and over an extended frequency range (0.1-1000 Hz) using both optical tweezers and conventional rheometry. Good agreement between the high frequency dynamic behaviour (optical tweezers) and the viscoelastic properties at low frequency (rheometry) was found. We also found that, in apparent contradiction with polyelectrolyte solution theory, HA solution behaves as liquid-like viscoelastic fluid (G'>G') even at concentrations higher than the entanglement concentration where a weak-gel behavior should be expected.     

Nonlinear Elastic and Viscoelastic Deformation of the Human Red Blood Cell with Optical Tweezers

Studies of the deformation characteristics of single biological cells can offer insights into the connections among mechanical state, biochemical response and the onset and progression of diseases. Deformation imposed by optical tweezers provides a useful means for the study of single cell mechanics under a variety of well-controlled stress-states. In this paper, we first critically review recent advances in the study of single cell mechanics employing the optical tweezers method, and assess its significance and limitations in comparison to other experimental tools. We then present new experimental and computational results on shape evolution, force--extension curves, elastic properties and viscoelastic response of human red blood cells subjected to large elastic deformation using optical tweezers. Potential applications of the methods examined here to study diseased cells are also briefly addressed.     

Fast fluorescence laser tracking microrheometry, II: quantitative studies of cytoskeletal mechanotransduction

Fluorescence laser tracking microrheometry (FLTM) is what we believe to be a novel method able to assess the local, frequency-dependent mechanical properties of living cells with nanometer spatial sensitivity at speeds up to 50 kHz. In an earlier article, we described the design, development, and optimization phases of the FLTM before reporting its performances in a variety of viscoelastic materials. In the work presented here, we demonstrate the suitability of FLTM to study local cellular rheology and obtain values for the storage and loss moduli G′(ω) and G″(ω) of fibroblasts consistent with past literature. We further establish that chemically induced cytoskeletal disruption is accompanied by reduced cellular stiffness and viscosity. Next, we provide a systematic study of some experimental variables that may critically influence microrheology measurements. First, we interrogate and justify the relevance of bead endocytosis as a method of cellular internalization of 1-μm probes in FLTM. Second, we show that as sample temperature increases, FLTM findings are elevated toward higher frequencies. Third, we confirm that relevant bead sizes (1 and 2 μm) have no effect on FLTM measurements. Fourth, we report the lack of influence of bead coatings (antiintegrin, antitransferrin, antidystroglycan, or uncoated tracers were surveyed) on their rheological readouts. Finally, we demonstrate the potential of FLTM in studying how substratum rigidity regulates cellular rheological properties. Interestingly, multiple, coupled strain relaxation mechanisms can be observed separated by two plateau moduli. Although these observations can be partly explained by rheological theories describing entangled actin filaments, there is a clear need to extend existing microrheology models to the cytoskeleton, including potentially important factors such as network geometry and remodeling.     

Dictyostelium cells' cytoplasm as an active viscoplastic body

We applied a recently developed microrheology technique based on colloidal magnetic tweezers to measure local viscoelastic moduli and active forces in cells of Dictyostelium discoideum. The active transport of nonmagnetic beads taken up by phagocytosis was analyzed by single particle tracking, which allowed us to measure the length of straight steps and the corresponding velocities of the movements. The motion consists of a superposition of nearly straight long-range steps (step length in the micrometer range) and local random walks (step widths about 0.1 microm). The velocities for the former type of motion range from 1 to 3 microm/s. They decrease with increasing bead size and are attributed to rapid active transport along microtubuli. The short-range local motions exhibit velocities of less than 0.5 microm/s and reflect the internal dynamics of the cytoplasm. Viscoelastic response curves were measured by application of force pulses with amplitudes varying between 50 pN and 400 pN. Analysis of the response curves in terms of mechanical equivalent circuits yielded cytoplasmic viscosities varying between 10 and 350 Pa s. Simultaneous analysis of the response curves and of the bead trajectories showed that the motion of the beads is determined by the local yield stress within the cytoplasmic scaffold and cisternae, which varies between sigma = 30 Pa and 250 Pa. The motion of intracellular particles is interpreted in terms of viscoplastic behavior and the apparent viscosity is a measure of the reciprocal rate of bond breakage within the cytoplasmatic network. The viscoelastic moduli are interpreted as dynamic quantities which depend sensitively on the amplitude of the forces, and the rate of bond breakage is determined by the Arrhenius-Kramers law with the activation energy being reduced by the work performed by the applied force. In agreement with previous work, we provide evidence that the myosin II-deficient cells exhibit higher yield stresses, suggesting that the function of myosin II as a cross-linker is taken over by the other (non-active) cross-linkers.     

Influence of some physicochemical factors on the viscosity of aqueous levan solutions of Zymomonas mobilis

Zymomonas mobilis strain 113 “S” produces levan – an extracellular, viscous, biologically active, non-toxic fructose polymer with a unique structure and extraordinary properties. This polysaccharide was isolated at two different degrees of purity by alcohol precipitation from aqueous solutions and was characterized with respect to some rheological properties and stability of viscous solutions. The effects of temperature, pH and salt concentration on the viscosity of 1–3% levan solutions were examined. The viscosity of levan solutions was found to be quite stable and reversible at room temperature over a wide range of pH from 4 to 11. The viscosity was slightly affected by increased salt concentration. Levan solutions were rather stable at high temperatures (up to 70°C, 1 h, pH 6), where the viscosity could be almost completerly restored (up to 80–100%). Therefore, the degradation of the polymer structure under these conditions is probably insignificant. Temperatures of 70–100°C with a pH of less than 3.5 caused irreversible degradation of the levan structure. The above-mentioned properties of levan, obtained from Zymomonas mobilis 113 “S”, demonstrated the potential for the development of various therapeutic forms of pharmacologically-active levan and their application in medicine as well as in the food and other industries.     

The Role of Vimentin Intermediate Filaments in Cortical and Cytoplasmic Mechanics

The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin^−/− mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.     

Ion-Dependent Dynamics of DNA Ejections for Bacteriophage λ

We studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage λ. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counterions is an important control parameter. These measurements show that the mobility of ejecting DNA is independent of ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection, and confirm that this effect occurs using optical tweezers.