Mechanics of living cells measured by laser tracking microrheology

To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity.     

Digital holographic microtomography for high‐resolution refractive index mapping of live cells

Quantification of three‐dimensional (3D) refractive index (RI) with sub‐cellular resolution is achieved by digital holographic microtomography (DHμT) using quantitative phase images measured at multiple illumination angles. The DHμT system achieves sensitive and fast phase measurements based on iterative phase extraction algorithm and asynchronous phase shifting interferometry without any phase monitoring or active control mechanism. A reconstruction algorithm, optical diffraction tomography with projection on convex sets and total variation minimization, is implemented to substantially reduce the number of angular scattered fields needed for reconstruction without sacrificing the accuracy and quality of the reconstructed 3D RI distribution. Tomogram of a living CA9‐22 cell is presented to demonstrate the performance of the method. Further, a statistical analysis of the average RI of the nucleoli, the nucleus excluding the nucleoli and the cytoplasm of twenty CA9‐22 cells is performed. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress

Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and pathophysiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. The exact time scales of these mechanical adaptations, however, remain unknown. We used particle-tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that ECs become significantly more compliant as early as 30 s after a step change in shear stress from 0 to 10 dyn/cm² followed by recovery of viscoelastic parameters within 4 min of shearing, even though shear stress was maintained. After ECs were sheared for 5 min, return of shear stress to 0 dyn/cm² in a stepwise manner did not result in any further rheological adaptation. Average vesicle displacements were used to determine time-dependent cell deformation and macrorheological parameters by fitting creep function to a linear viscoelastic liquid model. Characteristic time and magnitude for shear-induced deformation were 3 s and 50 nm, respectively. We conclude that ECs rapidly adapt their mechanical properties in response to shear stress, and we provide the first macrorheological parameters for time-dependent deformations of ECs to a physiological forcing function. Such studies provide insight into pathologies such as atherosclerosis, which may find their origins in EC mechanics. viscoelasticity; atherosclerosis; cell mechanics; particle tracking; mechanotransduction     

Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells

Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell “softness” is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.     

Labeling live cells by copper-catalyzed alkyne--azide click chemistry

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, optimized for biological molecules in aqueous buffers, has been shown to rapidly label mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine developed by Bertozzi, followed by CuAAC ligation using sodium ascorbate and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA), gave rise to abundant covalent attachment of dye-alkyne reactants. THPTA serves both to accelerate the CuAAC reaction and to protect the cells from damage by oxidative agents produced by the Cu-catalyzed reduction of oxygen by ascorbate, which is required to maintain the metal in the active +1 oxidation state. This procedure extends the application of this fastest of azide-based bioorthogonal reactions to the exterior of living cells.     

Alteration of red blood cell microrheology by anti-tumor chemotherapy drugs

The aim of this study was to estimate effects of some chemotherapy drugs on the elasticity and deformability of the membrane of a red blood cell (RBC). It was found that incubation of red blood cells (RBCs) with cisplatin or epoetin alpha led to considerable (by 10–17%; p < 0.05) increase in the RBC deformability and that cisplatin could activate tyrosine protein kinases (TPKs). Preincubation of RBCs with a specific inhibitor of EGF-R and Src kinase, lavendustin A, almost completely prevented the cisplatin effect. Tyrosine phosphatase inhibitor, sodium orthovanadate, increased the RBC deformability (p < 0.05). This effect was also abandoned by lavendustin A. To test a hypothesis on the involvement of protein kinases of mature RBCs in control of their membrane elasticity, the cells were incubated with phorbol 12-myristate 13-acetate (PMA) activating protein kinase Cα (PKCα). PMA increased the RBC deformability only moderately (by 8%, p < 0.05) and the effect was canceled by nonselective and selective PKC inhibitors staurosporin and 4-(1-methylindol-3-yl)maleimide hydrochloride. Erythropoietin is known to inhibit the nonselective cation channels of the RBC membrane; however, preincubation of the cells with verapamil did not cancel the increase in their deformability. Hence, this increase in deformability could be a result of the action of tyrosine protein kinases, the more so that this effect was almost completely canceled by lavendustion A. The results suggest that the presence of functionally active protein kinases and phosphatases in the membranes of mature RBC makes them a target for the addressed effects of signal molecules, including some chemotherapy drugs, causing consecutive alterations in the RBC membrane elasticity, microrheological properties, and transport potential.     

Measuring DNA content in live cells by fluorescence microscopy

Background

Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.

Results

Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm’s accurate assessment of DNA content was validated by parallel flow cytometric studies.

Conclusions

This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

     

High-throughput RNAi screening by time-lapse imaging of live human cells

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.     

Intracellular microrheology of motile Amoeba proteus

The motility of Amoeba proteus was examined using the technique of passive particle tracking microrheology, with the aid of newly developed particle tracking software, a fast digital camera, and an optical microscope. We tracked large numbers of endogeneous particles in the amoebae, which displayed subdiffusive motion at short timescales, corresponding to thermal motion in a viscoelastic medium, and superdiffusive motion at long timescales due to the convection of the cytoplasm. Subdiffusive motion was characterized by a rheological scaling exponent of 3/4 in the cortex, indicative of the semiflexible dynamics of the actin fibers. We observed shear-thinning in the flowing endoplasm, where exponents increased with increasing flow rate; i.e., the endoplasm became more fluid-like. The rheology of the cortex is found to be isotropic, reflecting an isotropic actin gel. A clear difference was seen between cortical and endoplasmic layers in terms of both viscoelasticity and flow velocity, where the profile of the latter is close to a Poiseuille flow for a Newtonian fluid.     

DWS microrheology of a linear polysaccharide

Diffusing wave spectroscopy has been used to measure the rheological behavior of pullulan (M(w) = 1 x 10(5)) aqueous solutions up to concentration of 40 g/dL. It was found that these solutions were mainly viscous, with the loss modulus G' higher than the elastic modulus G'. The plot of the specific viscosity eta(sp) as a function of pullulan concentration showed two critical concentrations c = 4 g/dL and c = 15 g/dL. For c < c, eta( sp) approximately c(1.25+/-0.05); for c < c < c, eta( sp) approximately c(2+/-0.05); and for c > c, eta( sp) approximately c(4.5+/-0.5). These results are in very good agreement with those reported in the literature.     

Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking

By monitoring the thermally driven displacements of imbedded polystyrene microspheres via video fluorescence microscopy, we quantified the microstructural and micromechanical heterogeneities of wheat gliadin suspensions. We found that the degree of heterogeneity of the suspensions, as measured by the width and skewness of the microspheres' mean squared displacement (MSD) distribution, increased dramatically over a narrow range of gliadin concentrations. The ensemble-averaged MSD of a 250 mg/mL gliadin suspension exhibited a power-law behavior scaling linearly with time, a behavior similar to that observed for a homogeneous aqueous glycerol solution. However, the MSD distribution was wider and more asymmetric than for glycerol. With increasing concentration of gliadin, the ensemble-averaged MSD rapidly displayed a plateau at small time scales, the MSD distribution became wider and more asymmetric, and the local viscoelastic moduli extracted from multiple-particle-tracking measurements showed an increasingly wide range.     

Probing molecular processes in live cells by quantitative multidimensional microscopy

Modern light microscopy has become a most powerful analytical tool for studying molecular processes in live cells. Recent advances in sample preparation, microscope design and image processing allow the generation of "multidimensional" data, simultaneously reporting the three-dimensional distribution and concentrations of several different molecules within cells and tissues at multiple time points with sub-micron spatial resolution and sub-second temporal resolution. Thus, molecular interactions and processes that were approached by biochemical analyses in vitro can now be directly monitored in live cells. Here, we address different aspects of multidimensional microscopy and, in particular, image quantification and the characterization of molecular dynamics, as applied to the study of cell adhesion.     

Imaging of RNA in live cells

Fluorescence microscopy and molecular tagging technologies have ushered in a new era in our understanding of protein localization and function in cells. This review summarizes recent efforts to extend some of these methods (and to create new ones) to imaging of RNA in live cells. Both fluorescent proteins and hybridization probes allow noncovalent labeling of specific RNA molecules with fluorescent dyes that allow detection and tracking in real time.     

High-resolution nonlinear optical imaging of live cells by second harmonic generation

By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.     

Chemical mapping of mammalian cells by atom probe tomography

In atom probe tomography (APT), a technique that has been used to determine 3D maps of ion compositions of metals and semiconductors at sub-nanometer resolutions, controlled emissions of ions can be induced from needle-shaped specimens in the vicinity of a strong electric field. Detection of these ions in the plane of a position sensitive detector provides two-dimensional compositional information while the sequence of ion arrival at the detector provides information in the third dimension. Here we explore the use of APT technology for imaging biological specimens. We demonstrate that it is possible to obtain 3D spatial distributions of cellular ions and metabolites from unstained, freeze-dried mammalian cells. Multiple peaks were reliably obtained in the mass spectrum from tips with diameters of ~50 nm and heights of ~200 nm, with mass-to-charge ratios (m/z) ranging from 1 to 80. Peaks at m/z 12, 23, 28 and 39, corresponding to carbon, sodium, carbonyl and potassium ions respectively, showed distinct patterns of spatial distribution within the cell. Our studies establish that APT could become a powerful tool for mapping the sub-cellular distribution of atomic species, such as labeled metabolites, at 3D spatial resolutions as high as ~1 nm.