Sapun H. Parekh

Reversible stress softening of actin networks

The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.

Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

The Effect of 3D Hydrogel Scaffold Modulus on Osteoblast Differentiation and Mineralization Revealed by Combinatorial Screening

Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell-material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus ( approximately 10 kPa to approximately 300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of approximately 225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.

Unilamellar vesicle formation and encapsulation by microfluidic jetting

Compartmentalization of biomolecules within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of membrane-enclosed solutions of proteins, DNA, and other biologically active compounds have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with solution from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles containing solutions of unrestricted composition and molecular weight. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solutions inside of GUVs creates new opportunities for the study and use of compartmentalized biomolecular systems in science, industry, and medicine.

Loading history determines the velocity of actin-network growth.

Directional polymerization of actin filaments in branched networks is one of the most powerful force-generating systems in eukaryotic cells. Growth of densely cross-linked actin networks drives cell crawling, intracellular transport of vesicles and organelles, and movement of intracellular pathogens such as Listeria monocytogenes. Using a modified atomic force microscope (AFM), we obtained force–velocity (Fv) measurements of growing actin networks in vitro until network elongation ceased at the stall force. We found that the growth velocity of a branched actin network against increasing forces is load-independent over a wide range of forces before a convex decline to stall. Surprisingly, when force was decreased on a growing network, the velocity increased to a value greater than the previous velocity, such that two or more stable growth velocities can exist at a single load. These results demonstrate that a single Fv relationship does not capture the complete behaviour of this system, unlike other molecular motors in cells, because the growth velocity depends on loading history rather than solely on the instantaneous load.

Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells.

The mechanical rigidity of cells and adhesion forces between cells are important in various biological processes, including cell differentiation, proliferation and tissue organization. Atomic force microscopy has emerged as a powerful tool to quantify the mechanical properties of individual cells and adhesion forces between cells. Here we demonstrate an instrument that combines atomic force microscopy with a side-view fluorescent imaging path that enables direct imaging of cellular deformation and cytoskeletal rearrangements along the axis of loading. With this instrument, we directly observed cell shape under mechanical load, correlated changes in shape with force-induced ruptures and imaged formation of membrane tethers during cell-cell adhesion measurements. Additionally, we observed cytoskeletal reorganization and stress-fiber formation while measuring the contractile force of an individual cell. This instrument can be a useful tool for understanding the role of mechanics in biological processes.

Label-Free Cellular Imaging by Broadband Coherent Anti-Stokes Raman Scattering Microscopy

Raman microspectroscopy can provide the chemical contrast needed to characterize the complex intracellular environment and macromolecular organization in cells without exogenous labels. It has shown a remarkable ability to detect chemical changes underlying cell differentiation and pathology-related chemical changes in tissues but has not been widely adopted for imaging, largely due to low signal levels. Broadband coherent anti-Stokes Raman scattering (B-CARS) offers the same inherent chemical contrast as spontaneous Raman but with increased acquisition rates. To date, however, only spectrally resolved signals from the strong CH-related vibrations have been used for CARS imaging. Here, we obtain Raman spectral images of single cells with a spectral range of 600-3200 cm⁻¹, including signatures from weakly scattering modes as well as CH vibrations. We also show that B-CARS imaging can be used to measure spectral signatures of individual cells at least fivefold faster than spontaneous Raman microspectroscopy and can be used to generate maps of biochemical species in cells. This improved spectral range and signal intensity opens the door for more widespread use of vibrational spectroscopic imaging in biology and clinical diagnostics.

Liquid flow along a solid surface reversibly alters interfacial chemistry

Monitoring water interfaces in motion Water behaves differently at interfaces—where it meets the air, or a solid surface—than it does in the middle of the liquid. Past laboratory studies of this phenomenon have mainly focused on still samples, despite the fact that in natural settings such as rivers and rain, the water moves along the surfaces. Lis et al. used a microfluidics apparatus and a spectroscopy technique called sum frequency generation to study the effects of flow on aqueous chemistry at silica and fluorite surfaces (see the Perspective by Waychunas). The flow of fresh water along the surfaces disrupts the equilibrium of dissolved ions, substantially changing the surface charge and the molecular orientation of the water at the interface. Science, this issue p. 1138; see also p. 1094 A combination of microfluidics and surface-specific spectroscopy enables the study of flow effects at aqueous interfaces. [Also see Perspective by Waychunas] In nature, aqueous solutions often move collectively along solid surfaces (for example, raindrops falling on the ground and rivers flowing through riverbeds). However, the influence of such motion on water-surface interfacial chemistry is unclear. In this work, we combine surface-specific sum frequency generation spectroscopy and microfluidics to show that at immersed calcium fluoride and fused silica surfaces, flow leads to a reversible modification of the surface charge and subsequent realignment of the interfacial water molecules. Obtaining equivalent effects under static conditions requires a substantial change in bulk solution pH (up to 2 pH units), demonstrating the coupling between flow and chemistry. These marked flow-induced variations in interfacial chemistry should substantially affect our understanding and modeling of chemical processes at immersed surfaces.

Near shot-noise limited hyperspectral stimulated Raman scattering spectroscopy using low energy lasers and a fast CMOS array

We demonstrate near shot-noise limited hyperspectral stimulated Raman scattering (SRS) spectroscopy using oscillator-only excitation conditions. Using a fast CMOS camera synchronized to an acousto-optic modulator and subtracting subsequent frames acquired at up to 1 MHz frame rates, we demonstrate demodulation and recovery of the SRS spectrum. Surprisingly, we observe that the signal-to-noise of SRS spectra is invariant at modulation frequencies down to 2.5 kHz. Our approach allows for a direct comparison of SRS with coherent anti-Stokes Raman scattering (CARS) spectroscopy under identical experimental conditions. Our findings suggest that hyperspectral SRS imaging with shot-noise limited performance at biologically compatible excitation energies is feasible after minor modifications to fast frame-rate CMOS array technology.

Optimized continuum from a photonic crystal fiber for broadband time-resolved coherent anti-Stokes Raman scattering.

We demonstrate an optimization of continuum generation in a commercially available photonic crystal fiber and show that this continuum can be used to simultaneously measure vibrational dephasing times over an unprecedented frequency range of Raman modes. The dephasing time measurement is based on 2-pulse 3-color coherent anti-Stokes Raman scattering (CARS), and requires a continuum pulse that is coherent over a broad spectral bandwidth. We demonstrate that a continuum with the required characteristics can be generated from a photonic crystal fiber by appropriately conditioning the chirp of the excitation pulse and controlling its pulse energy. We are able to simultaneously measure vibrational dephasing times of multiple Raman modes (covering 500 cm(-1) to 3100 cm(-1)) of acetonitrile and benzonitrile using the optimized continuum with broadband time-resolved CARS.