Single-cell level LasR-mediated quorum sensing response of Pseudomonas aeruginosa to pulses of signal molecules.

Quorum sensing (QS) is a communication form between bacteria via small signal molecules that enables global gene regulation as a function of cell density. We applied a microfluidic mother machine to study the kinetics of the QS response of Pseudomonas aeruginosa bacteria to additions and withdrawals of signal molecules. We traced the fast buildup and the subsequent considerably slower decay of a population-level and single-cell-level QS response. We applied a mathematical model to explain the results quantitatively. We found significant heterogeneity in QS on the single-cell level, which may result from variations in quorum-controlled gene expression and protein degradation. Heterogeneity correlates with cell lineage history, too. We used single-cell data to define and quantitatively characterize the population-level quorum state. We found that the population-level QS response is well-defined. The buildup of the quorum is fast upon signal molecule addition. At the same time, its decay is much slower following signal withdrawal, and the quorum may be maintained for several hours in the absence of the signal. Furthermore, the quorum sensing response of the population was largely repeatable in subsequent pulses of signal molecules.

3D-printed ultra-small Brownian viscometers.

Measuring viscosity in volumes smaller than a microliter is a challenging endeavor. A new type of microscopic viscometers is presented to assess the viscosity of Newtonian liquids. Micron-sized flexible polymer cantilevers are created by two-photon polymerization direct laser writing. Because of the low stiffness and high elasticity of the polymer material the microcantilevers exhibit pronounced Brownian motion when submerged in a liquid medium. By imaging the cantilever's spherically shaped end, these fluctuations can be tracked with high accuracy. The hydrodynamic resistance of the microviscometer is determined by fitting the power spectral density of the measured fluctuations with a theoretical frequency dependence. Validation measurements in water-glycerol mixtures with known viscosities reveal excellent linearity of the hydrodynamic resistance to viscosity, allowing for a simple linear calibration. The stand-alone viscometer structures have a characteristic size of a few tens of microns and only require a very basic external instrumentation in the form of microscopic imaging at moderate framerates (~ 100 fps). Thus, our results point to a practical and simple to use ultra-low volume viscometer that can be integrated into lab-on-a-chip devices.

Optically Actuated Soft Microrobot Family for Single-Cell Manipulation.

Precisely controlled manipulation of nonadherent single cells is often a pre-requisite for their detailed investigation. Optical trapping provides a versatile means for positioning cells with submicrometer precision or measuring forces with femto-Newton resolution. A variant of the technique, called indirect optical trapping, enables single-cell manipulation with no photodamage and superior spatial control and stability by relying on optically trapped microtools biochemically bound to the cell. High-resolution 3D lithography enables to prepare such cell manipulators with any predefined shape, greatly extending the number of achievable manipulation tasks. Here, it is presented for the first time a novel family of cell manipulators that are deformable by optical tweezers and rely on their elasticity to hold cells. This provides a more straightforward approach to indirect optical trapping by avoiding biochemical functionalization for cell attachment, and consequently by enabling the manipulated cells to be released at any time. Using the photoresist Ormocomp, the deformations achievable with optical forces in the tens of pN range and present three modes of single-cell manipulation as examples to showcase the possible applications such soft microrobotic tools can offer are characterized. The applications describe here include cell collection, 3D cell imaging, and spatially and temporally controlled cell-cell interaction.

Synergistic induction of blood-brain barrier properties.

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/ß-catenin signaling and inhibition of the TGF-ß pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/ß-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Assessing the Viscoelasticity of Photopolymer Nanowires Using a Three-Parameter Solid Model for Bending Recovery Motion.

Photopolymer nanowires prepared by two-photon polymerization direct laser writing (TPP-DLW) are the building blocks of many microstructure systems. These nanowires possess viscoelastic characteristics that define their deformations under applied forces when operated in a dynamic regime. A simple mechanical model was previously used to describe the bending recovery motion of deflected nanowire cantilevers in Newtonian liquids. The inverse problem is targeted in this work; the experimental observations are used to determine the nanowire physical characteristics. Most importantly, based on the linear three-parameter solid model, we derive explicit formulas to calculate the viscoelastic material parameters. It is shown that the effective elastic modulus of the studied nanowires is two orders of magnitude lower than measured for the bulk material. Additionally, we report on a notable effect of the surrounding aqueous glucose solution on the elasticity and the intrinsic viscosity of the studied nanowires made of Ormocomp.

Optically Manipulated Microtools to Measure Adhesion of the Nanoparticle-Targeting Ligand Glutathione to Brain Endothelial Cells.

Targeting nanoparticles as drug delivery platforms is crucial to facilitate their cellular entry. Docking of nanoparticles by targeting ligands on cell membranes is the first step for the initiation of cellular uptake. As a model system, we studied brain microvascular endothelial cells, which form the anatomical basis of the blood-brain barrier, and the tripeptide glutathione, one of the most effective targeting ligands of nanoparticles to cross the blood-brain barrier. To investigate this initial docking step between glutathione and the membrane of living brain endothelial cells, we applied our recently developed innovative optical method. We present a microtool, with a task-specific geometry used as a probe, actuated by multifocus optical tweezers to characterize the adhesion probability and strength of glutathione-coated surfaces to the cell membrane of endothelial cells. The binding probability of the glutathione-coated surface and the adhesion force between the microtool and cell membrane was measured in a novel arrangement: cells were cultured on a vertical polymer wall and the mechanical forces were generated laterally and at the same time, perpendicularly to the plasma membrane. The adhesion force values were also determined with more conventional atomic force microscopy (AFM) measurements using functionalized colloidal probes. The optical trapping-based method was found to be suitable to measure very low adhesion forces (≤ 20 pN) without a high level of noise, which is characteristic for AFM measurements in this range. The holographic optical tweezers-directed functionalized microtools may help characterize the adhesion step of nanoparticles initiating transcytosis and select ligands to target nanoparticles.

Single-Cell Elasticity Measurement with an Optically Actuated Microrobot.

A cell elasticity measurement method is introduced that uses polymer microtools actuated by holographic optical tweezers. The microtools were prepared with two-photon polymerization. Their shape enables the approach of the cells in any lateral direction. In the presented case, endothelial cells grown on vertical polymer walls were probed by the tools in a lateral direction. The use of specially shaped microtools prevents the target cells from photodamage that may arise during optical trapping. The position of the tools was recorded simply with video microscopy and analyzed with image processing methods. We critically compare the resulting Young's modulus values to those in the literature obtained by other methods. The application of optical tweezers extends the force range available for cell indentations measurements down to the fN regime. Our approach demonstrates a feasible alternative to the usual vertical indentation experiments.

The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel.

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

A transition to stable one-dimensional swimming enhances E. coli motility through narrow channels.

Living organisms often display adaptive strategies that allow them to move efficiently even in strong confinement. With one single degree of freedom, the angle of a rotating bundle of flagella, bacteria provide one of the simplest examples of locomotion in the living world. Here we show that a purely physical mechanism, depending on a hydrodynamic stability condition, is responsible for a confinement induced transition between two swimming states in E. coli. While in large channels bacteria always crash onto confining walls, when the cross section falls below a threshold, they leave the walls to move swiftly on a stable swimming trajectory along the channel axis. We investigate this phenomenon for individual cells that are guided through a sequence of micro-fabricated tunnels of decreasing cross section. Our results challenge current theoretical predictions and suggest effective design principles for microrobots by showing that motility based on helical propellers provides a robust swimming strategy for exploring narrow spaces.

Multiview microscopy of single cells through microstructure-based indirect optical manipulation.

Fluorescent observation of cells generally suffers from the limited axial resolution due to the elongated point spread function of the microscope optics. Consequently, three-dimensional imaging results in axial resolution that is several times worse than the transversal. The optical solutions to this problem usually require complicated optics and extreme spatial stability. A straightforward way to eliminate anisotropic resolution is to fuse images recorded from multiple viewing directions achieved mostly by the mechanical rotation of the entire sample. In the presented approach, multiview imaging of single cells is implemented by rotating them around an axis perpendicular to the optical axis by means of holographic optical tweezers. For this, the cells are indirectly trapped and manipulated with special microtools made with two-photon polymerization. The cell is firmly attached to the microtool and is precisely manipulated with 6 degrees of freedom. The total control over the cells' position allows for its multiview fluorescence imaging from arbitrarily selected directions. The image stacks obtained this way are combined into one 3D image array with a multiview image processing pipeline resulting in isotropic optical resolution that approaches the lateral diffraction limit. The presented tool and manipulation scheme can be readily applied in various microscope platforms.

Brownian fluctuations and hydrodynamics of a microhelix near a solid wall.

We combine two-photon lithography and optical tweezers to investigate the Brownian fluctuations and propeller characteristics of a microfabricated helix. From the analysis of mean squared displacements and time correlation functions we recover the components of the full mobility tensor. We find that Brownian motion displays correlations between angular and translational fluctuations from which we can directly measure the hydrodynamic coupling coefficient that is responsible for thrust generation. By varying the distance of the microhelices from a no-slip boundary we can systematically measure the effects of a nearby wall on the resistance matrix. Our results indicate that a rotated helix moves faster when a nearby no-slip boundary is present, providing a quantitative insight on thrust enhancement in confined geometries for both synthetic and biological microswimmers.

Invariance properties of bacterial random walks in complex structures.

Motile cells often explore natural environments characterized by a high degree of structural complexity. Moreover cell motility is also intrinsically noisy due to spontaneous random reorientations and speed fluctuations. This interplay of internal and external noise sources gives rise to a complex dynamical behavior that can be strongly sensitive to details and hard to model quantitatively. In striking contrast to this general picture we show that the mean residence time of swimming bacteria inside artificial complex microstructures is quantitatively predicted by a generic invariance property of random walks. We find that while external shape and internal disorder have dramatic effects on the distributions of path lengths and residence times, the corresponding mean values are constrained by the sole free surface to perimeter ratio. As a counterintuitive consequence, bacteria escape faster from structures with higher density of obstacles due to the lower accessible surface.

An optical reaction micro-turbine.

To any energy flow there is an associated flow of momentum, so that recoil forces arise every time an object absorbs or deflects incoming energy. This same principle governs the operation of macroscopic turbines as well as that of microscopic turbines that use light as the working fluid. However, a controlled and precise redistribution of optical energy is not easy to achieve at the micron scale resulting in a low efficiency of power to torque conversion. Here we use direct laser writing to fabricate 3D light guiding structures, shaped as a garden sprinkler, that can precisely reroute input optical power into multiple output channels. The shape parameters are derived from a detailed theoretical analysis of losses in curved microfibers. These optical reaction micro-turbines can maximally exploit light's momentum to generate a strong, uniform and controllable torque.

Dynamic density shaping of photokinetic E. coli.

Many motile microorganisms react to environmental light cues with a variety of motility responses guiding cells towards better conditions for survival and growth. The use of spatial light modulators could help to elucidate the mechanisms of photo-movements while, at the same time, providing an efficient strategy to achieve spatial and temporal control of cell concentration. Here we demonstrate that millions of bacteria, genetically modified to swim smoothly with a light controllable speed, can be arranged into complex and reconfigurable density patterns using a digital light projector. We show that a homogeneous sea of freely swimming bacteria can be made to morph between complex shapes. We model non-local effects arising from memory in light response and show how these can be mitigated by a feedback control strategy resulting in the detailed reproduction of grayscale density images.

Light controlled 3D micromotors powered by bacteria.

Self-propelled bacteria can be integrated into synthetic micromachines and act as biological propellers. So far, proposed designs suffer from low reproducibility, large noise levels or lack of tunability. Here we demonstrate that fast, reliable and tunable bio-hybrid micromotors can be obtained by the self-assembly of synthetic structures with genetically engineered biological propellers. The synthetic components consist of 3D interconnected structures having a rotating unit that can capture individual bacteria into an array of microchambers so that cells contribute maximally to the applied torque. Bacterial cells are smooth swimmers expressing a light-driven proton pump that allows to optically control their swimming speed. Using a spatial light modulator, we can address individual motors with tunable light intensities allowing the dynamic control of their rotational speeds. Applying a real-time feedback control loop, we can also command a set of micromotors to rotate in unison with a prescribed angular speed.

Active dynamics of colloidal particles in time-varying laser speckle patterns.

Colloidal particles immersed in a dynamic speckle pattern experience an optical force that fluctuates both in space and time. The resulting dynamics presents many interesting analogies with a broad class of non-equilibrium systems like: active colloids, self propelled microorganisms, transport in dynamical intracellular environments. Here we show that the use of a spatial light modulator allows to generate light fields that fluctuate with controllable space and time correlations and a prescribed average intensity profile. In particular we generate ring-shaped random patterns that can confine a colloidal particle over a quasi one-dimensional random energy landscape. We find a mean square displacement that is diffusive at both short and long times, while a superdiffusive or subdiffusive behavior is observed at intermediate times depending on the value of the speckles correlation time. We propose two alternative models for the mean square displacement in the two limiting cases of a short or long speckles correlation time. A simple interpolation formula is shown to account for the full phenomenology observed in the mean square displacement across the entire range from fast to slow fluctuating speckles.

Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells.

We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and non-specifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation.

Optically Trapped Surface-Enhanced Raman Probes Prepared by Silver Photoreduction to 3D Microstructures.

3D microstructures partially covered by silver nanoparticles have been developed and tested for surface-enhanced Raman spectroscopy (SERS) in combination with optical tweezers. The microstructures made by two-photon polymerization of SU-8 photoresist were manipulated in a dual beam optical trap. The active area of the structures was covered by a SERS-active silver layer using chemically assisted photoreduction from silver nitrate solutions. Silver layers of different grain size distributions were created by changing the photoreduction parameters and characterized by scanning electron microscopy. The structures were tested by measuring the SERS spectra of emodin and hypericin.

Holographic multi-focus 3D two-photon polymerization with real-time calculated holograms.

Two-photon polymerization enables the fabrication of micron sized structures with submicron resolution. Spatial light modulators (SLM) have already been used to create multiple polymerizing foci in the photoresist by holographic beam shaping, thus enabling the parallel fabrication of multiple microstructures. Here we demonstrate the parallel two-photon polymerization of single 3D microstructures by multiple holographically translated foci. Multiple foci were created by phase holograms, which were calculated real-time on an NVIDIA CUDA GPU, and displayed on an electronically addressed SLM. A 3D demonstrational structure was designed that is built up from a nested set of dodecahedron frames of decreasing size. Each individual microstructure was fabricated with the parallel and coordinated motion of 5 holographic foci. The reproducibility and the high uniformity of features of the microstructures were verified by scanning electron microscopy.

Optical forces through guided light deflections.

Optical trapping and manipulation typically relies on shaping focused light to control the optical force, usually on spherical objects. However, one can also shape the object to control the light deflection arising from the light-matter interaction and, hence, achieve desired optomechanical effects. In this work we look into the object shaping aspect and its potential for controlled optical manipulation. Using a simple bent waveguide as example, our numerical simulations show that the guided deflection of light efficiently converts incident light momentum into optical force with one order-of-magnitude improvement in the efficiency factor relative to a microbead, which is comparable to the improvement expected from orthogonal deflection with a perfect mirror. This improvement is illustrated in proof-of-principle experiments demonstrating the optical manipulation of two-photon polymerized waveguides. Results show that the force on the waveguide exceeds the combined forces on spherical trapping handles. Furthermore, it shows that static illumination can exert a constant force on a moving structure, unlike the position-dependent forces from harmonic potentials in conventional trapping.