Size-Based Sorting of Emulsion Droplets in Microfluidic Channels Patterned with Laser-Ablated Guiding Tracks.

We demonstrate an autonomous, high-throughput mechanism for sorting of emulsion droplets with different sizes concurrently flowing in a microfluidic Hele-Shaw channel. The aqueous droplets of varying radii suspended in olive oil are separated into different streamlines across the channel upon interaction with a shallow (depth ∼ 700 nm) inclined guiding track ablated into the polydimethylsiloxane-coated surface of the channel with focused femtosecond laser pulses. Specifically, the observed differences in the droplet trajectories along the guiding track arise due to the different scaling of the confinement force attracting the droplets into the track, fluid drag, and wall friction, with the droplet radius. In addition, the distance traveled by the droplets along the track also depends on the track width, with wider tracks providing more stable droplet guiding for any given droplet size. We systematically study the influence of the droplet size and velocity on the trajectory of the droplets in the channel and analyze the sensitivity of size-based droplet sorting for varying flow conditions. The droplet guiding and sorting experiments are complemented by modeling of the droplet motion in the channel flow using computational fluid dynamics simulations and a previously developed model of droplet guiding. Finally, we demonstrate a complete separation of droplets produced by fusion of two independent droplet streams at the inlet of the Hele-Shaw channel from unfused daughter droplets. The presented droplet sorting technique can find applications in the development of analytical and preparative microfluidic protocols.

Analysis of Bacteriophage-Host Interaction by Raman Tweezers.

Bacteriophages, or "phages" for short, are viruses that replicate in bacteria. The therapeutic and biotechnological potential of phages and their lytic enzymes is of interest for their ability to selectively destroy pathogenic bacteria, including antibiotic-resistant strains. Introduction of phage preparations into medicine, biotechnology, and food industry requires a thorough characterization of phage-host interaction on a molecular level. We employed Raman tweezers to analyze the phage-host interaction of Staphylococcus aureus strain FS159 with a virulent phage JK2 (=812K1/420) of the Myoviridae family and a temperate phage 80α of the Siphoviridae family. We analyzed the timeline of phage-induced molecular changes in infected host cells. We reliably detected the presence of replicating phages in bacterial cells within 5 min after infection. Our results lay the foundations for building a Raman-based diagnostic instrument capable of real-time, in vivo, in situ, nondestructive characterization of the phage-host relationship on the level of individual cells, which has the potential of importantly contributing to the development of phage therapy and enzybiotics.

Mechanical Contact Spectroscopy: Characterizing Nanoscale Adhesive Contacts via Thermal Forces.

The adhesion of micro- and nanoparticles to solid substrates immersed in liquids is a problem of great scientific and technological importance. However, the quantitative characterization of such nanoscale adhesive contacts without rupturing them still presents a major experimental challenge. In this article, we introduce mechanical contact spectroscopy (MCS), an experimental technique for the nondestructive probing of particle adhesion in liquid environments. With MCS, the strength of adhesive contacts is inferred from residual position fluctuations of adherent particles excited by thermal forces. In particular, the strength of adhesion is correlated with the standard deviation of the particle lateral position x, with smaller position standard deviations [Formula: see text] indicating higher adhesive strength. For a given combination of particles, substrate, and immersion medium, the adhesion is characterized by the mechanical contact spectrum, which is a histogram of ξ values obtained from tracking an ensemble of adherent particles. Because the energy of thermal excitation at room temperature is very small in comparison to the typical total energy of adhesive contacts, the studied contacts remain in equilibrium during the measurement. Using MCS, we study the adhesion of micrometer-sized particles to planar solid substrates under a wide range of environmental conditions, including liquid immersion media of varying ionic strength and adhesion substrates with different chemical functionality of their surfaces. These experiments provide evidence that MCS is capable of reproducibly detecting minute changes in the particle-substrate work of adhesion while at the same time covering the range of adhesive contact strength relevant in the context of surface chemistry, biology, and microfabrication.

Effects of Infrared Optical Trapping on Saccharomyces cerevisiae in a Microfluidic System.

Baker's yeast (Saccharomyces cerevisiae) represents a very popular single-celled eukaryotic model organism which has been studied extensively by various methods and whose genome has been completely sequenced. It was also among the first living organisms that were manipulated by optical tweezers and it is currently a frequent subject of optical micromanipulation experiments. We built a microfluidic system for optical trapping experiments with individual cells and used it for the assessment of cell tolerance to phototoxic stress. Using optical tweezers with the wavelength of 1064 nm, we trapped individual Saccharomyces cerevisiae cells for 15 min and, subsequently, observed their stress response in specially designed microfluidic chambers over time periods of several hours by time-lapse video-microscopy. We determined the time between successive bud formations after the exposure to the trapping light, took account of damaged cells, and calculated the population doubling period and cell areas for increasing trapping power at a constant trapping time. Our approach represents an attractive, versatile microfluidic platform for quantitative optical trapping experiments with living cells. We demonstrate its application potential by assessing the limits for safe, non-invasive optical trapping of Saccharomyces cerevisiae with infrared laser light.

Spectral tuning of lasing emission from optofluidic droplet microlasers using optical stretching.

We introduce tunable optofluidic microlasers based on active optical resonant cavities formed by optically stretched, dye-doped emulsion droplets confined in a dual-beam optical trap. To achieve tunable dye lasing, optically pumped droplets of oil dispersed in water are stretched by light in the dual-beam trap. Subsequently, resonant path lengths of whispering gallery modes (WGMs) propagating in the droplet are modified, leading to shifts in the microlaser emission wavelengths. Using this technique, we present all-optical, almost reversible spectral tuning of the lasing WGMs and show that the direction of tuning depends on the position of the pump beam focus on the droplet. In addition, we study the effects of temperature changes on the spectral position of lasing WGMs and demonstrate that droplet heating leads to red-tuning of the droplet lasing wavelength.

Size stabilization of surface-supported liquid aerosols using tapered optical fiber coupling.

We demonstrate long-term size stabilization of surface-supported liquid aerosols of salt-water. Single tapered optical fibers were used to couple the light from independent heating and probe lasers into individual microdroplets that were kept on a superhydrophobic surface in a high-humidity chamber. Size stabilization of microdroplets resulted from competition between resonant absorption of the infrared heating laser by a microdroplet whispering gallery mode and water condensation in the sample chamber. Microdroplet size was continuously monitored using the tunable red probe laser. Thanks to the narrow linewidth of the heating laser, stabilization of the 110 µm radius of a microdroplet with a precision down to 0.54 nm was achieved for a period of 410 s.

Video-based tracking of single molecules exhibiting directed in-frame motion.

Trajectories of individual molecules moving within complex environments such as cell cytoplasm and membranes or semiflexible polymer networks provide invaluable information on the organization and dynamics of these systems. However, when such trajectories are obtained from a sequence of microscopy images, they can be distorted due to the fact that the tracked molecule exhibits appreciable directed motion during the single-frame acquisition. We propose a new model of image formation for mobile molecules that takes the linear in-frame motion into account and develop an algorithm based on the maximum likelihood approach for retrieving the position and velocity of the molecules from single-frame data. The position and velocity information obtained from individual frames are further fed into a Kalman filter for interframe tracking of molecules that allows prediction of the trajectory of the molecule and further improves the precision of the position and velocity estimates. We evaluate the performance of our algorithm by calculations of the Cramer-Rao Lower Bound, simulations, and model experiments with a piezo-stage. We demonstrate tracking of molecules moving as fast as 7 pixels/frame (12.6 µm/s) within a mean error of 0.42 pixel (37.43 nm).

Probing microscopic wetting properties of superhydrophobic surfaces by vibrated micrometer-sized droplets.

We determine contact angles of micrometer-sized NaCl-water droplets on superhydrophobic surfaces by analyzing their lowest-order axisymmetric vibrational resonances driven by vertical oscillations of the surface. Fluorescence spectra of the dye-doped droplets excited by laser light feature whispering-gallery modes (WGMs) whose spectral widths depend on the droplet vibration amplitude, thus enabling precise measurements of the droplet mechanical resonant frequency. Following droplet size determination by WGM mode-matching, we calculate the contact angles from the dependence of the measured mechanical resonant frequency on the droplet size for two surfaces with different superhydrophobicity levels, and find a good correlation with the values measured by direct imaging of millimeter-sized droplets.

Optically Transportable Optofluidic Microlasers with Liquid Crystal Cavities Tuned by the Electric Field.

Liquid crystal microdroplets with readily adjustable optical properties have attracted considerable attention for building reconfigurable optofluidic microsystems for sensing, imaging, and light routing applications. In this quest, development of active optical microcavities serving as versatile integrated sources of coherent light and ultra-sensitive environmental sensors has played a prominent role. Here, we study transportable optofluidic microlasers reversibly tunable by an external electric field, which are based on fluorophore-doped emulsion droplets of radial nematic liquid crystals manipulated by optical tweezers in microfluidic chips with embedded liquid electrodes. Full transparency of the electrodes formed by a concentrated electrolyte solution allows for applying an electric field to the optically trapped droplets without undesired heating caused by light absorption. Taking advantage of independent, precise control over the electric and thermal stimulation of the lasing liquid crystal droplets, we characterize their spectral tuning response at various optical trapping powers and study their relaxation upon a sudden decrease in the trapping power. Finally, we demonstrate that sufficiently strong applied electric fields can induce fully reversible phase transitions in the trapped droplets even below the bulk melting temperature of the used liquid crystal. Our observations indicate viability of creating electrically tunable, optically transported microlasers that can be prepared on-demand and operated within microfluidic chips to implement integrated microphotonic or sensing systems.

Raman microspectroscopy of individual algal cells: sensing unsaturation of storage lipids in vivo.

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm(-1) (cis C═C stretching mode) and 1,445 cm(-1) (CH(2) scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.

Detecting sequential bond formation using three-dimensional thermal fluctuation analysis.

We present a novel experimental method that solves two key problems in nondestructive mechanical studies of small biomolecules at the single-molecule level, namely the confirmation of single-molecule conditions and the discrimination against nonspecific binding. A biotin-avidin ligand-receptor couple is spanned between a glass slide and a 1 microm latex particle using short linker molecules. Optical tweezers are used to initiate bond formation and to follow the particle's thermal position fluctuations with nanometer spatial and microsecond temporal resolution. Here we show that each step in the specific binding process leads to an abrupt change in the magnitude of the particle's thermal position fluctuations, allowing us to count the number of bonds formed one by one. Moreover, three-dimensional position histograms calculated from the particle's fluctuations can be separated into well-defined categories reflecting different binding conditions (single specific, multiple specific, nonspecific). Our method brings quantitative mechanical single-molecule studies to the majority of proteins, paving the way for the investigation of a wide range of phenomena at the single-molecule level.

Light at work: the use of optical forces for particle manipulation, sorting, and analysis.

We review the combinations of optical micro-manipulation with other techniques and their classical and emerging applications to non-contact optical separation and sorting of micro- and nanoparticle suspensions, compositional and structural analysis of specimens, and quantification of force interactions at the microscopic scale. The review aims at inspiring researchers, especially those working outside the optical micro-manipulation field, to find new and interesting applications of these methods.

Primitive and committed human hematopoietic progenitor cells interact with primary murine neural cells and are induced to undergo self-renewing cell divisions.

OBJECTIVE: Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined. METHODS: CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens. RESULTS: The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC. CONCLUSION: Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.

A new type of microphotoreactor with integrated optofluidic waveguide based on solid-air nanoporous aerogels.

In this study, we developed a new type of microphotoreactor based on an optofluidic waveguide with aqueous liquid core fabricated inside a nanoporous aerogel. To this end, we synthesized a hydrophobic silica aerogel monolith with a density of 0.22 g cm-3 and a low refractive index of 1.06 that-from the optical point of view-effectively behaves like solid air. Subsequently, we drilled an L-shaped channel within the monolith that confined both the aqueous core liquid and the guided light, the latter property arising due to total internal reflection of light from the liquid-aerogel interface. We characterized the efficiency of light guiding in liquid-filled channel and-using the light delivered by waveguiding-we carried out photochemical reactions in the channel filled with aqueous solutions of methylene blue dye. We demonstrated that methylene blue could be efficiently degraded in the optofluidic photoreactor, with conversion increasing with increasing power of the incident light. The presented optofluidic microphotoreactor represents a versatile platform employing light guiding concept of conventional optical fibres for performing photochemical reactions.

In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities.

Fluorescent proteins are indispensable for selective, quantitative visualization of localization, dynamics, and interactions of key molecular constituents of live cells. Incorporation of fluorescent proteins into an optical cavity can lead to a significant increase in fluorescence signal levels due to stimulated emission and light amplification in the cavity, forming a laser with biological gain medium. Utilization of lasing emission from fluorescent biological molecules can then greatly enhance the performance of fluorescence-based biosensors benefiting from the high sensitivity of non-linear lasing processes to small perturbations in the cavity and the gain medium. Here we study optofluidic biolasers that exploit active liquid optical resonators formed by surface-supported aqueous microdroplets containing purified yellow fluorescent protein or a suspension of live E. coli bacterial cells expressing the fluorescent protein. We first demonstrate lasing in fluorescent protein solutions at concentrations as low as 49 µM. Subsequently, we show that a single fluorescent bacterial cell of micrometre size confined in a droplet-based cavity can serve as a laser gain medium. Aqueous droplet microcavities allow the maintenance of the bacterial cells under conditions compatible with unimpeded growth. Therefore, our results also suggest a direct route to microscopic sources of laser light with self-regenerating gain media.

Preparation and characterization of superhydrophobic surfaces based on hexamethyldisilazane-modified nanoporous alumina.

Superhydrophobic nanoporous anodic aluminum oxide (alumina) surfaces were prepared using treatment with vapor-phase hexamethyldisilazane (HMDS). Nanoporous alumina substrates were first made using a two-step anodization process. Subsequently, a repeated modification procedure was employed for efficient incorporation of the terminal methyl groups of HMDS to the alumina surface. Morphology of the surfaces was characterized by scanning electron microscopy, showing hexagonally ordered circular nanopores with approximately 250 nm in diameter and 300 nm of interpore distances. Fourier transform infrared spectroscopy-attenuated total reflectance analysis showed the presence of chemically bound methyl groups on the HMDS-modified nanoporous alumina surfaces. Wetting properties of these surfaces were characterized by measurements of the water contact angle which was found to reach 153.2 ± 2°. The contact angle values on HMDS-modified nanoporous alumina surfaces were found to be significantly larger than the average water contact angle of 82.9 ± 3° on smooth thin film alumina surfaces that underwent the same HMDS modification steps. The difference between the two cases was explained by the Cassie-Baxter theory of rough surface wetting.

Direct measurement of the nonconservative force field generated by optical tweezers.

The force field of optical tweezers is commonly assumed to be conservative, neglecting the complex action of the scattering force. Using a novel method that extracts local forces from trajectories of an optically trapped particle, we measure the three-dimensional force field experienced by a Rayleigh particle with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is nonconservative with the nonconservative component increasing radially away from the optical axis, in agreement with the Gaussian beam model of the optical trap.

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length.

Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length l(p). Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of l(p) on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of l(p) for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 mum, we find a systematic increase of l(p) from 110 to 5,035 mum. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature.

Simplified description of optical forces acting on a nanoparticle in the Gaussian standing wave.

We study the axial force acting on dielectric spherical particles smaller than the trapping wavelength that are placed in the Gaussian standing wave. We derive analytical formulas for immersed particles with relative refractive indices close to unity and compare them with the numerical results obtained by generalized Lorenz-Mie theory (GLMT). We show that the axial optical force depends periodically on the particle size and that the equilibrium position of the particle alternates between the standing-wave antinodes and nodes. For certain particle sizes, gradient forces from the neighboring antinodes cancel each other and disable particle confinement. Using the GLMT we compare maximum axial trapping forces provided by the Gaussian standing-wave trap (SWT) and single-beam trap (SBT) as a function of particle size, refractive index, and beam waist size. We show that the SWT produces axial forces at least ten times stronger and permits particle confinement in a wider range of refractive indices and beam waists compared with those of the SBT.

Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging.

We show that the position of a fluorescent nanoparticle can be measured in three dimensions with subnanometer precision and 100-ms temporal resolution by use of standard epifluorescence video imaging in off-focus mode. The particle can be tracked without feedback in a volume of at least 40 microm x 60 microm x 3 microm. With the technique presented, the structure-mobility relationship of 216-nm latex particle in a porous polymer network was studied in three dimensions.