Enhancing the Strength of an Optical Trap by Truncation

Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances.     

Precise Adsorption Method for Measuring the Percentage of Dead Bacterial Cells

It was observed that Freundlich's law is obeyed when methylene blue is adsorbed from different aqueous solutions by mixtures of dead and live bacterial cells. A simple and precise colorimetric method was developed for determining the percentage of dead cells.     

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.     

A Simple Technique Based on a Single Optical Trap for the Determination of Bacterial Swimming Pattern

Bacterial motility is associated to a wide range of biological processes and it plays a key role in the virulence of many pathogens. Here we describe a method to distinguish the dynamic properties of bacteria by analyzing the statistical functions derived from the trajectories of a bacterium trapped by a single optical beam. The approach is based on the model of the rotation of a solid optically trapped sphere. The technique is easily implemented in a biological laboratory, since with only a small number of optical and electronic components a simple biological microscope can be converted into the required analyzer. To illustrate the functionality of this method, we probed several serovar Typhimurium mutants that differed from the wild-type with respect to their swimming patterns. In a further application, the motility dynamics of the Typhimurium mutant were characterized.     

Evaluation of the Hydrophobicity of Bacterial Cells by Measuring Their Adherence to Chloroform Drops

A simple method for measuring the hydrophobicity of bacterial cells is proposed, which is based on the spectrophotometric evaluation of cell adherence to chloroform drops in a biphasic water–chloroform mixture.     

Theoretical Calculation of Bending Stiffness of Alveolar Wall

The bending stiffness of the alveolar wall is theoretically analyzed in this study through analytical modeling. First, the alveolar wall facet and its characteristics were geometrically simplified and then modeled using known physical laws. Bending stiffness is shown to be dependent on alveolar wall thickness, density, Poisson’s ratio and speed of the longitudinal wave. The normal bending stiffness of the alveolar wall was further determined. For the adult human, the normal bending stiffness is calculated to be 71.0–414.7 nNm, while for the adult mouse it is 1.9–30.0 nNm. The results of this study can be used as a reference for future pulmonary emphysema and fibrosis studies, as the bending stiffness of alveolar wall will be lower and higher, respectively; than the theoretically determined normal values.     

Frequency of Dividing Cells as an Estimator of Bacterial Productivity

It has recently been proposed that the frequency of dividing bacterial cells (FDC) can be used to predict growth rates of natural aquatic bacterial assemblages. We have examined the relationship between FDC and growth rate in bacteria from southern-temperate, coastal marine waters by using incubation under conditions of manipulated nutrient availability and exclusion of bacterivores. The regression of the natural logarithm of bacterial instantaneous growth rate (μ) on FDC resulted in a better fit than regression of untransformed μ on FDC. The regression equation was ln μ = 0.299FDC − 4.961. The coefficient of variation for predicted ln μ at mean FDC was 7%. The range of FDC-estimated bacterial instantaneous generation times for coastal Georgia waters was 12 to 68 h, and range of calculated bacterial production rates was 0.6 to 17.6 mg of C·m⁻³· h⁻¹. Unresolved problems of and suggested improvements on the FDC method of predicting growth rate are discussed.     

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.     

Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.     

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system^{1, 2}; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis³ have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions⁴. Radiation pressure was first observed and applied to optical tweezer systems in 1970^{5, 6}, and was first used to control biological specimens in 1987⁷. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena^8-13.We describe a method¹⁴ that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals^{15, 16} (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.     

Single-Molecule Vibrational Spectroscopy Adds Structural Resolution to the Optical Trap

The ability to apply forces on single molecules with an optical trap is combined with the endogenous structural resolution of Raman spectroscopy in an article in this issue, and applied to measure the Raman spectrum of ds-DNA during force-extension.The resounding success of single-molecule biophysical techniques has encouraged the development of additional tools for more detailed exploration. The unique ability of single-molecule methods to apply force and torque, to disentangle heterogeneity, and to watch equilibrium kinetics would pair beautifully with the ultrafast time resolution and atomistic structural sensitivity of vibrational spectroscopy. However, the weak signal levels endemic to vibrations have left them mostly in the domain of bulk spectroscopy; cross-sections for Raman scattering are typically 10¹⁴ times smaller than for fluorescence emission. In this issue, Rao et al. (1) overcome this gap using surface-enhanced Raman spectroscopy (SERS) (2,3) to add vibrational spectroscopic resolution to their optical trap. In this experiment, a single DNA strand is brought into the near-field vicinity of a silver nanoparticle-coated silica bead that enhances its Raman scattering, and the spectrum is recorded as the DNA is extended in the optical trap. The authors find that applying force shifts the phosphate-stretching vibrational frequency. Molecular dynamics and density functional theory calculations were used to explain these results by showing that external load applied to the DNA backbone induces Ångstrom-level displacements in the P-O bonds.This work is immediately relevant to the communities interested in DNA mechanics and single-molecule Raman spectroscopy. While the authors’ results may refine our structural models for DNA in the low-force regime (1–9 pN), the ongoing debate about the molecular nature of the transition into overstretched DNA (≥65 pN) (4) would be well served by additional structural resolution. For the SERS community, the optical trap provides a fantastic control as it allows one to unambiguously verify that a single-molecule is probed and systematically control its distance and orientation to the metal surface, which may finally resolve long-standing mysteries about the mechanism of SERS. Ideally, both methods will be advanced in concert at the expense of coercing as much information as possible out of a single molecule.While this work is groundbreaking, the real excitement is in its potential. One limitation in most implementations of both single-molecule force and fluorescence spectroscopy is acute sensitivity to distance changes >5 nm, which diminishes upon approaching the subnanometer scale. Raman scattering and infrared absorption vibrational spectroscopies offer a complementary distance sensitivity as molecular oscillators sense their local environment and couple to one another on scales of ∼0.1 nm; see Fig. 1 for a comparison. The optical trap can now be used to initiate specific structural changes to be probed by SERS. In such mechanistic studies, one benefits from the fact that the vibrational spectrum is an endogenous probe, arising from oscillations in all the different bonds present (enzyme as well as substrate), that directly encodes the kinetics and dynamics of structural changes. Such a detailed view of hydrogen-bond rearrangements, covalent-bond formation/breaking, and symmetry changes can offer subtle details that are impossible to tag with fluorophores or directly monitor via a force measurement. As vibrational spectroscopy is rapidly approaching the molecular fingerprinting level with DNA base resolution (5) and protein identification (6), there is an optimistic future for this apparently new multiplexed technique across the various divisions of biophysics.Open in a separate windowFigure 1(A) Examples of mesoscopic structural changes typically underlying single-molecule experiments, such as unfolding of DNA and proteins; translocation of enzymes on a scaffold such as the motor proteins, dynein and kinesin, and replication proteins; and binding of substrates such as ATP and FAD (7,8). (B) Examples of microscopic structural changes probed by bulk vibrational spectroscopy, which may complement single-molecule studies such as hydrogen bonding, isomerization, subtle secondary structural changes such as α-helix rotation and β-sheet reordering, and ligand-binding geometry and kinetics (9–12).     

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-μm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of α₁-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces.MS is the method of choice for identification of peptides in digests of biological samples based on the power of MS to detect the chemically well defined masses of both peptides and their fragments produced by processes such as CID. This high level of structural specificity is also critical in improving peptide (and protein) quantitation because it overcomes the well known problems inherent in classical immunoassays related to limited antibody specificity, dynamic range, and multiplexability. In principle, a quantitative peptide assay using MRM¹ detection in a triple quadrupole mass spectrometer should have nearly absolute structural specificity, a dynamic range of ∼1e+4, and the ability to multiplex measurements of hundreds of peptides per sample (1). These properties suggest that MS-based methods could ultimately replace classical immunoassay technologies in many research and clinical applications.An important limitation of present peptide MRM measurements is sensitivity. The most sensitive widely used quantitative MS platforms use nanoflow chromatography and ESI to deliver trace amounts of peptides to the mass spectrometer. However, these processes are limited in the total amount of peptide that can be applied while retaining maximum sensitivity (typically limited to ∼1 μg of total peptide sample, i.e. the product obtained from digesting ∼14 nl of plasma). The lower cutoff for detecting proteins in a digest of unfractionated plasma by this approach appears to be in the neighborhood of 1–20 μg/ml plasma concentration, which would restrict analysis to the top 100 or so proteins in plasma (1).The sensitivity of MS assays can be substantially increased by fractionating the sample at the level of intact proteins, the tryptic peptides derived from them, or both. For example, immunodepletion of the six most abundant plasma proteins, removes ∼85% of the protein mass (2) and results in an increase of ∼7-fold in the signal-to-noise of MRM measurements of peptides from the remaining proteins after digestion (1). Similarly chromatographic fractionation by strong cation exchange provides another major improvement in sensitivity (3). However, increased sample fractionation brings with it the disadvantages of increased cost and time, the risk of losing specific components, and the continued requirement for very high resolution (lengthy, low throughput) reversed phase nanoflow chromatography en route to the ESI source.An alternative fractionation approach, used in the SISCAPA method, enriches specific target peptides through capture by anti-peptide antibodies, thus circumventing these disadvantages for preselected targets (4). In its initial implementation, SISCAPA used very small (∼10-nl) columns of POROS chromatography support carrying covalently bound rabbit antibodies and provided ∼100-fold enrichment of target peptides with respect to others (4). These columns were, like immunoaffinity depletion columns (2), recyclable many times. However, the potential for sample-to-sample carryover, limitations in the amount of sample digest that could be pumped over nanoaffinity columns at flow rates slow enough to permit peptide binding, and limited flexibility in changing and multiplexing antibodies were problematic. This led us to explore an alternative approach using magnetic beads as the antibody support (5). In this case, the binding reaction can be carried out off line, allowing equilibrium binding; the magnetic beads can be removed from the digest sample and washed; and the bound peptides can be eluted in 96-well plates either manually or using automated equipment such as a KingFisher Magnetic Particle Processor (ThermoFisher). One potential pitfall remains in the handling of eluted peptides. If the anti-peptide antibodies have very high selectivity, as desired in the SISCAPA approach, then in the case of low abundance peptides, only a very small amount of peptide will be eluted from the antibody. Such small amounts of peptide are easily lost through irreversible binding to the walls of vessels such as 96-well plate wells, and the smaller the amount of peptide (i.e. the more specific the capture), the worse the problem may be.To address this issue, we report here a hybrid approach in which peptide binding occurs off line (to equilibrium), whereas the subsequent washing and elution steps are carried out within a capillary that forms part of the nanoflow LC system, thus ensuring that peptide eluted from the antibodies on the beads will not be “lost” between elution and the ESI source. Although there is extensive literature on macroscopic and microfluidic devices for manipulating magnetic beads (6–8) we were unable to find components adaptable to the small scales and high pressures required for integration into nanoflow HPLC. We therefore developed a novel “bead trap” device that satisfies the following requirements: 1) the need to retain beads in a “trap” region against the flow of liquid (loading, wash, and elution buffers for example) in a vessel of capillary dimensions, 2) the need to ensure that beads do not escape from the trap region to contaminate downstream apparatus or columns, 3) the need to ensure that beads are effectively mixed with the flowing fluids (required for efficient washing and elution), and 4) the need to ensure that all beads can be efficiently ejected from the trap region in preparation for a subsequent cycle. The device provides multiple sequential magnetic trapping regions capable of sweeping commonly used 2.8- and 1-μm magnetic beads against liquid flow to prevent escape of beads through the trapping device (i.e. the second downstream trapping zone captures beads swept by the liquid stream past the first trap and so on). In addition, the bead trap device allows the movement of these trapping regions to agitate the trapped bead mass and mix it with fluids flowing past. Finally the device allows reversal of the sweeping action to effectively eject beads from the trap into the fluid stream. The bead trap capillary can be plumbed at various points in conventional nanoflow LC systems (e.g. in place of a sample loop or connecting tube), and the device can be controlled directly by the LC-MS/MS instrument software through contact closures. We show that the bead trap provides an effective method of implementing SISCAPA experiments.     

Bending Stiffness Depends on Curvature of Ternary Lipid Mixture Tubular Membranes

Lipid and protein sorting and trafficking in intracellular pathways maintain cellular function and contribute to organelle homeostasis. Biophysical aspects of membrane shape coupled to sorting have recently received increasing attention. Here we determine membrane tube bending stiffness through measurements of tube radii, and demonstrate that the stiffness of ternary lipid mixtures depends on membrane curvature for a large range of lipid compositions. This observation indicates amplification by curvature of cooperative lipid demixing. We show that curvature-induced demixing increases upon approaching the critical region of a ternary lipid mixture, with qualitative differences along two roughly orthogonal compositional trajectories. Adapting a thermodynamic theory earlier developed by M. Kozlov, we derive an expression that shows the renormalized bending stiffness of an amphiphile mixture membrane tube in contact with a flat reservoir to be a quadratic function of curvature. In this analytical model, the degree of sorting is determined by the ratio of two thermodynamic derivatives. These derivatives are individually interpreted as a driving force and a resistance to curvature sorting. We experimentally show this ratio to vary with composition, and compare the model to sorting by spontaneous curvature. Our results are likely to be relevant to the molecular sorting of membrane components in vivo.     

Formation of Starting Plasma Flow in an Open Trap Using Arc Plasma Gun

Plasma Physics Reports - The system is described for the formation of the low-temperature starting plasma flow in the GOL-NB trap. The starting plasma is a target for capturing heating neutral...     

Rapid Immunocapture of Pseudomonas putida Cells from Lake Water by Using Bacterial Flagella

Monoclonal antibodies to Pseudomonas putida Paw340 cells were produced. In an enzyme-linked immunosorbent assay (ELISA) against whole bacterial cells, a hybridoma cell line termed MLV1 produced a monoclonal antibody that reacted with P. putida Paw340 but showed no cross-reaction with 100 medical isolates and 150 aquatic isolates. By ELISA, immunogold electron microscopy, and Western blot (immunoblot) analysis, MLV1 antibody was found to react with purified bacterial flagella. The surfaces of magnetic polystyrene beads were coated with MLV1 antibody. By mixing MLV1 antibody-coated beads with lake water samples containing the target P. putida host, bead-cell complexes which could be recovered by attraction towards a magnet were formed. Prevention of nonspecific attachment of cells to the beads required the incorporation of detergents in the isolation protocol. These detergents affected colony-forming ability; however, the cells remained intact for direct detection. When reisolated by standard cultural methods, approximately 20% of the initial target population was recovered. Since the beads and bead-cell complexes were recovered in a magnetic field, target bacteria were separated from other lake water organisms and from particulate material which was not attracted towards the magnet and were thereby enriched. This method may now provide a useful system for recovering recombinant bacteria selectively from environmental samples.     

Characterizing Organelles in Live Stem Cells Using Label-Free Optical Diffraction Tomography

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells—namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell—based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.     

Phenanthrene Biodegradation in Soils Using an Antarctic Bacterial Consortium

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in Antarctic soils is limited by low temperatures, lack of adequate levels of nutrients, low number of PAH-tolerant members in the autochthonous microbiota and low bioavailability of contaminants. In the present work, microcosms systems (performed in 1-L glass flasks containing Antarctic soil supplemented with 1744 ppm of phenanthrene) were used to study (i) the effect of biostimulation with a complex organic source of nutrients (fish meal) combined with a surfactant (Brij 700); (ii) the effect of bioaugmentation with a psychrotolerant PAH-degrading bacterial consortium (M10); (iii) the effect of the combination of both strategies. The authors found that combination of biostimulation and bioaugmentation caused a significant removal (46.6%) of phenanthrene after 56 days under Antarctic environmental conditions. When bioaugmentation or biostimulation were applied separately, nonsignificant reduction in phenanthrene concentration was observed. Microtox test showed a low increase in toxicity only in the most efficient system. Results proved that “in situ” bioremediation process of phenanthrene-contaminated soils is possible in Antarctic stations. In addition, inoculation with a psychrotolerant PAH-degrading bacterial consortium in association with a mix of fish meal and a high-molecular-weight surfactant improved phenanthrene removal and should be the selected strategy when the number of hydrocarbons degrading bacteria in the target soil is low.     

Measuring the Characteristic Topography of Brain Stiffness with Magnetic Resonance Elastography

Purpose

To develop a reliable magnetic resonance elastography (MRE)-based method for measuring regional brain stiffness.

Methods

First, simulation studies were used to demonstrate how stiffness measurements can be biased by changes in brain morphometry, such as those due to atrophy. Adaptive postprocessing methods were created that significantly reduce the spatial extent of edge artifacts and eliminate atrophy-related bias. Second, a pipeline for regional brain stiffness measurement was developed and evaluated for test-retest reliability in 10 healthy control subjects.

Results

This technique indicates high test-retest repeatability with a typical coefficient of variation of less than 1% for global brain stiffness and less than 2% for the lobes of the brain and the cerebellum. Furthermore, this study reveals that the brain possesses a characteristic topography of mechanical properties, and also that lobar stiffness measurements tend to correlate with one another within an individual.

Conclusion

The methods presented in this work are resistant to noise- and edge-related biases that are common in the field of brain MRE, demonstrate high test-retest reliability, and provide independent regional stiffness measurements. This pipeline will allow future investigations to measure changes to the brain’s mechanical properties and how they relate to the characteristic topographies that are typical of many neurologic diseases.