Procedure for automated low uncertainty assessment of empty cavity mode frequencies in Fabry-Pérot cavity based refractometry.

A procedure for automated low uncertainty assessment of empty cavity mode frequencies in Fabry-Pérot cavity based refractometry that does not require access to laser frequency measuring instrumentation is presented. It requires a previously well-characterized system regarding mirror phase shifts, Gouy phase, and mode number, and is based on the fact that the assessed refractivity should not change when mode jumps take place. It is demonstrated that the procedure is capable of assessing mode frequencies with an uncertainty of 30 MHz, which, when assessing pressure of nitrogen, corresponds to an uncertainty of 0.3 mPa.

Realization of the pascal based on argon using a Fabry-Perot refractometer.

Based on a recent experimental determination of the static polarizability and a first-principle calculation of the frequency-dependent dipole polarizability of argon, this work presents, by using a Fabry-Perot refractometer operated at 1550 nm, a realization of the SI unit of pressure, the pascal, for pressures up to 100 kPa, with an uncertainty of [(1.0 mPa)2 + (5.8 × 10-6P)2 + (26 × 10-12P2)2]1/2. The work also presents a value of the molar polarizability of N2 at 1550 nm and 302.9146 K of 4.396572(26) × 10-6 m3/mol, which agrees well with previously determined ones.

Demonstration of a Transportable Fabry-Pérot Refractometer by a Ring-Type Comparison of Dead-Weight Pressure Balances at Four European National Metrology Institutes.

Fabry-Pérot-based refractometry has demonstrated the ability to assess gas pressure with high accuracy and has been prophesized to be able to realize the SI unit for pressure, the pascal, based on quantum calculations of the molar polarizabilities of gases. So far, the technology has mostly been limited to well-controlled laboratories. However, recently, an easy-to-use transportable refractometer has been constructed. Although its performance has previously been assessed under well-controlled laboratory conditions, to assess its ability to serve as an actually transportable system, a ring-type comparison addressing various well-characterized pressure balances in the 10-90 kPa range at several European national metrology institutes is presented in this work. It was found that the transportable refractometer is capable of being transported and swiftly set up to be operational with retained performance in a variety of environments. The system could also verify that the pressure balances used within the ring-type comparison agree with each other. These results constitute an important step toward broadening the application areas of FP-based refractometry technology and bringing it within reach of various types of stakeholders, not least within industry.

In situ determination of the penetration depth of mirrors in Fabry-Perot refractometers and its influence on assessment of refractivity and pressure.

A procedure is presented for in situ determination of the frequency penetration depth of coated mirrors in Fabry-Perot (FP) based refractometers and its influence on the assessment of refractivity and pressure. It is based on assessments of the absolute frequency of the laser and the free spectral range of the cavity. The procedure is demonstrated on an Invar-based FP cavity system with high-reflection mirrors working at 1.55 µm. The influence was assessed with such a low uncertainty that it does not significantly contribute to the uncertainties (k = 2) in the assessment of refractivity (<8 × 10-13) or pressure of nitrogen (<0.3 mPa).

The Short-Term Performances of Two Independent Gas Modulated Refractometers for Pressure Assessments.

Refractometry is a powerful technique for pressure assessments that, due to the recent redefinition of the SI system, also offers a new route to realizing the SI unit of pressure, the Pascal. Gas modulation refractometry (GAMOR) is a methodology that has demonstrated an outstanding ability to mitigate the influences of drifts and fluctuations, leading to long-term precision in the 10-7 region. However, its short-term performance, which is of importance for a variety of applications, has not yet been scrutinized. To assess this, we investigated the short-term performance (in terms of precision) of two similar, but independent, dual Fabry-Perot cavity refractometers utilizing the GAMOR methodology. Both systems assessed the same pressure produced by a dead weight piston gauge. That way, their short-term responses were assessed without being compromised by any pressure fluctuations produced by the piston gauge or the gas delivery system. We found that the two refractometer systems have a significantly higher degree of concordance (in the 10-8 range at 1 s) than what either of them has with the piston gauge. This shows that the refractometry systems under scrutiny are capable of assessing rapidly varying pressures (with bandwidths up to 2 Hz) with precision in the 10-8 range.

Invar-based refractometer for pressure assessments.

Gas modulation refractometry (GAMOR) is a methodology that can mitigate fluctuations and drifts in refractometry. This can open up for the use of non-conventional cavity spacer materials. In this paper, we report a dual-cavity system based on Invar that shows better precision for assessment of pressure than a similar system based on Zerodur. This refractometer shows for empty cavity measurements, up to 104 s, a white noise response (for N2) of 3 mPa s1/2. At 4303 Pa, the system has a minimum Allan deviation of 0.34 mPa (0.08 ppm) and a long-term stability (24 h) of 0.7 mPa. This shows that the GAMOR methodology allows for the use of alternative cavity materials.

Realizing Large-Area Arrays of Semiconducting Fullerene Nanostructures with Direct Laser Interference Patterning.

We present a laser interference patterning method for the facile fabrication of large-area and high-contrast arrays of semiconducting fullerene nanostructures, which does not rely on a tedious application of sacrificial photoresists or photomasks. A solution-deposited phenyl-C61-butyric acid methyl ester (PCBM) fullerene thin film is exposed to a spatially modulated illumination intensity, as realized by a two-beam laser interference. The PCBM molecules exposed to strong intensity are photochemically transformed into a low-solubility dimeric state, so that the nontransformed PCBM molecules can be selectively removed in a subsequent solution-based development step. Following brief exposure to green laser light (λ = 532 nm, t = 5 s, p = 0.17 W cm-2) in the designed two-beam interference setup, and a 1 min development in a tuned acetone-chloroform solution, we realize well-defined and ordered PCBM nanostripe patterns with a fwhm line width of ∼200 nm and a repetition rate of ∼2.900 lines mm-1 over a large area of 1 cm2. We demonstrate that a desired high contrast is effectuated because the initial PCBM-dimer transformation rate is dependent on the square of the illumination intensity. The semiconducting functionality of the patterned fullerene is verified in a field-effect transistor experiment, where a typical PCBM nanostripe featured an electron mobility of 5.3 × 10-3 cm2 V-1 s-1 and an on/off ratio of 3 × 103.

Cooke-Triplet tweezers: more compact, robust, and efficient optical tweezers.

We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke-Triplet configuration, allows for continuous beam magnifications of 2-10×, and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke-Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 µm beads shows that the Cooke-Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman-Green interferometer integrated in the Cooke-Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.

Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

Biomechanical and structural features of CS2 fimbriae of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC.

Tethered cells in fluid flows--beyond the Stokes' drag force approach.

Simulations of tethered cells in viscous sub-layers are frequently performed using the Stokes' drag force, but without taking into account contributions from surface corrections, lift forces, buoyancy, the Basset force, the cells' finite inertia, or added mass. In this work, we investigate to what extent such contributions, under a variety of hydrodynamic conditions, influence the force at the anchor point of a tethered cell and the survival probability of a bacterium that is attached to a host by either a slip or a catch bond via a tether with a few different biomechanical properties. We show that a consequence of not including some of these contributions is that the force to which a bond is exposed can be significantly underestimated; in general by ∼32-46%, where the influence of the surface corrections dominate (the parallel and normal correction coefficients contribute ∼5-8 or ∼23-26%, respectively). The Basset force is a major contributor, up to 20%, for larger cells and shear rates. The lift force and inertia contribute when cells with radii >3 µm have shear rates >2000 s(-1). Buoyancy contributes significantly for cells with radii >3 µm for shear rates <10 s(-1). Since the lifetime of a bond depends strongly on the force, both the level of approximation and the biomechanical model of the tether significantly affect the survival probability of tethered bacteria. For a cell attached by a FimH-mannose bond and an extendable tether with a shear rate of 3000 s(-1), neglecting the surface correction coefficients or the Basset force can imply that the survival probability is overestimated by more than an order of magnitude. This work thus shows that in order to quantitatively assess bacterial attachment forces and survival probabilities, both the fluid forces and the tether properties need to be modeled accurately.

Rigid multibody simulation of a helix-like structure: the dynamics of bacterial adhesion pili.

We present a coarse-grained rigid multibody model of a subunit assembled helix-like polymer, e.g., adhesion pili expressed by bacteria, that is capable of describing the polymer's force-extension response. With building blocks representing individual subunits, the model appropriately describes the complex behavior of pili expressed by the gram-negative uropathogenic Escherichia coli bacteria under the action of an external force. Numerical simulations show that the dynamics of the model, which include the effects of both unwinding and rewinding, are in good quantitative agreement with the characteristic force-extension response as observed experimentally for type 1 and P pili. By tuning the model, it is also possible to reproduce the force-extension response in the presence of anti-shaft antibodies, which dramatically changes the mechanical properties. Thus, the model and results in this work give enhanced understanding of how a pilus unwinds under the action of external forces and provide a new perspective of the complex bacterial adhesion processes.

Cell shape identification using digital holographic microscopy.

We present a cost-effective, simple, and fast digital holographic microscopy method based upon Rayleigh-Sommerfeld backpropagation for identification of the geometrical shape of a cell. The method was tested using synthetic hologram images generated by ray-tracing software and from experimental images of semitransparent spherical beads and living red blood cells. Our results show that, by only using the real part of the back-reconstructed amplitude, the proposed method can provide information of the geometrical shape of the object and at the same time accurately determine the axial position of the object under study. The proposed method can be used in flow chamber assays for pathophysiological studies where fast morphological changes of cells are studied in high numbers and at different heights.

The shaft of the type 1 fimbriae regulates an external force to match the FimH catch bond.

Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli to host cells. It has been hypothesized that due to their ability to uncoil under exposure to force, fimbriae can reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work, we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbria varies as a bacterium is affected by a time-dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time-dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ∼120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbria shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments.

Helix-like biopolymers can act as dampers of force for bacteria in flows.

Biopolymers are vital structures for many living organisms; for a variety of bacteria, adhesion polymers play a crucial role for the initiation of colonization. Some bacteria express, on their surface, attachment organelles (pili) that comprise subunits formed into stiff helix-like structures that possess unique biomechanical properties. These helix-like structures possess a high degree of flexibility that gives the biopolymers a unique extendibility. This has been considered beneficial for piliated bacteria adhering to host surfaces in the presence of a fluid flow. We show in this work that helix-like pili have the ability to act as efficient dampers of force that can, for a limited time, lower the load on the force-mediating adhesin-receptor bond on the tip of an individual pilus. The model presented is applied to bacteria adhering with a single pilus of either of the two most common types expressed by uropathogenic Escherichia coli, P or type 1 pili, subjected to realistic flows. The results indicate that for moderate flows (~25 mm/s) the force experienced by the adhesin-receptor interaction at the tip of the pilus can be reduced by a factor of ~6 and ~4, respectively. The uncoiling ability provides a bacterium with a "go with the flow" possibility that acts as a damping. It is surmised that this can be an important factor for the initial part of the adhesion process, in particular in turbulent flows, and thereby be of use for bacteria in their striving to survive a natural defense such as fluid rinsing actions.