Bacterial adhesion to surfaces mediated by specific adhesion organelles that promote infections, as exemplified by the pili of uropathogenic E. coli, is studied mostly at the level of cell-cell interactions and thereby reflects the averaged behavior of multiple pili. The role of pilus rod structure has therefore only been estimated from the outcome of experiments involving large numbers of organelles at the same time. It has, however, lately become clear that the biomechanical behavior of the pilus shafts play an important, albeit hitherto rather unrecognized, role in the adhesion process. For example, it has been observed that shafts from two different strains, even though they are similar in structure, result in large differences in the ability of the bacteria to adhere to their host tissue. However, in order to identify all properties of pilus structures that are of importance in the adhesion process, the biomechanical properties of pili must be assessed at the single-molecule level. Due to the low range of forces of these structures, until recently it was not possible to obtain such information. However, with the development of force-measuring optical tweezers (FMOT) with force resolution in the low piconewton range, it has lately become possible to assess forces mediated by individual pili on single living bacteria in real time. FMOT allows for a more or less detailed mapping of the biomechanical properties of individual pilus shafts, in particular those that are associated with their elongation and contraction under stress. This Mi- nireview presents the FMOT technique, the biological model system, and results from assessment of the biomechanical properties of bacterial pili. The information retrieved is also compared with that obtained by atomic force microscopy.
A model of the elongation of P pili expressed by uropathogenic Escherichia coli exposed to stress is presented. The model is based upon the sticky chain concept, which is based upon Hooke’s law for elongation of the layer-to-layer and head-to-tail bonds between neighboring units in the PapA rod and a kinetic description of the opening and closing of bonds, described by rate equations and an energy landscape model. It provides an accurate description of the elongation behavior of P pili under stress and supports a hypothesis that the PapA rod shows all three basic stereotypes of elongation/unfolding: elongation of bonds in parallel, the zipper mode of unfolding, and elongation and unfolding of bonds in series. The two first elongation regions are dominated by a cooperative bond opening, in which each bond is influenced by its neighbor, whereas the third region can be described by individual bond opening, in which the bonds open and close randomly. A methodology for a swift extraction of model parameters from force-versus-elongation measurements performed under equilibrium conditions is derived. Entities such as the free energy, the stiffness, the elastic elongation, the opening length of the various bonds, and the number of PapA units in the rod are determined.
Surface organelles (so-called pili) expressed on the bacterial membrane mediate the adhesion of Escherichia coli causing urinary tract infection. These pili possess some extraordinary elongation properties that are assumed to allow a close bacterium-to-host contact even in the presence of shear forces caused by urine flow. The elongation properties of P pili have therefore been assessed for low elongation speeds (steady-state conditions). This work reports on the behavior of P pili probed by dynamic force spectroscopy. A kinetic model for the unfolding of a helixlike chain structure is derived and verified. It is shown that the unfolding of the quaternary structure of the PapA rod takes place at a constant force that is almost independent of elongation speed for slow elongations (up to ~0.4 μm/s), whereas it shows a dynamic response with a logarithmic dependence for fast elongations. The results provide information about the energy landscape and reaction rates. The bond length and thermal bond opening and closure rates for the layer-to-layer bond have been assessed to ~0.76 nm, ~0.8 Hz, and ~8 GHz, respectively. The results also support a previously constructed sticky-chain model for elongation of the PapA rod that until now had been experimentally verified only under steady-state conditions.
A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic E. coli (UPEC) under strain are presented. The instrumentation is presented in some detail. Special emphasis is given to measures taken to reduce the influence of noise and drifts in the system and from the surrounding, which makes long term force measurements possible. Individual P pili from UPEC bacteria were used as a biological model system for repetitive unfolding and refolding cycles of bacterial fimbriae under equilibrium conditions. P pili have evolved into a three-dimensional helix-like structure, the PapA rod, that can be successively and significantly elongated and/or unfolded when exposed to external forces. The instrumentation is used for characterization of the force-vs.-elongation response of the PapA rod of individual P pili, with emphasis on the long time stability of the forced unfolding and refolding of the helical structure of the PapA rod. The results show that the PapA rod is capable of withstanding extensive strain, leading to a complete unfolding of the helical structure, repetitive times during the life cycle of a bacterium without any noticeable alteration of the mechanical properties of the P pili. This function is believed to be importance for UPEC bacteria in vivo since it provides a close contact to a host cell (which is an initial step of invasion) despite urine cleaning attempts.
Optical tweezers (OT) are a technique that, by focused laser light, can both manipulate micrometer sized objects and measure minute forces (in the pN range) in biological systems. The technique is therefore suitable for assessment of bacterial adhesion on an individual adhesin-receptor and single attachment organelle (pili) level. This chapter summarizes the use of OT for assessment of adhesion mechanisms of both non-piliated and piliated bacteria. The latter include the important helix-like pili expressed by uropathogenic Escherichia coli (UPEC), which have shown to have unique and intricate biomechanical properties. It is conjectured that the large flexibility of this type of pili allows for a redistribution of an external shear force among several pili, thereby extending the adhesion lifetime of bacteria. Systems with helix-like adhesion organelles may therefore act as dynamic biomechanical machineries, enhancing the ability of bacteria to withstand high shear forces originating from rinsing flows such as in the urinary tract. This implies that pili constitute an important virulence factor and a possible target for future anti-microbial drugs.
Many types of bacterium express micrometer-long attachment organelles (so-called pili) whose role is to mediate adhesion to host tissue. Until recently, little was known about their function in the adhesion process. Force-measuring optical tweezers (FMOT) have since then been used to unravel the biomechanical properties of various types of pili, primarily those from uropathogenic E. coli, in particular their force-vs.-elongation response, but lately also some properties of the adhesin situated at the distal end of the pilus. This knowledge provides an understanding of how piliated bacteria can sustain external shear forces caused by rinsing processes, e.g., urine flow. It has been found that many types of pilus exhibit unique and complex force-vs.-elongation responses. It has been conjectured that their dissimilar properties impose significant differences in their ability to sustain external forces and that different types of pilus therefore have dissimilar predisposition to withstand different types of rinsing conditions. An understanding of these properties is of high importance since it can serve as a basis for finding new means to combat bacterial adhesion, including that caused by antibiotic-resistance bacteria. This work presents a review of the current status of the assessment of biophysical properties of individual pili on single bacteria exposed to strain/stress, primarily by the FMOT technique. It also addresses, for the first time, how the elongation and retraction properties of the rod couple to the adhesive properties of the tip adhesin.
Many types of bacterium express micrometer-long attachment organelles (so called pili) whose role is to mediate adhesion to host tissue. Until recently, little was known about their function in the adhesion process. Forcemeasuring ptical tweezers (FMOT) have since then been used to unravel the iomechanical properties of various types of pili, primarily those from uropathogenic E. coli, in particular their force-vs.-elongation response, but lately also some properties of the adhesin situated and the distal end of the pilus. This knowledge provides an understanding of how piliated bacteria can sustain external shear forces caused by rinsing processes, e.g. urine flow. It has been found that anytypes of pilus exhibit unique and complex force-vs.-elongation responses. It has been conjectured that their dissimilar properties impose significant differences in their ability to sustain external forces and that different types of pilus therefore have dissimilar predisposition to withstand different types of rinsing conditions. An understanding of these properties is of high importance since it can serve as a basis for finding new means to combat bacterial adhesion, including that caused by antibiotic-resistance bacteria. This work presents a review of the current status of the assessment of biophysical properties of individual pili on single bacteria exposed to strain/stress, primarily by the FMOT technique. It also addresses, for the first time, how the elongation and retraction properties of the rod couple to the adhesive properties of the tip adhesin.
Noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) is a sensitive technique for detection of molecular species in gas phase. It is based on a combination of frequency modulation for reduction of noise and cavity enhancement for prolongation of the interaction length between the light and a sample. It is capable of both Doppler-broadened and sub-Doppler detection with absorption sensitivity down to the 10−12 and 10−14 Hz−1/2 cm−1 range, respectively. This chapter provides a thorough description of the basic principles and the performance of the technique.
Noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) is a powerful technique for detection of molecular compounds in gas phase that is based on a combination of two important concepts: frequency modulation spectroscopy (FMS) for reduction of noise, and cavity enhancement, for prolongation of the interaction length between the light and the sample. Due to its unique properties, it has demonstrated unparalleled detection sensitivity when it comes to detection of molecular constituents in the gas phase. However, despite these, it has so far not been used for detection of atoms, i.e. for elemental analysis. The present work presents an assessment of the expected performance of Doppler-broadened (Db) NICE-OHMS for analytical atomic spectrometry, then referred to as noise-immune cavity-enhanced analytical atomic spectrometry (NICE-AAS). After a description of the basic principles of Db-NICE-OHMS, the modulation and detection conditions for optimum performance are identified. Based on a previous demonstrated detection sensitivity of Db-NICE-OHMS of 5×10−12 cm−1 Hz−1∕2 (corresponding to a single-pass absorbance of 7×10−11 over 10 s), the expected limits of detection (LODs) of Hg and Na by NICE-AAS are estimated. Hg is assumed to be detected in gas phase directly while Na is considered to be atomized in a graphite furnace (GF) prior to detection. It is shown that in the absence of spectral interferences, contaminated sample compartments, and optical saturation, it should be feasible to detect Hg down to 10 zg/cm3 (10 fg/m3 or 10-5 ng/m3), which corresponds to 25 atoms/cm3, and Na down to 0.5 zg (zg = zeptogram = 10-21 g), representing 50 zg/mL (parts-per-sextillion, pps, 1:1021) in liquid solution (assuming a sample of 10 µL) or solely 15 atoms injected into the GF, respectively. These LODs are several orders of magnitude lower (better) than any previous laser-based absorption technique previously has demonstrated under atmospheric pressure conditions. It is prophesied that NICE-AAS could provide such high detection sensitivity that the instrumentation should not, by itself, be the limiting factor of an assessment of elemental abundance; the accuracy of an assessment would then instead be limited by concomitant species, e.g. originating from the handling procedures of the sample or the environment.
Gas modulation refractometry (GAMOR) is a methodology for assessment of gas refractivity, molar density, and pressure that, by a rapid gas modulation, exhibits a reduced susceptibility to various types of disturbances. Although previously demonstrated experimentally, no detailed analysis of its ability to reduce the pickup of drifts has yet been given. This work provides an explication of to what extent modulated refractometry in general, and GAMOR in particular, can reduce drifts, predominantly those of the cavity lengths, gas leakages, and outgassing. It is indicated that the methodology is insensitive to the linear parts of so-called campaign-persistent drifts and that it has a significantly reduced susceptibility to others. This makes the methodology suitable for high-accuracy assessments and out-of-laboratory applications.
The main purpose of this article is to describe the theory, instrumentation, and analytical performance of laser analytical atomic spectrometry, focusing on absorption, fluorescence, and ionization techniques. The structure of the article is such that it first provides an outline of the theory of light-matter interactions that prevail for each type of technique as well as of the most common modulation techniques, primarily, wavelength modulation (WM). This is followed by a section that covers the most important types of instrumentation used. The article finally provides a detailed discussion on the specific instrumentation, mode of operation, use, performance, and applications (both analytical and diagnostic) of each technique. In terms of laser atomic absorption, the focus of the discussion is on the use of approaches to enhance the detection capability by either reducing the amount of noise (as is done by the wavelength modulation absorption spectrometry (WMAS) technique) or by providing an extended interaction length (giving rise to cavity-enhanced absorption spectrometry (CEAS) techniques in general, and cavity ring-down spectrometry (CRDS) in particular). Laser atomic fluorescence techniques (often referred to as laser-excited atomic fluorescence spectrometry (LEAFS)) as well as laser ionization techniques (termed either laser-enhanced ionization (LEI) or resonance ionization spectrometry (RIS)) are also covered in some detail. It is shown that several of these techniques are capable of detecting a variety of elements in liquid samples down to low picogram per milliliter (pg mL−1) or parts-per-trillion (ppt) concentrations and in low femtogram amounts. Also other properties of the techniques, e.g. selectivity and linear dynamic range (LDR), are in general superior to those of conventional (nonlaser-based techniques).
An expression for the peak-to-peak sub-Doppler optical phase shift of two counter-propagating modes of light, to which the noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) dispersion signal is proportional, valid for arbitrary degree of saturation, is derived.
For low degrees of saturation it agrees with the expression for weakly saturating (ws) conditions, [(1+S)-1/2-(1+2S)-1/2]α0/2, where S is the degree of saturation and α0 the unsaturated peak absorption.
However, the new expression, which can be written as 0.45S(1+S)-1α0/2, does not predict a distinct maximum as the ws-expression does; instead it predicts an optical phase shift that increases monotonically with S and levels off to 0.45α0/2 for large S. This alters the optimum conditions for the sub-Doppler NICE-OHMS technique and improves its shot-noise-limited detectability.
The new expression is based upon the same explicit assumptions as the ws-expression but not the Kramers-Kronig’s relations, which are not valid for nonlinear responses, and is supported by experimental results up to S = 100. The new expression is expected to be valid for all techniques measuring sub-Doppler dispersion signals
This work examines absorption spectrometry by narrowband light in gaseous media with arbitrary optical thickness when the light induces optical saturation or optical pumping. Two quantities are defined: the observed absorbance, Aobs, and the true absorbance, Atrue. The former is the absorbance that is measured under the existing conditions, whereas the latter represents the absorbance one would measure if the light acted solely as a probe of the populations of the various levels, and it is therefore directly proportional to the concentration or density of absorbers. A general integral equation for the propagation of light in media of arbitrary optical thickness in which the light influences the populations of the levels involved is derived. This expression is transcendental in the observed absorbance and cannot be solved analytically. It is shown that an analytical expression can be derived by investigating the inverse relationship, i.e., Atrue = f(Aobs). Inasmuch as collision and Doppler broadened media react differently to optical saturation, they are considered separately. It is shown that a nonlinear response results if the medium is optically saturated (or pumped) and not optically thin. Expressions for the error introduced if the technique of standard additions is uncritically applied to such a system are derived.
Gas modulation refractometry is a technique for assessment of gas refractivity, density, and pressure that, by a rapid modulation of the gas, provides a means to significantly reduce the pickup of fluctuations. Although its unique feature has previously been demonstrated, no detailed explication or analysis of this ability has yet been given. This work provides a theoretical explanation, in terms of the length of the modulation cycle, of the extent to which gas modulation can reduce the pickup of fluctuations. It is indicated that a rapid modulation can significantly reduce the influence of fluctuations with Fourier frequencies lower than the inverse of the modulation cycle length, which often are those that dominate. The predictions are confirmed experimentally.
A technique for high-precision and high-accuracy assessment of both gas molar (and number) density and pressure, Gas Modulation Refractometry (GAMOR), is presented. The technique achieves its properties by assessing refractivity as a shift of a directly measurable beat frequency by use of Fabry-Perot cavity (FPC) based refractometry utilizing the Pound-Drever-Hall laser locking technique. Conventional FPC-based refractometry is, however, often limited by fluctuations and drifts of the FPC. GAMOR remedies this by an additional utilization of a gas modulation methodology, built upon a repeated filling and evacuation of the measurement cavity together with an interpolation of the empty cavity responses. The procedure has demonstrated an ability to reduce the influence of drifts in a non-temperature stabilized dual-FPC (DFPC)-based refractometry system, when assessing pressure, by more than three orders of magnitude. When applied to a DFPC system with active temperature stabilization, it has demonstrated, for assessment of pressure of N2 at 4304 Pa at room temperature, which corresponds to a gas molar density of 1.7 × 10−6 mol/cm3, a sub-0.1 ppm precision (i.e. a resolution of 0.34 mPa). It is claimed that the ability to assess gas molar density is at least as good as so far has been demonstrated for pressure (i.e. for the molar density addressed, a resolution of at least 1.2 × 10−13 mol/cm3). It has recently been argued that the methodology should be capable of providing an accuracy that is in the low ppm range. These levels of precision and accuracy are unprecedented among laser-based techniques for detection of atomic and molecular species. Since the molar polarizability of He can be calculated by ab initio quantum mechanical calculations with sub-ppm accuracy, it can also be used as a primary or semi-primary standard of both gas molar (and number) density and pressure.
It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.
A number of biomechanical properties of various types of pili expressed by Escherichia coli, predominantly their force–vs.–elongation behavior, have previously been assessed in detail on a single pilus level. In vivo, however, bacteria bind in general to host cells by a multitude of pili, which presumably provides them with adhesion properties that differs from those of single pili. Based upon the previously assessed biomechanical properties of individual pili, this work presents a theoretical analysis of the adhesion properties of multipili–attaching bacteria expressing helix–like pili exposed to an external force. Expressions for the adhesion lifetime of dual– and multipili–attaching bacteria are derived and their validity is verified by Monte Carlo simulations. It is shown that the adhesion lifetime of a multipili–binding bacterium depends to a large degree on the cooperativity of the attaching pili, which, in turn, depends strongly on their internal biomechanical properties, in particular their helix–like structure and its ability to elongate, which, in turn, depend on the intrinsic properties of the bonds, e.g. their lengths and activation energies. It is shown, for example, that a decrease in the length of a layer–to–layer bond in the rod of P pili, expressed by E. coli, by 50 % leads to a decrease in the adhesion lifetime of a bacterium attaching by 10 pili and exposed to a force of 500 pN by three orders of magnitude. The results indicate moreover that the intrinsic properties of the rod for this particular type of pili are optimized for multipili attachment under a broad range of external forces and presumably also to its in vivo environment. Even though the results presented in this work apply quantitatively to one type of pilus, they are assumed to apply qualitatively to all helix–like pili systems expressing slip bonds.
P pili are fimbrial adhesion organelles expressed by uropathogenic Escherichia coli in the upper urinary tract. They constitute a stiff helix-like polymer consisting of a number of subunits joined by head-to-tail bonds. The elongation and retraction properties of individual P pili exposed to strain have been modeled by Monte Carlo (MC) simulations. The simulation model is based upon a three-state energy landscape that deforms under an applied force. Bond opening and closure are modeled by Bells theory while the elongation of the linearized part of the pilus is described by a worm-like chain model. The simulations are compared with measurements made by force measuring optical tweezers. It was found that the simulations can reproduce pili elongation as well as retraction, under both equilibrium and dynamic conditions, including entropic effects. It is shown that the simulations allow for an assessment of various model parameters, e.g. the unfolding force, energy barrier heights, and various distances in the energy landscape, including their stochastic spread that analytical models are unable to do. The results demonstrate that MC simulations are useful to model elongation and retraction properties of P pili, and therefore presumably also other types of pili, exposed to strain and/or stress. MC simulations are particularly suited for description of helix-like pili since these have an intricate self-regulating mechanical elongation behavior that makes analytical descriptions non-trivial when dynamic processes are studied, or if additional interactions in the rod or the behavior of the adhesion tip needs to be modeled.
Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of approximately 100 pNs, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25+/-1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.
Uropathogenic Escherichia coli (UPEC) expressvarious kinds of organelles, so-called pili or fimbriae, thatmediate adhesion to host tissue in the urinary tract throughspecific receptor-adhesin interactions. The biomechanicalproperties of these pili have been considered important forthe ability of bacteria to withstand shear forces from rinsingurine flows. Force-measuring optical tweezers have beenused to characterize individual organelles of F1C typeexpressed by UPEC bacteria with respect to such properties.Qualitatively, the force-versus-elongation response wasfound to be similar to that of other types of helix-like piliexpressed by UPEC, i.e., type 1, P, and S, with force-inducedelongation in three regions, one of which represents theimportant uncoiling mechanism of the helix-like quaternarystructure. Quantitatively, the steady-state uncoiling forcewas assessed as 26.4 ±1.4 pN, which is similar to those ofother pili (which range from 21 pN for SI to 30 pN for type 1).The corner velocity for dynamic response (1,400 nm/s) wasfound to be larger than those of the other pili (400–700 nm/sfor S and P pili, and 6 nm/s for type 1). The kinetics werefound to be faster, with a thermal opening rate of 17 Hz, afew times higher than S and P pili, and three orders ofmagnitude higher than type 1. These data suggest that F1Cpili are, like P and S pili, evolutionarily selected to primarilywithstand the conditions expressed in the upper urinary tract.
Bacterial adhesion organelles, known as fimbria or pili, are expressed by Gram–positive as well as Gram–negative bacteria families. These appendages play a key role in the first steps of the invasion and infection processes, and they therefore provide bacteria with pathogenic abilities. To improve the knowledge of pili-mediated bacterial adhesion to host cells and how these pili behave under the presence of an external force, we first characterize, using force measuring optical tweezers, open coil-like T4 pili expressed by Gram–positive Streptococcus pneumoniae with respect to their biomechanicalproperties. It is shown that their elongation behavior can be well described by the worm-like chain model and that they possess a large degree of flexibility. Their properties are then compared with those of helix-like pili expressed by Gram–negative uropathogenic Escherichia coli (UPEC), which have different pili architecture. The differences suggest that these two types of pili have distinctly dissimilar mechanisms to adhere and sustain external forces. Helix-like pili expressed by UPEC bacteria adhere to host cells by single adhesins located at the distal end of the pili while their helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow and to increase the cooperativity of the pili ensemble. Open coil-like pili expressed by S. pneumoniae adhere to cells by a multitude of adhesins distributed along the pili. It is hypothesized that these two types of pili represent different strategies of adhering to host cells in the presence of external forces. When exposed to significant forces, bacteria expressing helix-like pili remain attached bydistributing the external force among a multitude of pili, whereas bacteria expressing open coil-like pili sustain large forces primarily by their multitude of binding adhesins.
We combine an external cavity diode laser with noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) using current modulation. With a finesse of 1600, we demonstrate noise equivalent absorption sensitivities of 4.1 x 10(-10) cm(-1) Hz(-1/2), resulting in sub-ppbv detection limits for Doppler-broadened transitions of CH4 at 6132.3 cm(-1), C2H2 at 6578.5 cm(-1) and HCN at 6541.7 cm(-1). The system is used for hydrogen cyanide detection from sweet almonds.
The detection sensitivity of phase-modulated techniques such as frequency-modulation spectroscopy (FMS) and noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) is often limited by etalon background signals. It has previously been shown that the impact of etalons can be reduced by the use of etalon-immune distances (EIDs), i.e., by separating the surfaces that give rise to etalons by a distance of q. L-m, where L-m is given by c/2n nu(m), where, in turn, n and nu(m) are the index of refraction between the components that make up the etalon (thus most often that of air) and the modulation frequency, respectively, and where q is an integer (i.e., 1, 2, 3,.) or half-integer (i.e., 1/2, 1, 3/2,.) for the dispersion and absorption modes of detection, respectively. An etalon created by surfaces separated by an EID will evade detection and thereby not contribute to any background signal. The concept of EIDs in FMS and NICE-OHMS is in this work demonstrated, scrutinized, and discussed in some detail. It is shown that the influence of EIDs on the absorption and dispersion modes of detection is significantly different; signals detected at the dispersion phase are considerably less sensitive to deviations from exact EID conditions than those detected at the absorption phase. For example, the FM background signal from an etalon whose length deviates from an EID by 2.5% of L-m (e.g., by 1 cm for an L-m of 40 cm), detected in dispersion, is only 9% of that in absorption. This makes the former mode of detection the preferred one whenever a sturdy immunity against etalons is needed or when optical components with parallel surfaces (e.g., lenses, polarizers, or beam splitters) are used. The impact of the concept of EIDs on NICE-OHMS is demonstrated by the use of Allan-Werle plots.
Doppler broadened noise-immune cavity-enhanced optical heterodyne molecular spectrometry (Db-NICE-OHMS) has been scrutinized with respect to modulation and demodulation conditions (encompassing the modulation frequency,nu(m), the modulation index, beta, and the detection phase, theta), the cavity length, L, and the modulation order, k (defined as nu(m)/nu(FSR), where nu(FSR) is the free-spectral range of the cavity), primarily in the Doppler limit but also for two specific situations in the Voigt regime (for equal Doppler and homogeneous width and for purely Lorentzian broadened transitions), both in the absence and presence of optical saturation (the latter for the case in which the homogeneous broadening is smaller than the modulation frequency). It is found that, for a system with a given cavity length, the optimum conditions (i.e., those that produce the largest NICE-OHMS signal) for an unsaturated transition in the Doppler limit comprise nu(m)/Gamma(D) = 1.6 (where Gamma(D) is the half-width at half-maximum of the Doppler width of the transition), beta = 1.3, and theta = 0.78 pi. It is also found that the maximum is rather broad; the signal takes 95% of its maximum value for modulation frequencies in the entire 0.4 less than or similar to nu(m)/Gamma(D) less than or similar to 2.4 range. When optical saturation sets in, theta shifts toward the dispersion phase. The optimum conditions encompass k > 1 whenever L > 0.35L(D) and 2.6L(D) for the dispersion and absorption modes of detection, respectively [where L-D is a characteristic length given by c/(2 Gamma(D))]. Similar conditions are found under pressure broadened conditions.
A fiber-laser-based noise-immune cavity-enhanced optical heterodyne molecular spectrometry (FL-NICE-OHMS) system for white-noise-limited Doppler-broadened detection down to 5.6 x 10(-12) cm(-1) Hz(-1/2) is demonstrated. The system is based on a previous FL-NICE-OHMS instrumentation in which the locking of the laser frequency to a cavity mode has been improved by the use of an acousto-optic modulator (AOM) and provision of a more stable environment by the employment of a noise-isolating enclosed double-layer table, a temperature regulation of the laboratory, and an ultra-high-vacuum (UHV) gas system. White-noise behavior up to 10 s provides the instrument with a minimum detectable on-resonance absorbance per unit length of 1.8 x 10(-12) cm(-1) and a relative single-pass absorption (Delta I/I) of 7.2 x 10(-11). The system was applied to detection of acetylene on a transition at 1531.588 nm, yielding a detection sensitivity of C2H2 in atmospheric pressure gas of 4 ppt (measured over 10 s). (C) 2012 Optical Society of America
To reduce the complexity of fiber-laser-based noise-immune cavity-enhanced optical heterodyne molecular spectrometry, a system incorporating a fiber-coupled optical circulator to deflect the cavity-reflected light for laser stabilization has been realized. Detection near the shot-noise limit has been demonstrated for both Doppler-broadened and sub-Doppler signals, yielding a lowest detectable absorption and optical phase shift of 2.2 x 10(-12) cm(-1) and 4.0 x 10(-12) cm(-1), respectively, both for a 10 s integration time, where the former corresponds to a detection limit of C2H2 of 5 ppt. (C) 2014 Optical Society of America
The dependence of Doppler broadened noise-immune cavity-enhanced optical heterodyne molecular spectrometry (NICE-OHMS) on the modulation index, beta, has been investigated experimentally on C2H2 and CO2, both in the absence and the presence of optical saturation. It is shown that the maximum signals are obtained for beta that produce more than one pair of sidebands: in the Doppler limit and for the prevailing conditions (unsaturated transition and the pertinent modulation frequency and Doppler widths) around 1 and 1.4 for the dispersion and absorption detection phases, respectively. The results verify predictions given in an accompanying work. It is also shown that there is no substantial broadening of the NICE-OHMS signal for beta < 1. The use of beta of unity has yielded a Db-NICE-OHMS detection sensitivity of 4.9 x 10(-12) cm(-1) Hz(-1/2), which is the lowest (best) value so far achieved for NICE-OHMS based on a tunable laser. The number of sidebands that needs to be included in fits of the line-shape function to obtain good accuracy has been assessed. It is concluded that it is enough to consider three pairs of sidebands whenever the systematic errors in a concentration assessment should be below 1% when beta < 2 are used and <1 parts per thousand for beta < 1.5.
The potential of fiber-laser-based sub-Doppler noise-immune cavity-enhanced optical heterodyne molecular spectrometry for trace gas detection is scrutinized. The non-linear dependence of the on-resonance sub-Doppler dispersion signal on the intracavity pressure and power is investigated and the optimum conditions with respect to these are determined. The linearity of the signal strength with concentration is demonstrated and the dynamic range of the technique is discussed. Measurements were performed on C2H2 at 1531 nm up to degrees of saturation of 100. The minimum detectable sub-Doppler optical phase shift was 5 x 10-11 cm-1 Hz-1/2, corresponding to a partial pressure of C2H2 of 1 x 10-12 atm for an intracavity pressure of 20 mTorr, and a concentration of 10 ppb at 400 mTorr.
The influence of optical saturation on noise-immune cavity-enhanced optical heterodyne molecular spectrometry (NICE-OHMS) signals from purely Doppler-broadened transitions is investigated experimentally. It is shown that the shape and the strength of the dispersion signal are virtually unaffected by optical saturation, whereas the strength of the absorption signal decreases as (1+G+-1)-1/2, where G+-1 is the degree of saturation induced by the sideband of the frequency modulated triplet, in agreement with theoretical predictions. This implies, first of all, that Doppler-broadened NICE-OHMS is affected less by optical saturation than other cavity enhanced techniques but also that it exhibits nonlinearities in the power and pressure dependence for all detection phases except pure dispersion. A methodology for assessments of the degree of saturation and the saturation power of a transition from Doppler-broadened NICE-OHMS signals is given. The implications of optical saturation for practical trace species detection by Doppler-broadened NICE-OHMS are discussed.
A thorough analysis of the shape and strength of Doppler-broadened wavelength modulated noise-immune cavity-enhanced optical heterodyne molecular spectroscopy signals is presented and their dependence on modulation frequency, modulation amplitude and detection phase is investigated in detail. The conditions that maximize the on-resonance signal are identified. The analysis is based on the standard frequency modulation spectroscopy formalism and the Fourier description of wavelength modulation spectroscopy and verified by fits to experimental signals from C2H2 and CO2 measured at 1531 nm. In addition, the line strengths of two CO2 transitions in the v2→3v1+v2+v3 hot band [Pe(7) and Pe(9)] were found to differ by ~20% from those given in the HITRAN database.
Optical-optical double-resonance spectroscopy with a continuous wave pump and frequency comb probe allows measurement of sub-Doppler transitions to highly excited molecular states over a wide spectral range with high frequency accuracy. We report on assessment and characterization of sub-Doppler double-resonance transitions in methane measured using a 3.3-μm continuous wave optical parametric oscillator as a pump and a 1.67-μm frequency comb as a probe. The comb spectra were recorded using a Fourier transform spectrometer with comb-mode-limited resolution. With the pump tuned to nine different transitions in the ν3 fundamental band, we detected 36 ladder-type transitions to the 3ν3 overtone band region, and 18 V-type transitions to the 2ν3 overtone band. We describe in detail the experimental approach and the pump stabilization scheme, which currently limits the frequency accuracy of the measurement. We present the data analysis procedure used to extract the frequencies and intensities of the probe transitions for parallel and perpendicular relative pump-probe polarization. We compare the center frequencies and relative intensities of the ladder-type transitions to theoretical predictions from the TheoReTS and ExoMol line lists, demonstrating good agreement with TheoReTS.
We report the first measurement of sub-Doppler molecular response using a frequency comb by employing the comb as a probe in optical-optical double-resonance spectroscopy. We use a 3.3 μm continuous wave pump and a 1.67 μm comb probe to detect sub-Doppler transitions to the 2ν3 and 3ν3 bands of methane with ∼1.7 MHz center frequency accuracy. These measurements provide the first verification of the accuracy of theoretical predictions from highly vibrationally excited states, needed to model the high-temperature spectra of exoplanets. Transition frequencies to the 3ν3 band show good agreement with the TheoReTS line list.
We use a 3.3 µm continuous wave optical parametric oscillator as a pump and a 1.67 µm frequency comb as a probe to record 36 sub-Doppler double-resonance transitions in the 3v3 band of methane (including 26 previously unreported) with ~1.5 MHz center frequency accuracy.
We use a 3.3 μm continuous wave optical parametric oscillator as a pump and a 1.67 μm frequency comb as a probe to record 36 sub-Doppler double-resonance transitions in the 3v3 band of methane (including 26 previously unreported) with ∼1.5 MHz center frequency accuracy.
A theoretical investigation of the influence of optical pumping on wavelength modulation absorption spectrometry (WMAS) signals from collision broadened atoms and molecules is presented. General expressions for the nf-WMAS signal from atomic and molecular systems, modeled as three-level systems that can accommodate both optical saturation and optical pumping, are derived by the use of a previously developed Fourier series-based formalism in combination with rate equations solved under steady-state conditions. The expressions are similar to those describing the nf-WMAS signal from two-level systems that can accommodate optical saturation [Schmidt FM, Foltynowicz A, Gustafsson J, Axner O, WMAS from optically saturated collision-broadened transitions. JQSRT 2005;94:225–54], the difference being the value of the saturation flux, wherefore the general parametric dependence of WMAS signals from optically pumped systems is the same as that from optically saturated systems. The additional effect of optical pumping on the WMAS signal is investigated for three typical cases: molecules or atoms in an ordinary atmosphere, atoms in an inert atmosphere, and atoms or molecules possessing metastable states. The possibility to describe any of these systems with a two-level model is investigated.
As a result of a combination of an external cavity and modulation techniques, noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) is one of the most sensitive absorption techniques, capable of reaching close-to-shot-noise sensitivities, down to 5×10-13 fractional absorption at 1 s averaging. Due to its ability to provide sub-Doppler signals from weak molecular overtone transitions, the technique was first developed for frequency standard applications. It has since then also found use in fields of molecular spectroscopy of weak overtone transitions and trace gas detection. This paper describes the principles and the unique properties of NICE-OHMS. The historical background, the contributions of various groups, as well as the performance and present status of the technique are reviewed. Recent progress is highlighted and the future potential of the technique for trace species detection is discussed.
A compact noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) system based on a narrow linewidth distributed-feedback laser and fiber-coupled acousto-optic and electro-optic modulators has been developed. Measurements of absorption and dispersion signals have been performed at pressures up to 1/3 atmosphere on weak acetylene transitions at 1551 nm. Multiline fitting routines were implemented to obtain transition parameters, i.e., center frequencies, linestrengths, and pressure broadening coefficients. The signal strength was shown to be linear with pressure and concentration, and independent of detection phase. The minimum detectable on-resonance absorption with a cavity with a finesse of 460 was 2 × 10−10 cm−1 for 1 minute of integration time.
Noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) based on a fiber-coupled electro-optic modulator (EOM) provides a compact and versatile experimental setup. It has, however, been limited by background signals originating from an imbalance of the phase modulated triplet created by a cross-coupling between the principal axes of the polarization maintaining fibers and the extraordinary axis of the EOM. Two strategies for reducing these background signals are investigated: (i) using an EOM with a titanium diffused waveguide, in which the balance of the triplet is controlled by active feedback, and (ii) using an EOM with a proton exchanged waveguide that does not support light propagation along the ordinary axis. It is shown that both approaches significantly reduce drifts and noise in the system. Using a cavity with a finesse of 5700, an absorption sensitivity of 3: 2 x 10(-12) cm(-1) in 1 min of integration time (i.e., 1: 8 x 10(-11) cm(-1) Hz(-1/2)) is demonstrated for Doppler-broadened detection, the lowest reported so far for Doppler-broadened NICE-OHMS. For sub-Doppler detection, a minimum detectable optical phase shift of 1: 3 x 10(-12) cm(-1) in 400s of integration time is obtained. (C) 2011 Optical Society of America
A transportable refractometer for assessment of kPa pressures with ppm level precision is presented. It is based on the GAs MOdulation Refractometry (GAMOR) methodology, making it resistant to fluctuations and drifts. At the National Metrology Institute at RISE, Sweden, the system assessed pressures in the 4.3 - 8.7 kPa range with sub-ppm precision (0.5 - 0.9 ppm). The system was thereafter disassembled, packed, and transported 1040 km to Umeå University, where it, after unpacking and reassembling, demonstrated a similar precision (0.8 - 2.1 ppm). This shows that the system can be disassembled, packed, transported, unpacked, and reassembled with virtually unchanged performance.
Using a transportable Fabry-Pérot cavity refractometer, a circular comparison of existing primary standards at several national metrology institutes is currently underway. This paper provides information about the refractometer, the preparation for the comparison, and the transportation procedure.
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.
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.