Mutation supply and the repeatability of selection for antibiotic resistance.

Whether evolution can be predicted is a key question in evolutionary biology. Here we set out to better understand the repeatability of evolution, which is a necessary condition for predictability. We explored experimentally the effect of mutation supply and the strength of selective pressure on the repeatability of selection from standing genetic variation. Different sizes of mutant libraries of antibiotic resistance gene TEM-1 ß-lactamase in Escherichia coli, generated by error-prone PCR, were subjected to different antibiotic concentrations. We determined whether populations went extinct or survived, and sequenced the TEM gene of the surviving populations. The distribution of mutations per allele in our mutant libraries followed a Poisson distribution. Extinction patterns could be explained by a simple stochastic model that assumed the sampling of beneficial mutations was key for survival. In most surviving populations, alleles containing at least one known large-effect beneficial mutation were present. These genotype data also support a model which only invokes sampling effects to describe the occurrence of alleles containing large-effect driver mutations. Hence, evolution is largely predictable given cursory knowledge of mutational fitness effects, the mutation rate and population size. There were no clear trends in the repeatability of selected mutants when we considered all mutations present. However, when only known large-effect mutations were considered, the outcome of selection is less repeatable for large libraries, in contrast to expectations. We show experimentally that alleles carrying multiple mutations selected from large libraries confer higher resistance levels relative to alleles with only a known large-effect mutation, suggesting that the scarcity of high-resistance alleles carrying multiple mutations may contribute to the decrease in repeatability at large library sizes.

On the importance of image formation optics in the design of infrared spectroscopic imaging systems.

Infrared spectroscopic imaging provides micron-scale spatial resolution with molecular contrast. While recent work demonstrates that sample morphology affects the recorded spectrum, considerably less attention has been focused on the effects of the optics, including the condenser and objective. This analysis is extremely important, since it will be possible to understand effects on recorded data and provides insight for reducing optical effects through rigorous microscope design. Here, we present a theoretical description and experimental results that demonstrate the effects of commonly-employed cassegranian optics on recorded spectra. We first combine an explicit model of image formation and a method for quantifying and visualizing the deviations in recorded spectra as a function of microscope optics. We then verify these simulations with measurements obtained from spatially heterogeneous samples. The deviation of the computed spectrum from the ideal case is quantified via a map which we call a deviation map. The deviation map is obtained as a function of optical elements by systematic simulations. Examination of deviation maps demonstrates that the optimal optical configuration for minimal deviation is contrary to prevailing practice in which throughput is maximized for an instrument without a sample. This report should be helpful for understanding recorded spectra as a function of the optics, the analytical limits of recorded data determined by the optical design, and potential routes for optimization of imaging systems.

Rapid spectral-domain localization.

We present a method to dynamically image structures at nanometer spatial resolution with far-field instruments. We propose the use of engineered nanoprobes with distinguishable spectral responses and the measurement of coherent scattering, rather than fluorescence. Approaches such as PALM/STORM have relied on the rarity of emission events in time to distinguish signals from distinct probes. By distinguishing signals in the spectral domain, we enable the acquisition of data in a multiplex fashion and thus circumvent the fundamental problem of slow data acquisition of current techniques. The described method has the potential to image dynamic systems with a spatial resolution only limited to the size of the scattering probes.

Coherence effects in Mie scattering.

The scattering of a partially coherent beam by a deterministic, spherical scatterer is studied. In particular, the Mie scattering by a Gaussian Schell-model beam is analyzed. Expressions are derived for (a) the extinguished power, (b) the radiant intensity of the scattered field, and (c) the encircled energy in the far field. It is found that the radiant intensity and the encircled energy in the far field depend on the degree of coherence of the incident beam, whereas the extinguished power does not.

In vivo Raman spectroscopy for bladder cancer detection using a superficial Raman probe compared to a nonsuperficial Raman probe.

Raman spectroscopy is promising as a noninvasive tool for cancer diagnosis. A superficial Raman probe might improve the classification of bladder cancer, because information is gained solely from the diseased tissue and irrelevant information from deeper layers is omitted. We compared Raman measurements of a superficial to a nonsuperficial probe, in bladder cancer diagnosis. Two-hundred sixteen Raman measurements and biopsies were taken in vivo from at least one suspicious and one unsuspicious bladder location in 104 patients. A Raman classification model was constructed based on histopathology, using a principal-component fed linear-discriminant-analysis and leave-one-person-out cross-validation. The diagnostic ability measured in area under the receiver operating characteristics curve was 0.95 and 0.80, the sensitivity was 90% and 85% and the specificity was 87% and 88% for the superficial and the nonsuperficial probe, respectively. We found inflammation to be a confounder and additionally we found a gradual transition from benign to low-grade to high-grade urothelial carcinoma. Raman spectroscopy provides additional information to histopathology and the diagnostic value using a superficial probe.

The Pancharatnam-Berry phase for non-cyclic polarization changes.

We present a novel setup that allows the observation of the geometric phase that accompanies polarization changes in monochromatic light beams for which the initial and final states are different (so-called non-cyclic changes). This Pancharatnam-Berry phase can depend in a linear or in a nonlinear fashion on the orientation of the optical elements, and sometimes the dependence is singular. Experimental results that confirm these three types of behavior are presented. The observed singular behavior may be applied in the design of optical switches.

Experimental demonstration of an intensity minimum at the focus of a laser beam created by spatial coherence: application to the optical trapping of dielectric particles.

In trying to manipulate the intensity distribution of a focused field, one typically uses amplitude or phase masks. Here we explore an approach, namely, varying the state of spatial coherence of the incident field. We experimentally demonstrate that the focusing of a Bessel-correlated beam produces an intensity minimum at the geometric focus rather than a maximum. By varying the spatial coherence width of the field, which can be achieved by merely changing the size of an iris, it is possible to change this minimum into a maximum in a continuous manner. This method can be used, for example, in novel optical trapping schemes, to selectively manipulate particles with either a low or high index of refraction.

Effects of spatial coherence on the angular distribution of radiant intensity generated by scattering on a sphere.

In the analysis of light scattering on a sphere it is implicitly assumed that the incident field is spatially fully coherent. However, under usual circumstances the field is partially coherent. We generalize the partial waves expansion method to this situation and examine the influence of the degree of coherence of the incident field on the radiant intensity of the scattered field in the far zone. We show that when the coherence length of the incident field is comparable to, or is smaller than, the radius of the sphere, the angular distribution of the radiant intensity depends strongly on the degree of coherence. The results have implications, for example, for scattering in the atmosphere and colloidal suspensions.

Geometric interpretation of the Pancharatnam connection and non-cyclic polarization changes.

If the state of polarization of a monochromatic light beam is changed in a cyclical manner, the beam acquires-in addition to the usual dynamic phase-a geometric phase. This geometric or Pancharatnam-Berry phase equals half the solid angle of the contour traced out on the Poincaré sphere. We show that such a geometric interpretation also exists for the Pancharatnam connection, the criterion according to which two beams with different polarization states are said to be in phase. This interpretation offers what is to our knowledge a new and intuitive method to calculate the geometric phase that accompanies non-cyclic polarization changes.

BIM-Sim: Interactive Simulation of Broadband Imaging Using Mie Theory.

Understanding the structure of a scattered electromagnetic (EM) field is critical to improving the imaging process. Mechanisms such as diffraction, scattering, and interference affect an image, limiting the resolution, and potentially introducing artifacts. Simulation and visualization of scattered fields thus plays an important role in imaging science. However, EM fields are high-dimensional, making them time-consuming to simulate, and difficult to visualize. In this paper, we present a framework for interactively computing and visualizing EM fields scattered by micro and nano-particles. Our software uses graphics hardware for evaluating the field both inside and outside of these particles. We then use Monte-Carlo sampling to reconstruct and visualize the three-dimensional structure of the field, spectral profiles at individual points, the structure of the field at the surface of the particle, and the resulting image produced by an optical system.

Test-retest reliability of skeletal muscle oxygenation measurements during submaximal cycling exercise in patients with chronic heart failure.

The potential purpose of near-infrared spectroscopy (NIRS) as a clinical application in patients with chronic heart failure (CHF) is the identification of limitations in O2 delivery or utilization during exercise. The objective of this study was to evaluate absolute and relative test-retest reliability of skeletal muscle oxygenation measurements in patients with CHF. Thirty patients with systolic heart failure (left ventricular ejection fraction 31 ± 8%) performed 6-min constant-load cycling tests at 80% of the anaerobic threshold (AT) with tissue saturation index (TSI) measurement at the vastus lateralis. Tests were repeated after 10 ± 5 days to evaluate reliability. Absolute reliability was assessed with limits of agreement (LoA, expressed as bias ± random error) and coefficients of variation (CV) for absolute values (LoA range: 0·4 ± 6·2% to 0·6 ± 7·9%; CV range: 4·7-7·1%), amplitudes (LoA range -0·5 ± 5·8% to -0·7 ± 6·8%; CV range: 26·2-42·1%), onset and recovery kinetics (mean response times; LoA 0·4 ± 9·5 s, CV 23·5% and LoA -5·8 ± 50·8 s, CV 67·4% respectively) and overshoot characteristics (CV range 45·7-208·6%). Relative reliability was assessed with intraclass correlation coefficients for absolute values (range 0·74-0·90), amplitudes (range 0·85-0·92), onset and recovery kinetics (0·53 and 0·51, respectively) and overshoot characteristics (range 0·17-0·74). In conclusion, absolute reliability of absolute values and onset kinetics seems acceptable for serial within-subject comparison, and as such, for evaluation of treatment effects. Absolute reliability of amplitudes and recovery kinetics is considered unsatisfactory. Relative reliability of absolute values and amplitudes is sufficient for purposes of physiological distinction between patients with CHF. Despite lower relative reliability, kinetics may still be useful for clinical application.

The influence of adipose tissue on spatially resolved near-infrared spectroscopy derived skeletal muscle oxygenation: the extent of the problem.

OBJECTIVE: Near-infrared spectroscopy (NIRS) measurements of tissue oxygen saturation (StO2) are useful for the assessment of skeletal muscle perfusion and function during exercise, however, they are influenced by overlying skin and adipose tissue. This study explored the extent and nature of the influence of adipose tissue thickness (ATT) on StO2. APPROACH: NIR spatially resolved spectroscopy (SRS) derived oxygenation was measured on vastus lateralis in 56 patients with chronic heart failure (CHF) and 20 healthy control (HC) subjects during rest and moderate intensity exercise with simultaneous assessment of oxygen uptake kinetics (τ [Formula: see text]). In vitro measurements were performed on a flow cell with a blood mixture with full oxygen saturation (100%), which was gradually decreased to 0% by adding sodium metabisulfite. Experiments were repeated with 2 mm increments of porcine fat layer between the NIRS device and flow cell up to 14 mm. MAIN RESULTS: Lower ATT, higher τ [Formula: see text], and CHF were independently associated with lower in vivo StO2 in multiple regression analysis, whereas age and gender showed no independent relationship. With greater ATT, in vitro StO2 was reduced from 100% to 74% for fully oxygenated blood and increased from 0% to 68% for deoxygenated blood. SIGNIFICANCE: This study shows that ATT independently confounds NIR-SRS derived StO2 by overestimating actual skeletal muscle oxygenation and by decreasing its sensitivity for deoxygenation. Because physiological properties (e.g. presence of disease and slowing of τ [Formula: see text]) also influence NIR-SRS, a correction based on optical properties is needed to interpret calculated values as absolute StO2.

Recovery of absorption spectra from Fourier transform infrared (FT-IR) microspectroscopic measurements of intact spheres.

An infrared spectrum recorded from a microscopic sample depends on spectral properties of the constituent material as well as on morphology. Many samples or domains within heterogeneous materials can be idealized as spheres, in which both scattering and absorption from the three-dimensional shape affect the recorded spectrum. Spectra recorded from such objects may be altered to such an extent that they bear little resemblance to spectra recorded from the bulk material; there are no methods, however, to reconcile the two from first principles. Here we provide the mathematical description of the optical physics underlying light-spherical sample interaction within an instrument. We use the developed analytical expressions to predict recorded data from spheres using Fourier transform infrared (FT-IR) spectroscopic imaging. Recorded spectra are shown to depend strongly on the size of the sphere as well as the optical arrangement of the instrument. Next, we present theory and experiments demonstrating the recovery of the complex refractive index of the material using data recorded from a sphere. The effects of the sample morphology on the measured spectra can be removed, and using the imaginary part of the index, the shape-independent IR absorption spectrum of the material is recovered.

Off-resonance surface-enhanced Raman spectroscopy from gold nanorod suspensions as a function of aspect ratio: not what we thought.

Design of nanoparticles for surface-enhanced Raman scattering (SERS) within suspensions is more involved than simply maximizing the local field enhancement. The enhancement at the nanoparticle surface and the extinction of both the incident and scattered light during propagation act in concert to determine the observed signal intensity. Here we explore these critical aspects of signal generation and propagation through experiment and theory. We synthesized gold nanorods of six different aspect ratios in order to obtain longitudinal surface plasmon resonances that incrementally spanned 600-800 nm. The Raman reporter molecule methylene blue was trap-coated near the surface of each nanorod sample, generating SERS spectra, which were used to compare Raman signals. The average number of reporter molecules per nanorod was quantified against known standards using electrospray ionization liquid chromatography mass spectrometry. The magnitude of the observed Raman signal is reported for each aspect ratio along with the attenuation due to extinction in suspension. The highest Raman signal was obtained from the nanorod suspension with a plasmon resonance blue-shifted from the laser excitation wavelength. This finding is in contrast to SERS measurements obtained from molecules dried onto the surface of roughened or patterned metal substrates where the maximum observed signal is near or red-shifted from the laser excitation wavelength. We explain these results as a competition between SERS enhancement and extinction, at the excitation and scattered wavelengths, on propagation through the sample.

Competition Between Extinction and Enhancement in Surface Enhanced Raman Spectroscopy.

Conjugated metallic nanoparticles are a promising means to achieve ultrasensitive and multiplexed sensing in intact three-dimensional samples, especially for biological applications, via surface enhanced Raman scattering (SERS). We show that enhancement and extinction are linked and compete in a collection of metallic nanoparticles. Counterintuitively, the Raman signal vanishes when nanoparticles are excited at their plasmon resonance, while increasing nanoparticle concentrations at off-resonance excitation sometimes leads to decreased signal. We develop an effective medium theory that explains both phenomena. Optimal choices of excitation wavelength, individual particle enhancement factor and concentrations are indicated. The same processes which give rise to enhancement also lead to increased extinction of both the illumination and the Raman scattered light. Nanoparticles attenuate the incident field (blue) and at the same time provide local enhancement for SERS. Likewise the radiation of the Raman-scattered field (green) is enhanced by the near-by sphere but extinguished by the rest of the spheres in the suspension on propagation.

Evolution of singularities in a partially coherent vortex beam.

We study the evolution of phase singularities and coherence singularities in a Laguerre-Gauss beam that is rendered partially coherent by letting it pass through a spatial light modulator. The original beam has an on-axis minumum of intensity--a phase singularity--that transforms into a maximum of the far-field intensity. In contrast, although the original beam has no coherence singularities, such singularities are found to develop as the beam propagates. This disappearance of one kind of singularity and the gradual appearance of another is illustrated with numerical examples.

Shaping the focal intensity distribution using spatial coherence.

The intensity and the state of coherence are examined in the focal region of a converging, partially coherent wave field. In particular, Bessel-correlated fields are studied in detail. It is found that it is possible to change the intensity distribution and even to produce a local minimum of intensity at the geometrical focus by altering the coherence length. It is also shown that, even though the original field is partially coherent, in the focal region there are pairs of points at which the field is fully correlated and pairs of points at which the field is completely incoherent. The relevance of this work to applications such as optical trapping and beam shaping is discussed.