Signal, noise and resolution in linear and nonlinear structured-illumination microscopy.

Structured-illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in-principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency-space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two-dimensional (2D) illumination patterns or sequentially rotated one-dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal-to-noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background. LAY DESCRIPTION: Structured-illumination microscopy (SIM) is a high-resolution light microscopy technique that allows imaging of fluorescence at a resolution about twice the classical diffraction limit. There are various ways that the illumination can be structured, but it is not obvious how the choice of illumination pattern affects the final image quality, especially in view of the noise. We present a detailed performance analysis considering two illumination techniques: sequential illumination with line-gratings that are shifted and rotated during image acquisition and two-dimensional (2D) illumination structures requiring only shift operations. Our analysis is based on analytical theory, supported by simulations of images considering noise. We also extend our analysis to a nonlinear variant of SIM, with which enhanced resolution can be achieved, limited only by noise. This includes nonlinear SIM based on the light-induced switching of the fluorescent molecules between a bright and a dark state. We find sequential illumination with line-gratings to be advantageous in ordinary (linear) SIM, whereas 2D patterns provides a slight signal-to-noise advantage under idealised conditions in nonlinear SIM if there is no nonswitching background.

Serological, genomic and structural analyses of the major mite allergen Der p 23.

BACKGROUND: Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein. OBJECTIVE: This study sought to assess the importance of Der p 23 among other Dermatophagoides allergens in a North American population and to determine the structure for functional characterization. METHODS: IgE binding to Der p 23, Der p 1, Der p 2, Der p 5, Der p 7 and Der p 8 was measured by ELISA. RNA-seq data from D. pteronyssinus were compared as estimates of allergen expression levels. The structure was analysed by X-ray crystallography and NMR. RESULTS: Despite a high prevalence of Der p 23, (75% vs. 87% and 79% for Der p 1 and Der p 2, respectively), the anti-Der p 23 IgE levels were relatively low. The patient response to the 6 allergens tested was variable (n = 47), but on average anti-Der p 1 and anti-Der p 2 together accounted for 85% of the specific IgE. In terms of abundance, the RNA expression level of Der p 23 is the lowest of the major allergens, thirty fold less than Der p 1 and sevenfold less than Der p 2. The structure of Der p 23 is a small, globular protein stabilized by two disulphide bonds, which is structurally related to allergens such as Blo t 12 that contain carbohydrate binding domains that bind chitin. Functional assays failed to confirm chitin binding by Der p 23. CONCLUSIONS AND CLINICAL RELEVANCE: Der p 23 accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2. The prevalence and amount of specific IgE to Der p 23 and Der p 2 are disproportionately high compared to the expression of other Dermatophagoides allergens.

Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.

Ultrafast transitions from solid to liquid and plasma states of graphite induced by x-ray free-electron laser pulses.

We used photon pulses from an x-ray free-electron laser to study ultrafast x-ray-induced transitions of graphite from solid to liquid and plasma states. This was accomplished by isochoric heating of graphite samples and simultaneous probing via Bragg and diffuse scattering at high time resolution. We observe that disintegration of the crystal lattice and ion heating of up to 5 eV occur within tens of femtoseconds. The threshold fluence for Bragg-peak degradation is smaller and the ion-heating rate is faster than current x-ray-matter interaction models predict.

Protease inhibitor levels in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Our previous study showed that protease inhibitors were attenuated by the periodontal pathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize that fewer protease inhibitors would be present in more advanced periodontal disease sites, where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor [secretory leukocyte protease inhibitor (SLPI), elastase-specific inhibitor (ELAFIN) and squamous cell carcinoma antigen (SCCA)] levels in gingival crevicular fluid and the number of P. gingivalis micro-organisms in subgingival plaque. MATERIAL AND METHODS: Plaque samples from subjects without (n = 18) and with moderate to advanced periodontitis (n = 41) were used to quantify P. gingivalis using real-time PCR. Protease inhibitor levels in the gingival crevicular fluid of all the subjects were determined by ELISA. RESULTS: P. gingivalis was detected in 68.3% of patients with periodontitis, while 16.7% of subjects without periodontitis had a detectable level of P. gingivalis. Patients with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p < 0.001) compared with control subjects without periodontitis. Secretory leukocyte protease inhibitor was also reduced (p < 0.05) in gingival crevicular fluid of periodontitis patients without a detectable level of P. gingivalis. Periodontitis patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations. CONCLUSION: The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may also help in finding pharmacological targets directed against periodontal inflammation.

Quantifying electron temperature distributions from time-integrated x-ray emission spectra.

K-shell x-ray emission spectroscopy is a standard tool used to diagnose the plasma conditions created in high-energy-density physics experiments. In the simplest approach, the emissivity-weighted average temperature of the plasma can be extracted by fitting an emission spectrum to a single temperature condition. It is known, however, that a range of plasma conditions can contribute to the measured spectra due to a combination of the evolution of the sample and spatial gradients. In this work, we define a parameterized model of the temperature distribution and use Markov Chain Monte Carlo sampling of the input parameters, yielding uncertainties in the fit parameters to assess the uniqueness of the inferred temperature distribution. We present the analysis of time-integrated S and Fe x-ray spectroscopic data from the Orion laser facility and demonstrate that while fitting each spectral region to a single temperature yields two different temperatures, both spectra can be fit simultaneously with a single temperature distribution. We find that fitting both spectral regions together requires a maximum temperature of 1310-70 +90 eV with significant contributions from temperatures down to 200 eV.

Damage mechanisms of MoN/SiN multilayer optics for next-generation pulsed XUV light sources.

We investigated the damage mechanism of MoN/SiN multilayer XUV optics under two extreme conditions: thermal annealing and irradiation with single shot intense XUV pulses from the free-electron laser facility in Hamburg - FLASH. The damage was studied "post-mortem" by means of X-ray diffraction, interference-polarizing optical microscopy, atomic force microscopy, and scanning transmission electron microscopy. Although the timescale of the damage processes and the damage threshold temperatures were different (in the case of annealing it was the dissociation temperature of Mo2N and in the case of XUV irradiation it was the melting temperature of MoN) the main damage mechanism is very similar: molecular dissociation and the formation of N2, leading to bubbles inside the multilayer structure.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity.

BACKGROUND: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.

Interaction of short x-ray pulses with low-Z x-ray optics materials at the LCLS free-electron laser.

Materials used for hard x-ray-free-electron laser (XFEL) optics must withstand high-intensity x-ray pulses. The advent of the Linac Coherent Light Source has enabled us to expose candidate optical materials, such as bulk B4C and SiC films, to 0.83 keV XFEL pulses with pulse energies between 1 µJ and 2 mJ to determine short-pulse hard x-ray damage thresholds. The fluence required for the onset of damage for single pulses is around the melt fluence and slightly lower for multiple pulses. We observed strong mechanical cracking in the materials, which may be due to the larger penetration depths of the hard x-rays.

Single shot damage mechanism of Mo/Si multilayer optics under intense pulsed XUV-exposure.

We investigated single shot damage of Mo/Si multilayer coatings exposed to the intense fs XUV radiation at the Free-electron LASer facility in Hamburg - FLASH. The interaction process was studied in situ by XUV reflectometry, time resolved optical microscopy, and "post-mortem" by interference-polarizing optical microscopy (with Nomarski contrast), atomic force microscopy, and scanning transmission electron microcopy. An ultrafast molybdenum silicide formation due to enhanced atomic diffusion in melted silicon has been determined to be the key process in the damage mechanism. The influence of the energy diffusion on the damage process was estimated. The results are of significance for the design of multilayer optics for a new generation of pulsed (from atto- to nanosecond) XUV sources.

Physical, chemical, and toxicological characterization of fibrillated forms of cellulose using an in vitro gastrointestinal digestion and co-culture model.

Fibrillated cellulose is a next-generation material in development for a variety of applications, including use in food and food-contact materials. An alternative testing strategy including simulated digestion was developed to compare the physical, chemical, and biological characteristics of seven different types of fibrillated cellulose, following European Food Safety Authority guidance. Fibrillated forms were compared to a conventional form of cellulose which has been used in food for over 85 years and has Generally Recognized as safe regulatory status in the USA. The physical and chemical characterization of fibrillated celluloses demonstrate that these materials are similar physically and chemically, which composed of the same fundamental molecular structure and exhibit similar morphology, size, size distribution, surface charge, and low levels of impurities. Simulated gastrointestinal and lysosomal digestions demonstrate that these physical and chemical similarities remain following exposure to conditions that mimic the gastrointestinal tract or intracellular lysosomes. A toxicological investigation with an advanced intestinal co-culture model found that exposure to each of the fibrillated and conventional forms of cellulose, in either the pristine or digested form at 0.4% by weight, showed no adverse toxicological effects including cytotoxicity, barrier integrity, oxidative stress, or inflammation. The results demonstrate the physical, chemical, and biological similarities of these materials and provide substantive evidence to support their grouping and ability to read-across data as part of a food safety demonstration.

Non-thermal desorption/ablation of molecular solids induced by ultra-short soft x-ray pulses.

We report the first observation of single-shot soft x-ray laser induced desorption occurring below the ablation threshold in a thin layer of poly (methyl methacrylate)--PMMA. Irradiated by the focused beam from the Free-electron LASer in Hamburg (FLASH) at 21.7 nm, the samples have been investigated by atomic-force microscope (AFM) enabling the visualization of mild surface modifications caused by the desorption. A model describing non-thermal desorption and ablation has been developed and used to analyze single-shot imprints in PMMA. An intermediate regime of materials removal has been found, confirming model predictions. We also report below-threshold multiple-shot desorption of PMMA induced by high-order harmonics (HOH) at 32 nm. Short-time exposure imprints provide sufficient information about transverse beam profile in HOH's tight focus whereas long-time exposed PMMA exhibits radiation-initiated surface ardening making the beam profile measurement infeasible.

In vivo determination of the pyridine nucleotide reduction charge by carbon-13 nuclear magnetic resonance spectroscopy.

An intracellular coenzyme has been observed by carbon-13 nuclear magnetic resonance spectroscopy. The pyridine nucleotides in Escherichia coli were specifically labeled with carbon-13 from the biosynthetic precursor, nicotinic acid. The intracellular redox status and metabolic transformations of the pyridine nucleotides were examined under a variety of conditions. A highly reduced nicotinamide adenine dinucleotide pool was observed under anaerobic conditions only in cells that were cultured aerobically on glycerol.

Nuclear magnetic resonance of rotational mobility of mouse hemoglobin labeled with (2-13C)histidine.

Carbon-13 nuclear magnetic resonance studies were made on mouse hemoglobin specifically labeled at the C-2 histidine position. Measurement of the spin lattice relaxation times of the label before and after hemolysis of the erythrocytes provides information on the intracellular fluid viscosities.

Measurement of Laser-Plasma Electron Density with a Soft X-ray Laser Deflectometer.

A soft x-ray laser (wavelength lambda = 15.5 nanometers) was used to create a moiré deflectogram of a high-density, laser-produced plasma. The use of deflectometry at this short wavelength permits measurement of the density spatial profile in a long-scalelength (3 millimeters), high-density plasma. A peak density of 3.2 x 10(21) per cubic centimeter was recorded.

Molecular dynamics simulations of temperature equilibration in dense hydrogen.

The temperature equilibration rate between electrons and protons in dense hydrogen has been calculated with molecular dynamics simulations for temperatures between 10 and 600eV and densities between 10;{20}cm;{-3}to10;{24}cm;{-3} . Careful attention has been devoted to convergence of the simulations, including the role of semiclassical potentials. We find that for Coulomb logarithms L greater, similar1 , a model by Gericke-Murillo-Schlanges (GMS) [D. O. Gericke, Phys. Rev. E 65, 036418 (2002)] based on a T -matrix method and the approach by Brown-Preston-Singleton [L. S. Brown, Phys. Rep. 410, 237 (2005)] agrees with the simulation data to within the error bars of the simulation. For smaller Coulomb logarithms, the GMS model is consistent with the simulation results. Landau-Spitzer models are consistent with the simulation data for L>4 .

Characteristics of focused soft X-ray free-electron laser beam determined by ablation of organic molecular solids.

A linear accelerator based source of coherent radiation, FLASH (Free-electron LASer in Hamburg) provides ultra-intense femtosecond radiation pulses at wavelengths from the extreme ultraviolet (XUV; lambda<100nm) to the soft X-ray (SXR; lambda<30nm) spectral regions. 25-fs pulses of 32-nm FLASH radiation were used to determine the ablation parameters of PMMA - poly (methyl methacrylate). Under these irradiation conditions the attenuation length and ablation threshold were found to be (56.9+/-7.5) nm and approximately 2 mJ*cm(-2), respectively. For a second wavelength of 21.7 nm, the PMMA ablation was utilized to image the transverse intensity distribution within the focused beam at mum resolution by a method developed here.

Serum hormones and lipoproteins in benign breast disease.

Seventeen young women with clinically confirmed mammary dysplasia and six age-matched controls were treated with alpha-tocopherol. Serum samples collected during the luteal phase of each woman at monthly intervals for the 4-month duration of the study were analyzed for serum luteinizing hormone, follicle-stimulating hormone, and prolactin concentrations by radioimmunoassay and for lipoprotein levels by a combination of precipitation, ultracentrifugation, and enzymatic techniques. Fifteen patients showed objective and subjective remission from disease. While prolactin levels did not change significantly, elevated levels of luteinizing and follicle-stimulating hormones were decreased to normal levels. Ratios of serum cholesterol to high-density lipoprotein cholesterol decreased; high-density lipoprotein and free cholesterol associated with low-density lipoproteins increased as a result of therapy. The results suggest that alpha-tocopherol may serve as an effective agent not only to treat patients with benign breast disease but also to normalize abnormal hormone and lipid levels in subjects at high risk for breast cancer.

Endocrine parameters and alpha-tocopherol therapy of patients with mammary dysplasia.

Patients with mammary dysplasia (17 patients) and controls (6 patients) were treated in a double-blind study with alpha-tocopherol acetate (600 units/day). Determination of serum alpha-tocopherol, estradiol, estriol., and progesterone were made from blood samples collected on Day 21 of the menstrual cycle before and during therapy. Eight-eight % of patients showed clinical response to therapy. Serum alpha-tocopherol concentrations rose after therapy in patients and controls. Serum estradiol and progesterone concentration were not statistically different in patients or controls after therapy, although patients showed a trend toward increased serum progesterone concentration. However, the ratio of progesterone to estradiol, which is abnormal in mammary dysplasia patients, rose from 30 +/- 7 (S.E.) to 53 +/- 11 in patients after alpha-tocopherol therapy (p less than 0.05). Control patients showed no significant change in progesterone/estradiol ratio. Results of this study indicate that alpha-tocopherol therapy may correct an abnormal progesterone/estradiol ratio in patients with mammary dysplasia, with implications on reducing future risk for malignant breast disease.

NMR study of the sites of human hemoglobin acetylated by aspirin.

Acetylation of hemoglobin by aspirin and other acetylating agents has been used to generate hemoglobin analogs with altered structural and functional properties, and may prove useful in the treatment of sickle cell disease. We have studied the acetylation of human hemoglobin using [1'-(13)C]acetylsalicylic acid in combination with two-dimensional HMQC and HSQC NMR analysis. The spectra of the acetylated hemoglobin exhibit a number of well resolved resonances. Several spectral assignment strategies were used: blocking the 2, 3-DPG binding site non-covalently with inositol hexaphosphate or covalently with a cross-linking agent, selective carbamylation of the N-terminal valine amino groups with cyanate, spin-labeling the hemoglobin at betaCys93, and analysis of a hemoglobin triple mutant: betaV1MH2DeltaK144R, in which betaLys144 is replaced by an arginine residue. These studies support the conclusion that the most rapidly acetylated residue is betaLys82 rather than betaLys144, as previously reported. Further, it is apparent that acetyl betaLys82 can give rise to several resonances due to additional acetylation of betaLys82' or other nearby residues. An additional assignment strategy involving comparison of the chemical shifts of the acetyl resonances observed for adducts of diamagnetic carbonmonoxyhemoglobin with the shifts observed in paramagnetic cyanomethemoglobin provides information about the location of the acetyl derivatives relative to the heme irons. This approach is limited, however, by the lack of well defined structural information for the lysine residues on the protein surface. Additional tentative assignments have also been made, using the above approaches.