Multisensor hyperspectral imaging approach for the microchemical analysis of ultramarine blue pigments.

This work presents a multisensor hyperspectral approach for the characterization of ultramarine blue, a valuable historical pigment, at the microscopic scale combining the information of four analytical techniques at the elemental and molecular levels. The hyperspectral images collected were combined in a single hypercube, where the pixels of the various spectral components are aligned on top of each other. Selected spectral descriptors have been defined to reduce data dimensionality before applying unsupervised chemometric data analysis approaches. Lazurite, responsible for the blue color of the pigment, was detected as the major mineral phase present in synthetic and good quality pigments. Impurities like pyrite were detected in lower quality samples, although the clear identification of other mineral phases with silicate basis was more difficult. There is no correlation between the spatial distribution of the bands arising in the Raman spectra of natural samples in the region 1200-1850 cm-1 and any of the transition metals or rare earth elements (REE). With this information, the previous hypothesis (based on bulk analysis) attributing these bands to luminescence emissions due to impurities of these elements must be revised. We propose the consideration of CO2 molecules trapped in the cages of the aluminosilicate structure of sodalite-type. Additionally, correlation between certain Raman features and the combined presence of Ca, P, and REE, in particular Nd, was detected for the lowest quality pigment. Our results highlight the usefulness of fusing chemical images obtained via different imaging techniques to obtain relevant information on chemical structure and properties.

Heteroresistant Bacteria Detected by an Extended Raman-Based Antibiotic Susceptibility Test.

Worldwide, multiresistant bacterial strains are emerging at unprecedented rates. This development seriously threatens the ability of humanity to treat even common infections, resulting in disability and death. Furthermore, this development endangers all medical achievements including cancer therapy or organ transplantations. Therefore, the World Health Organization has endorsed antimicrobial resistance as a great threat to humanity. To still allow effective treatment of patients, rapid, automated, and reliable antibiotic susceptibility testing (AST) of bacterial pathogens is essential. Thereby, speed and sensitivity of the AST results are crucial for improving patient care. Here, Raman spectroscopy as a nondestructive technique providing chemical-specific information is employed to monitor the deuterium uptake of metabolically active bacteria during antibiotic treatment, enabling fast and reliable AST. For this purpose, a bulk sample-preparation method was developed, allowing a high-throughput analysis of a significant number of cells. A protocol was developed for Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli) reference strains and was tested on 51 clinical isolates with well-characterized resistance phenotypes against ampicillin, ciprofloxacin, meropenem, and vancomycin. Borderline resistant and heteroresistant phenotypes were observed and further investigated. This is of critical importance as the sensitive detection of low-frequency heteroresistance in bacterial populations is a huge challenge. Such isolates seem susceptible but are resistant to treatment in vivo. Automatable analysis detects strong phenotypes within 3 h. On the basis of experimental and modeled data, heteroresistance is estimated to be detectable down to frequencies of 10-6 and investigated on clinical isolates as a proof-of-concept study, but requiring longer incubation time.

Identifying Dormant Growth State of Mycobacteria by Orthogonal Analytical Approaches on a Single Cell and Ensemble Basis.

Tuberculosis is currently the single most deadly infectious disease in the world and a public health priority as defined by WHO. Although the disease is in general curable, treatment success is hampered by the necessity of a long and side effect prone treatment. Low treatment efficiency may be partly due to the special growth states that mycobacteria enter to avoid being killed by antibiotics and to persist longer within the host. Such growth states have been recently defined as dormant or persistent. We produced dormant model-organism cultures using an acidification model and characterized those by a multilayered approach using mass spectrometry (MALDI-TOF), microscopy (SEM, Raman), and microbiological techniques (CFU, OD600, ATP-levels). With a fast and 96-well-adapted extraction protocol, mycobacteria could be inactivated and extracted for MALDI-TOF analysis. For the first time, we demonstrate growth-state-dependent changes in the mass signatures of the culture, allowing for a reliable differentiation of dormant state and exponential growth. We also demonstrate resuscitation from dormant state back to exponential growth. Viable mycobacteria were immobilized, and single organisms were analyzed individually by Raman microscopy. For single-cell Raman microscopy, Mycobacterium smegmatis cultures were fixed using a new fast and gentle single-step immobilization technique on a hydrophobic glass slide. We were able to distinguish single viable bacteria in the dormant state from their rapidly growing, genetically identical counterparts, identifying the growth state of the culture based on single-organism spectra. This allows for the separation of heterogeneous cultures depending on their growth state using the destruction-free optical method of Raman microscopy.

Rapid single-cell detection and identification of pathogens by using surface-enhanced Raman spectroscopy.

For the successful treatment of infections, real-time analysis and enhanced multiplex capacity, sensitivity and cost-effectiveness of the developed detection method are critical. In this work, surface-enhanced Raman scattering (SERS) was employed with the final aim of identification and discrimination of pathogenic bacteria, based on their detected SERS fingerprint at the single-cell level. Several genera of bacteria that are found in most of the isolated infections in bacteraemia were successfully identified in less than 5 minutes without the use of antibodies or other specific receptors. The key element of the SERS direct detection platform is the SERS substrate, which combines easy production at low costs with a high enhancement enabling single-cell detection. The innovative approach of detection required the in situ synthesis of silver nanoparticles (NPs), ensuring an intimate contact with the bacterial membrane. This protocol provided a good reproducibility of the single-cell SERS spectra and was successfully applied both on Gram-negative and Gram-positive microorganisms (E. coli, M. morganii, E. lactis, L. casei). Thus, a label-free SERS-based biosensor for pathogen detection was developed with low costs, minimal sample preparation, high-accuracy and a very short analysis time of less than 5 min, which is crucial for infection diagnosis.

Onset and progression of de novo donor-specific anti-human leukocyte antigen antibodies after BK polyomavirus and preemptive immunosuppression reduction.

BACKGROUND: BK polyomavirus (BKPyV) viremia/nephropathy and reduction in immunosuppression following viremia may increase the risk of alloimmune activation and allograft rejection. This study investigates the impact of BKPyV viremia on de novo donor anti-human leukocyte antigen (HLA)-specific antibodies (dnDSA). PATIENTS AND METHODS: All primary renal transplants at East Carolina University from March 1999 to December 2010, with at least 1 post-transplant BKPyV viral load testing, were analyzed. Patients were negative for anti-HLA antibodies to donor antigens (tested via single antigen beads) at transplantation and at first BKPyV testing. RESULTS: Nineteen of 174 patients (11%) tested positive for BKPyV viremia. Within 24 months of BKPyV viremia detection, 79% of BKPyV-viremic patients developed dnDSA. Only 20% of BKPyV viremia-persistent cases, compared to 86% of BKPyV viremia-resolved cases, developed dnDSA (P = 0.03). Poor allograft survival was evident in BKPyV viremia-persistent patients (60% failure by 2 years post BKPyV diagnosis) and in BKPyV viremia-resolved patients with dnDSA (5-year post BKPyV diagnosis allograft survival of 48%). CONCLUSIONS: Post-transplant BKPyV viremia and preemptive immunosuppression reduction is associated with high rates of dnDSA. When preemptively treating BKPyV viremia, dnDSA should be monitored to prevent allograft consequences.

Higher risk of kidney graft failure in the presence of anti-angiotensin II type-1 receptor antibodies.

Reports have associated non-HLA antibodies, specifically those against angiotensin II type-1 receptor (AT1R), with antibody-mediated kidney graft rejection. However, association of anti-AT1R with graft failure had not been demonstrated. We tested anti-AT1R and donor-specific HLA antibodies (DSA) in pre- and posttransplant sera from 351 consecutive kidney recipients: 134 with biopsy-proven rejection and/or lesions (abnormal biopsy group [ABG]) and 217 control group (CG) patients. The ABG's rate of anti-AT1R was significantly higher than the CG's (18% vs. 6%, p < 0.001). Moreover, 79% of ABG patients with anti-AT1R lost their grafts (vs. 0%, CG), anti-AT1R levels in 58% of those failed grafts increasing posttransplant. With anti-AT1R detectable before DSA, time to graft failure was 31 months-but 63 months with DSA detectable before anti-AT1R. Patients with both anti-AT1R and DSA had lower graft survival than those with DSA alone (log-rank p = 0.007). Multivariate analysis showed that de novo anti-AT1R was an independent predictor of graft failure in the ABG, alone (HR: 6.6), and in the entire population (HR: 5.4). In conclusion, this study found significant association of anti-AT1R with graft failure. Further study is needed to establish causality between anti-AT1R and graft failure and, thus, the importance of routine anti-AT1R monitoring and therapeutic targeting.

A wide spectral range photoacoustic aerosol absorption spectrometer.

A photoacoustic spectrometer for the measurement of aerosol absorption spectra, based on the excitation of a pulsed nanosecond optical parametrical oscillator (OPO), will be introduced. This spectrometer is working at ambient pressure and can be used to detect and characterize different classes of aerosols. The spectrometer features a spectral range of 410 to 2500 nm and a sensitivity of 2.5 × 10(-7) m(-1) at 550 nm. A full characterization of the system in the visible spectral range is demonstrated, and the potential of the system for near IR measurement is discussed. In the example of different kinds of soot particles, the performance of the spectrometer was assessed. As we demonstrate, it is possible to determine a specific optical absorption per particle by a combination of the new spectrometer with an aerosol particle counter.

Acoustical properties of selected tissue phantom materials for ultrasound imaging.

This note summarizes the characterization of the acoustic properties of four materials intended for the development of tissue, and especially breast tissue, phantoms for the use in photoacoustic and ultrasound imaging. The materials are agar, silicone, polyvinyl alcohol gel (PVA) and polyacrylamide gel (PAA). The acoustical properties, i.e., the speed of sound, impedance and acoustic attenuation, are determined by transmission measurements of sound waves at room temperature under controlled conditions. Although the materials are tested for application such as photoacoustic phantoms, we focus here on the acoustic properties, while the optical properties will be discussed elsewhere. To obtain the acoustic attenuation in a frequency range from 4 MHz to 14 MHz, two ultrasound sources of 5 MHz and 10 MHz core frequencies are used. For preparation, each sample is cast into blocks of three different thicknesses. Agar, PVA and PAA show similar acoustic properties as water. Within silicone polymer, a significantly lower speed of sound and higher acoustical attenuation than in water and human tissue were found. All materials can be cast into arbitrary shapes and are suitable for tissue-mimicking phantoms. Due to its lower speed of sound, silicone is generally less suitable than the other presented materials.

Visualisation of transient processes in biofilms by optical coherence tomography.

Biofilms occur in natural and engineered water systems. In technical processes, biofouling lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for the removal of organic and inorganic pollutants. For improvement of antifouling strategies and for process optimisation in wastewater treatment plants, analytical techniques for online monitoring of biofilms are needed. Optical coherence tomography (OCT) is a non-invasive optical tomography technique, which is increasingly applied in medical diagnostics. It reveals photon-reflecting structures in tissue with lateral and axial resolution in the range of 10 microm. In this paper, we demonstrate the capabilities of OCT for the monitoring of biofilm structures and their detachment. OCT is able to reveal spatially resolved structural information on biofilm without staining. A main focus of this work is set on the ability of OCT to monitor transient processes with temporal resolution in a second to minute scale. These key features of OCT allow online monitoring of biofilm growth and detachment in a flow channel. Three-dimensional (3D) imaging quality, spatial resolution, and temporally resolved profiling of biofilms are demonstrated. The results give rise to the hope that OCT may evolve to a standard tool for monitoring of biofilm density.

Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study.

Longitudinal studies were conducted over a five-year period for HLA antibodies on 493 sera tested from 54 kidney transplant patients. HLA single antigen beads were employed to establish donor specificity of the antibodies. Only 3 of 22 patients without antibodies rejected a graft in contrast to 17 out of 32 patients with posttransplant antibodies (p = 0.003). Using a serum creatinine value of 4.0 mg/dL as the cut-off for a failed graft, 4 of 22 patients without antibodies failed compared to 21 of 32 with antibodies (p = 0.0006). Among patients with donor-specific antibodies (DSA) 13 of 15 failed (p = 0.000004). Even among patients with non-donor specific antibodies (NDSA), 8 of 17 failed (p = 0.05). Among patients who could be identified as making de novo antibodies (since they developed antibodies while not having antibodies for more than six months after transplantation), 6 of 11 failed (p = 0.03). Sequential testing for HLA antibodies shows that antibodies appear prior to a rise in serum creatinine and subsequent graft failure. The very strong association between the production of HLA antibodies after transplantation and graft failure indicates the importance of monitoring for posttransplant HLA antibodies.

Potential of repairing ischemically damaged kidneys ex vivo.

Therapies that would accelerate recovery from ischemic injury could positively impact the number of kidneys procured from non-heart-beating donors. An acellular warm (32 degrees C) perfusion was used to deliver growth factors to canine kidneys damaged by 2 hours of warm ischemia. Fibroblast growth factors 1 and 2 were selected for activation of the tyrosine kinases because of their known receptor-specific binding in the kidney, metabolic regulation, and mitogenic effect. During 24 hours of ex vivo perfusion at near-normothermia, oxidative metabolism was sufficiently restored to the ischemically damaged tissue to support upregulation of cellular processes dependent on new synthesis. The junctional integrity protein, ZO-1 was used to determine recovery of cytoskeletal integrity. The upregulation of proliferating cell nuclear antigen was used as a marker for recovery of synthetic functions. This modulation of both injury and repair proteins in the damaged kidneys was dependent on new synthesis. The observed modulation resulting in normalization of the cytoskeletal integrity correlated with outcomes in that when the "repaired" kidneys were reimplanted, they provided life-sustaining function. In contrast, when warm ischemically damaged control kidneys without treatment, with subsequent hypothermic perfusion or warm perfused in the absence of growth factors, were reimplanted the result was nonviability. The results of this study suggest that the administration of growth factors during 24 hours of near-normothermic, acellular perfusion, in the absence of concordant inflammation, triggers pathways for new synthesis leading to cellular recovery rather than resulting in cell death.

Process analysis of biofilms by photoacoustic spectroscopy.

Biofilms are aggregates of microorganisms and biopolymers which occur at aqueous interfaces. Biofilms play an important role in the degradation of pollutants in natural water systems as well as in wastewater treatment plants. In this communication, the use of photoacoustic spectroscopy (PAS) as a new biofilm monitoring technique is presented. PAS combines features of optical spectroscopy and ultrasonic tomography and allows a depth-resolved analysis of optically and acoustically inhomogeneous media. For the first time, both biofilm and bulk liquid were monitored by photoacoustic sensor heads. In this way, sorption of suspended iron(III) oxide particles on the outer and inner surfaces of the biofilm could be observed on-line and in situ. Colloids can act as carriers of pollutants and influence stability and degradation efficiency of biofilms.

On-line monitoring of opaque liquids by photoacoustic spectroscopy.

A new photoacoustic sensor system for on-line monitoring of highly concentrated and optical opaque liquid samples is presented. The dyeing of textiles is performed with highly concentrated dye solutions with concentrations ranging from 50 mg L(-1) up to 40 g L(-1). For process optimization and control of the wastewater, an on-line monitoring of the dye concentration is needed. Optical transmission measurements allow the determination of the dye concentration in a relatively small range. Samples with concentrations in the upper mg L(-1) and g L(-1) range have to be diluted before the measurement due to their optical opacity. Additionally, light-scattering particles have a strong effect on the transmitted light intensity. By photoacoustic spectroscopy, concentrations in condensed matter can be determined over several orders of magnitude. Furthermore, scattering particles do not generate any photoacoustic signal.

Development and characterization of a mobile photoacoustic sensor for on-line soot emission monitoring in diesel exhaust gas.

Upcoming regulations for vehicle exhaust emission demand substantial reduction of particle emission in diesel exhaust. To achieve these emission levels, the car manufacturing industry is developing new combustion concepts and exhaust after-treatment techniques such as the use of catalysts and particle filters. Many of the state-of-the-art analytical instruments do not meet the required detection limits, in combination with a high temporal resolution necessary for engine optimization. This paper reports a new detection system and the first results of its application to on-line diesel exhaust soot measurements on a engine test bench (MAN diesel engine facility Nürnberg, Germany). The instrument is based on differential photoacoustic (PA) spectroscopy of black carbon aerosol. It contains two identical PA cells, one for the measurement of the aerosol particles and one which analyses the particle-free gas. Thus, a potential cross-sensitivity to gaseous absorbers in the exhaust gas can be excluded. The PA cells were characterized in a laboratory set-up, with water vapor as reference gas and artificial soot generated by a spark discharge generator. The detection limit was found to be 2 microg m(-3) BC (for diesel soot) with a sampling rate of 3 Hz. The temporal response of the system was found to be in the order of 1 s. After full characterization of the cells, the system was transferred into a mobile 19"-rack. Characterization of the mobile sensor system under real-world conditions was performed during several measurement campaigns at an engine test bench for heavy-duty diesel engines. Results for the limit of detection, the time resolution, accuracy, repeatability, and robustness of the sensor system are very promising with regards to a routine application of the system in engine development.

Biofilm monitoring by photoacoustic spectroscopy.

The use of photoacoustic spectroscopy (PAS) as a new biofilm monitoring technique is presented. Growth and detachment of biofilms at three different positions inside a flow channel were monitored by photoacoustic measurements in the visible spectral range (lambda = 532 nm). The experimental approach allows the investigation of the influence of various process parameters (e.g. pH or flow conditions) on growth and detachment of biofilms. In addition, the distribution of the attached biomass can be monitored by depth-resolved photoacoustic measurements.

A photoacoustic technique for depth-resolved in situ monitoring of biofilms.

Biofilms occur in natural and engineered water systems. Biofouling in technical processes lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for removal of organic an inorganic pollutants. For improvement of antifouling strategies and for process optimization in wastewater treatments plants, an analytical technique for online monitoring of biofilms is needed. In this article, a new setup for in situ monitoring of biofilms by photoacoustic spectroscopy is presented. To produce a biofilm, a mixture of microorganisms was grown in a nutrient solution inside a tube reactor. The content of the tube reactor was pumped through a flow channel, and biofilms were generated at the inner surfaces. Three photoacoustic sensor heads were integrated at different positions into the base plate of the flow channel. By photoacoustic spectroscopy, growth, thickness, and detachment of biofilms can be monitored on-line and nondestructively. Experiments presented in this article showed that the flow conditions influence the structure and thickness of biofilms. By changing the pH value, electrostatic interactions inside the biofilm matrix were influenced, and the subsequent detachment processes were observed online. The interaction of iron(III) oxide particles with biofilms led to particle adsorption on the outer and inner surfaces of the biofilm. Afterwards, biofilm flocs were sloughed off from the base biofilm.

Deletrious effect of prolonged cold ischemia on renal function.

The detrimental effect of prolonged cold ischemia (CI) on posttransplant renal function has long been recognized. However, the cellular consequences of CI have not been clearly defined. This study describes a model for the identification of CI-induced injury by evaluating ex-vivo renal metabolism and function prior to reperfusion. Small bovine kidneys were cold stored in Viaspan for 24-, 48-, 72-, and 96 h. Kidneys were then warm perfused (32 degrees C) using Exsangiunous Metabolic Support (EMS) technology and evaluated for oxidative metabolism, vascular dynamics and function. Oxygen consumption, vascular resistance, and diuresis were stable in kidneys with CI up to 48 h. After 72- and 96 h of CI, vascular resistance was increased while oxygen consumption and diuresis were reduced (P < 0.05). Glomerular filtration rate was diminished at CI greater than 24 h (P < 0.05). Results show that function was compromised with CI greater than 24 h and preceded the loss of cell viability following 48 h of CI.

Pretransplantation prognostic testing on damaged kidneys during ex vivo warm perfusion.

BACKGROUND: Further expansion of the donor pool with ischemically damaged kidneys will be predicated on the ability to develop prognostic testing. Using a well-established canine autotransplantation injury model, we assessed whether actual restoration of renal metabolism by ex vivo warm perfusion could be used to predict the status of an organ before transplantation. METHODS: Kidneys were subjected to 30 min of warm ischemia followed by 24 hr of static storage in ViaSpan at 4 degrees C. After warm ischemia and static storage the kidneys were transitioned to 3 hr of warm perfusion using Exsanguinous Metabolic Support technology. During this period, parameters indicative of renal metabolism and vascular function were used to predict outcomes prospectively. Parameters included measures of oxidative metabolism, perfusion characteristics, and vascular condition. A Viability Score (VS) was calculated as the sum of the three parameters mentioned above. Results were grouped by a VS>2 and a VS<2. RESULTS: A clear association between the severity and duration of graft dysfunction and the VS was observed. Organs with a VS>2 had a significantly milder period of acute tubular necrosis, with both a less severe rise in serum creatinine (mean of 4.4 vs. 11 mg/dl) and a shorter recovery period (mean of 8 vs. 18 days) than those with a VS<2. CONCLUSIONS: Results indicate the possibility of utilizing warm perfusion to evaluate kidneys before transplantation. The VS developed demonstrated efficacy in classifying the severity of the acute tubular necrosis and the occurrence of primary nonfunction, offering a sensitive assay for prospective organ testing.