Sequential Fluorescence Research Articles

In situ and denuder-based measurements of elemental and reactive gaseous mercury with analysis by laser-induced fluorescence – results from the Reno Atmospheric Mercury Intercomparison Experiment

Abstract. The University of Miami (UM) deployed a sequential two-photon laser-induced fluorescence (2P-LIF) instrument for the in situ measurement of gaseous elemental mercury, Hg(0), during the Reno Atmospheric Mercury Intercomparison Experiment (RAMIX) campaign. A number of extended sampling experiments, typically lasting 6–8 h but on one occasion extending to ∼ 24 h, were conducted, allowing the 2P-LIF measurements of Hg(0) concentrations to be compared with two independently operated instruments using gold amalgamation sampling coupled with cold vapor atomic fluorescence spectroscopic (CVAFS) analysis. At the highest temporal resolution, ∼ 5 min samples, the three instruments measured concentrations that agreed to within 10–25 %. Measurements of total mercury (TM) were made by using pyrolysis to convert total oxidized mercury (TOM) to Hg(0). TOM was then obtained by difference. Variability in the ambient Hg(0) concentration limited our sensitivity for measurement of ambient TOM using this approach. In addition, manually sampled KCl-coated annular denuders were deployed and analyzed using thermal dissociation coupled with single-photon LIF detection of Hg(0). The TOM measurements obtained were normally consistent with KCl denuder measurements obtained with two Tekran speciation systems and with the manual KCl denuder measurements but with very large uncertainty. They were typically lower than measurements reported by the University of Washington (UW) Detector for Oxidized Hg Species (DOHGS) system. The ability of the 2P-LIF pyrolysis system to measure TM was demonstrated during one of the manifold HgBr2 spikes but the results did not agree well with those reported by the DOHGS system. The limitations of the RAMIX experiment and potential improvements that should be implemented in any future mercury instrument intercomparison are discussed. We suggest that instrumental artifacts make a substantial contribution to the discrepancies in the reported measurements over the course of the RAMIX campaign. This suggests that caution should be used in drawing significant implications for the atmospheric cycling of mercury from the RAMIX results.

Abstract 1550: Multi-biomarker subtyping of circulating tumor cells using sequential fluorescence quenching

Abstract Background: Circulating tumor cells (CTCs) are rare but clinically valuable indicators of cancer status. However their clinical utility is limited to 2-3 positive fluorescent markers and 1 negative fluorescent marker. By contrast, tissue biopsies allow for numerous subtyping markers, yielding information about the tumor's biology and predicted treatment response. If CTC analysis is to be more useful, it must move beyond 2-3 identification markers. We describe a simple and inexpensive method to capture and identify CTCs using traditional fluorescence biomarkers with repeated restating of 9 unrelated fluorescent antibodies. In this study, we sought to subtype CTCs with the epithelial to mesenchymal-like phenotype (EMTCTCs) from pancreatic cancer samples first identified using a conventional CTC marker panel: Cytokeratin (CK), EpCAM, and CD45. We further subtyped these EMTCTCs with an immunosuppression therapy panel (PD-L1, CXCR4, PD-1) and then with a mesenchymal marker panel (CD14, CD34, Vimentin) to better interrogate the EMT phenotype. Alternative predictive and prognostic biomarkers may also be utilized. The order of staining does not affect the result. Our data demonstrate the ability to sequentially analyze, subtype and track 9 distinct cancer markers on each individual cell. Methods: We developed a method of fluorescence quenching using cell lines: A2058, MB231, MCF7, HUVEC, and LnCAP. The technique consists of the steps: quench, underivatize, amine strip and restain (“QUASR”). Cells isolated on CellSieveTM microfilters were stained using the CTC marker panel, after which the QUASR protocol was applied and the cells were stained with a second mesenchymal marker panel. After imaging, QUASR was repeated for a third immunotherapy marker panel, and cells imaged a third time. QUASR was then used on 12 pancreatic cancer patient blood samples previously identified with EMTCTCs provided by Medical College of Wisconsin. Results: No degradation was observed in cell surface, or intracellular markers for the 3 rounds of QUASR. There were 764 EMTCTCs identified as CK+/CD45- in the cancer patient samples, while EpCAM was positive in only 2% of these EMTCTCs. Post quenching, most EMTCTCs had additional mesenchymal phenotypes, i.e. 97% of cells were vimentin+ and 11% were CD34+. Expression of the immunotherapy markers were highly heterogeneous between patients, ranging from 0% to 100% positivity for PD-L1 and from 0% to 90% positivity for CXCR4. None of the CTCs were PD-1+ nor CD14+. Conclusions: Our data demonstrates that sequential multi-panel restaining of clinically applicable cancer biomarkers can provide a greater amount and broader variety of information from patient blood samples. The ability to analyze CTCs beyond simple enumeration will greatly enhance the clinical utility of blood based biopsies, as patient samples can now be screened for multiple prognostic and predictive biomarkers. Citation Format: Daniel L. Adams, Susan Tsai, Cha-Mei Tang, Steingrimur Stefansson. Multi-biomarker subtyping of circulating tumor cells using sequential fluorescence quenching. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1550.

Rapid quantitative determination of major and trace elements in silicate rocks and soils employing fused glass discs using wavelength dispersive X-ray fluorescence spectrometry

This paper introduces a calibration procedure and provides the data achieved for accuracy, precision, reproducibility and the detection limits for major (Si, Al, Fe, Mn, Mg, Ca, Na, K, Ti, P) and trace (Ba, Cr, Cu, Hf, La, Nb, Ni, Pb, Rb, Sr, Ta, Th, U, Y, Zn, Zr) elements in the routine analysis of geological and environmental samples. Forty-two rock and soil reference materials were used to calibrate and evaluate the analytical method using a sequential wavelength dispersive X-ray fluorescence spectrometer. Samples were prepared as fused glass discs and analysis performed with a total measuring time of thirty-one minutes. Another set of twelve independent reference materials were analyzed for the evaluation of accuracy.The detection limits and accuracy obtained for the trace elements (1–2mg/kg) are adequate both for geochemical exploration and environmental studies. The fitness for purpose of the results was also evaluated by the quality criteria test proposed by the International Global Geochemical Mapping Program (IGCP) from which it can be deduced that the method is adequate considering geochemical mapping application and accuracy obtained is within the expected interval of certified values in most cases.

Exploring metal detoxification and accumulation potential during vermicomposting of Tea factory coal ash: sequential extraction and fluorescence probe analysis.

Metal contamination from coal ashes (CAs) is widely recognized as a significant environmental concern. To learn more about metal detoxification and accumulation potential of earthworm species, metal-rich tea factory coal ashes (TFCA) were fed to Eisenia fetida and Lampito mauritii by employing a fluorescent tag detection method. Fascinatingly, on feeding fluorescence probed Zn and Cd along with cow dung to Eisenia fetida, the detection of the gut-proteins with a molecular mass higher than 100 kDa was a distinct evidence of metal binding. Significant increases were observed in the content of humified organic C [humic acid (HAC) and fulvic acid C (FAC)] and degree of humification during vermicomposting. Concurrently, considerably large amount of toxic metals (Cr, Cd, Pb, and Zn) was transformed from exchangeable to recalcitrant (organic matter and mineral bound) fractions. Moreover, total metal concentrations were reduced with high removal efficiency upon vermicomposting.

A molecular pathology method for sequential fluorescence in situ hybridization for multi-gene analysis at the single-cell level.

Multi-gene detection at the single-cell level is desirable to enable more precise genotyping of heterogeneous hematology and oncology samples. This study aimed to establish a single-cell multi-gene fluorescence in situ hybridization (FISH) method for use in molecular pathology analyses. Five fluorochromes were used to label different FISH gene probes, and 5 genes were detected using a five-color FISH protocol. After the first hybridization, the previous FISH probe set was stripped, and a second set of five-color FISH probes was used for rehybridization. After each hybridization, the fluorescence signals were recorded in 6 fluorescence filter channels that included DAPI, Spectrum Green™, Cy3™ v1, Texas Red, Cy5, and PF-415. A digital automatic relocation procedure was used to ensure that exactly the same microscopic field was studied in each stripping and hybridization cycle. By using this sequential stripping and rehybridization strategy, up to 20 genes can be detected within a single nucleus. In conclusion, a practical molecular pathology method was developed for analyzing multiple genes at the single-cell level.

A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant

The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.

Simultaneous three-dimensional velocimetry and thermometry in gaseous flows using the stereoscopic vibrationally excited nitric oxide monitoring technique.

We present a demonstration of the simultaneous measurement of spatially resolved three-component velocity and temperature in gaseous flow fields using a variant of the vibrationally excited nitric oxide monitoring (VENOM) technique, based on planar laser induced fluorescence and molecular tagging velocimetry methods. Three-component velocity determinations were derived from two-dimensional molecular tagging velocity measurements employing sequential fluorescence image pairs obtained simultaneously by two cameras in stereoscopic configuration. Probing two different rotational states of nitric oxide (X2∏, υ''=1), produced via fluorescence and collisional quenching from initial excitation to the A Σ+2 state, for the sequential velocimetry images allows simultaneous determination of the temperature field. Experimental measurements of velocity and temperature across an oblique shock result in mean values within 21 m/s for the three components of velocity and 20 K for planar temperature when compared to oblique shock calculations.

Reosseointegration Following Regenerative Therapy of Tissue-Engineered Bone in a Canine Model of Experimental Peri-Implantitis.

Due to the existence of inflammation and limited osteogenesis on the precontaminated implant surface, reosseointegration is difficult to realize by current therapies. Tissue-engineering strategy has been proved quite effective in intractable bone defect situation. This study was designed to see whether the adoption of tissue-engineered bone complex of adipose-derived stem cells (ASCs) and bone morphogenetic protein-2 (BMP-2) gene delivery would work efficiently in the correction of experimental peri-implantitis. All premolars in both side of mandibular were removed from six beagle canines three months before implant placement. Typical peri-implantitis were then induced by three month ligature placement. After the implementation of identical anti-bacterial and mechanical debridement therapy, the shaped peri-implant defect were stuffed with four groups of constructs, as A: beta tricalcium phosphate (β-TCP); B: β-TCP with ASCs; C: β-TCP with enhanced green fluorescent protein gene transduced ASCs (AdGFP-ASCs); and D: β-TCP with bone morphogenetic protein-2 gene-modified ASCs (AdBMP-2-ASCs). Systematic radiographic, micro-CT, and histomorphometrical assessments were performed. After six months of healing, more bone formation and reosseointegration was found around the implant of groups B and C than group A. And group D further promoted the new bone height and reosseointegration percentage. Moreover, sequential fluorescence labeling tells that group D exhibited the quickest and strongest bone formation on the cleaned implant surface during the entire observation period as compared to the other three groups. These data demonstrated that tissue engineered bone of ASCs, BMP-2 gene delivery, and β-TCP could exert powerful therapeutic effect on peri-implantitis as expected, which may suggest a feasible way to maintain the stability and masticatory function of dental implant.

Facile preparation of homogeneous and length controllable halloysite nanotubes by ultrasonic scission and uniform viscosity centrifugation

Halloysite nanotubes (HNTs) are natural “green” nanomaterials with tubular structures and have exciting potential applications in many fields. Preparation of homogeneous HNTs remains significant challenges in their fundamental studies and applications. Herein, we provide a method for preparing homogeneous and length controllable HNTs by the combination of ultrasonic treatment and two-step uniform viscosity centrifugation. Raw halloysite bundles have been successfully dispersed and cut by ultrasonic scission. Length controllable HNTs (140–240nm) can be prepared simply by changing ultrasonic treatment time or ultrasonic power. Short HNTs have been successfully separated from the sonicated suspension of raw HNTs by two-step uniform viscosity centrifugation. Transmission electron microscopy, X-ray diffraction and sequential X-ray fluorescence spectrometer measurements demonstrate that the purified HNTs are homogeneous with much less impurities. This simple, rapid, and effective method can be scaled up for industrial preparation of homogeneous and length controllable HNTs. The purified HNTs can be used as high value-added materials in many industrial fields and used as a carrier for a wide range of active agents, such as drugs, cosmetics, and catalysts. The separation method can be used to separate chemically synthesized nanotubes. In addition, a simple method for quick determination of the concentration of HNTs by UV–Vis spectrophotometer has been developed.

Cancer cell-targeted two-photon fluorescence probe for the real-time ratiometric imaging of DNA damage.

Real-time imaging of DNA damage in cancer cells could provide valuable information on the formation and development of cancer. Herein, a two-photon fluorescence probe was discovered. Through sequential ICT processes, it allows successful in vivo visualization of DNA damage in cancer cells by one/two-photon microscopic imaging or by the unaided eye and a hand-held ultraviolet lamp.

Development and Molecular Cytogenetic Identification of a Novel Wheat-Leymus mollis Lm#7Ns (7D) Disomic Substitution Line with Stripe Rust Resistance.

Leymus mollis (2n = 4x = 28, NsNsXmXm) possesses novel and important genes for resistance against multi-fungal diseases. The development of new wheat—L. mollis introgression lines is of great significance for wheat disease resistance breeding. M11003-3-1-15-8, a novel disomic substitution line of common wheat cv. 7182 –L. mollis, developed and selected from the BC1F5 progeny between wheat cv. 7182 and octoploid Tritileymus M47 (2n = 8x = 56, AABBDDNsNs), was characterized by morphological and cytogenetic identification, analysis of functional molecular markers, genomic in situ hybridization (GISH), sequential fluorescence in situ hybridization (FISH)—genomic in situ hybridization (GISH) and disease resistance evaluation. Cytological observations suggested that M11003-3-1-15-8 contained 42 chromosomes and formed 21 bivalents at meiotic metaphase I. The GISH investigations showed that line contained 40 wheat chromosomes and a pair of L. mollis chromosomes. EST-STS multiple loci markers and PLUG (PCR-based Landmark Unique Gene) markers confirmed that the introduced L. mollis chromosomes belonged to homoeologous group 7, it was designated as Lm#7Ns. While nulli-tetrasomic and sequential FISH-GISH analysis using the oligonucleotide Oligo-pSc119.2 and Oligo-pTa535 as probes revealed that the wheat 7D chromosomes were absent in M11003-3-1-15-8. Therefore, it was deduced that M11003-3-1-15-8 was a wheat–L. mollis Lm#7Ns (7D) disomic substitution line. Field disease resistance demonstrated that the introduced L. mollis chromosomes Lm#7Ns were responsible for the stripe rust resistance at the adult stage. Moreover, M11003-3-1-15-8 had a superior numbers of florets. The novel disomic substitution line M11003-3-1-15-8, could be exploited as an important genetic material in wheat resistance breeding programs and genetic resources.

Chimeric cells of maternal origin do not appear to be pathogenic in the juvenile idiopathic inflammatory myopathies or muscular dystrophy.

IntroductionMicrochimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues.MethodFluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls.ResultsMicrochimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm2 tissue) compared to controls (0.019 ± 0.007 cells/mm2 tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm2). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm2 versus 0 ± 0/mm2p = 0.003 and 0.043 ± 0.023/mm2 versus 0 ± 0/mm2p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype.ConclusionsMicrochimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.

Combining fluorescence and bioluminescence microscopy.

ABSTRACTBioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin—a substrate of luciferase in promoter assays of gene expression in cultured cells—confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492–506 nm and 524–578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol‐12‐myristate‐13‐acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. Microsc. Res. Tech. 78:715–722, 2015. © 2015 Wiley Periodicals, Inc.

Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers

The aim of this study was to assess whether there is a relationship between numerical chromosome abnormalities and certain segregation modes in spermatozoa from Robertsonian translocation carriers. A sequential fluorescence in-situ hybridization protocol based on two successive hybridization rounds was performed on sperm samples from one t(13;22) and ten t(13;14) carriers. Patient inclusion criteria included the presence of a positive interchromosomal effect (ICE). In the first round, numerical abnormalities for chromosomes 15/22, 18, 21, X and Y were analysed. In the second round, the segregation outcome of the rearranged chromosomes was evaluated in the numerically abnormal spermatozoa detected in the first round, as well as in randomly assessed spermatozoa. Aneuploid spermatozoa showed statistical differences in all segregation modes when compared with randomly assessed spermatozoa: alternate (50.7% versus 84.3%), adjacent (36.6% versus 14.6%) and 3:0 (10.2% versus 1%). Diploid/multiple disomic spermatozoa showed differences in alternate (3.7% versus 84.3%) and 3:0 (67.6% versus 1%). We concluded that in Robertsonian translocation carriers that exhibit ICE, numerically abnormal spermatozoa preferentially contain unbalanced segregation products. This might be explained by heterosynapsis acting as a rescue mechanism that would lead to aberrant recombination, which is a predisposing factor for non-disjunction events.

Spontaneous and divergent hexaploid triticales derived from common wheat × rye by complete elimination of D-genome chromosomes.

BackgroundHexaploid triticale could be either synthesized by crossing tetraploid wheat with rye, or developed by crossing hexaploid wheat with a hexaploid triticale or an octoploid triticale.Methodology/Principal FindingsHere two hexaploid triticales with great morphologic divergence derived from common wheat cultivar M8003 (Triticum aestivum L.) × Austrian rye (Secale cereale L.) were reported, exhibiting high resistance for powdery mildew and stripe rust and potential for wheat improvement. Sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) karyotyping revealed that D-genome chromosomes were completely eliminated and the whole A-genome, B-genome and R-genome chromosomes were retained in both lines. Furthermore, plentiful alterations of wheat chromosomes including 5A and 7B were detected in both triticales and additionally altered 5B, 7A chromosome and restructured chromosome 2A was assayed in N9116H and N9116M, respectively, even after selfing for several decades. Besides, meiotic asynchrony was displayed and a variety of storage protein variations were assayed, especially in the HMW/LMW-GS region and secalins region in both triticales.ConclusionThis study confirms that whole D-genome chromosomes could be preferentially eliminated in the hybrid of common wheat × rye, “genome shock” was accompanying the allopolyploidization of nascent triticales, and great morphologic divergence might result from the genetic variations. Moreover, new hexaploid triticale lines contributing potential resistance resources for wheat improvement were produced.

The parental origin correlates with the karyotype of human embryos developing from tripronuclear zygotes.

ObjectiveIt has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype.MethodsThe karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin.Results3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos.ConclusionThe differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.

Principles and examples of gel-based approaches for phosphoprotein analysis.

Methods for analyzing the phosphorylation status of proteins are essential to investigate in detail key cellular processes, including signal transduction and cell metabolism. The transience of this post-translational modification and the generally low abundance of phosphoproteins require specific enrichment and/or detection steps prior to analysis. Here, we describe three gel-based approaches for the analysis of differentially expressed phosphoproteins. These approaches comprise (1) the sequential fluorescence staining of two-dimensional (2-D) gels using Pro-Q(®) Diamond and SYPRO(®) Ruby dyes to visualize and quantify phosphoproteins in total cellular lysates as well as (2) affinity enrichment of phosphoproteins in conjunction with sequential fluorescence staining of the 2-D gels and (3) affinity enrichment of proteins prior to pre-electrophoretic fluorescence labeling and 2-D gel electrophoresis.

A non-symmetric pillar[5]arene based on triazole-linked 8-oxyquinolines as a sequential sensor for thorium(IV) followed by fluoride ions.

A novel non-symmetric pillar[5]arene bearing triazole-linked 8-oxyquinolines at one rim was synthesized and demonstrated as a sequential fluorescence sensor for thorium(iv) followed by fluoride ions with high sensitivity and selectivity.

Deployment of a sequential two-photon laser-induced fluorescence sensor for the detection of gaseous elemental mercury at ambient levels: fast, specific, ultrasensitive detection with parts-per-quadrillion sensitivity

Abstract. The operation of a laser-based sensor for gas-phase elemental mercury, Hg(0), is described. It utilizes sequential two-photon laser excitation with detection of blue-shifted laser-induced fluorescence (LIF) to provide a highly specific detection scheme that precludes detection of anything other than atomic mercury. It has high sensitivity, fast temporal resolution, and can be deployed for in situ measurements in the open atmosphere with essentially no perturbation of the environment. An ambient sample can also be pulled through a fluorescence cell, allowing for standard addition calibrations of the concentration. No type of preconcentration is required and there appears to be no significant interferences from other atmospheric constituents, including gas-phase oxidized mercury species. As a consequence, it is not necessary to remove oxidized mercury, commonly referred to as reactive gaseous mercury (RGM), from the air sample. The instrument has been deployed as part of an instrument intercomparison and compares well with conventional instrumentation that utilizes preconcentration on gold followed by analysis using cold-vapor atomic fluorescence spectroscopy (CVAFS). Currently, the achievable detection sensitivity is ~ 15 pg m−3 (~ 5 × 104 atoms cm−3, ~ 2 ppq) at a sampling rate of 0.1 Hz, i.e., averaging 100 shots with a 10 Hz laser system. Preliminary results are described for a 50 Hz instrument that utilizes a modified excitation sequence and has monitored ambient elemental mercury with an effective sampling rate of 10 Hz. Additional work is required to produce the precision necessary to perform eddy correlation measurements. Addition of a pyrolysis channel should allow for the measurement of total gaseous mercury (TGM) and hence RGM (by difference) with good sensitivity and time resolution.

Tissue engineering of a vascularized bone graft of critical size with an osteogenic and angiogenic factor-based in vivo bioreactor.

Engineering a large vascularized bone graft is a much greater challenge than engineering small bone tissues. Although this is essentially feasible through an osteogenic factor-based in vivo bioreactor technique, the ossification needs improving. This study was aimed to investigate the possibility and efficacy of ectopic cultivation of sizeable bone grafts with large angiogenic and osteogenic factor-loaded natural bovine bone mineral (NBBM) scaffolds. For this purpose, six groups of sizeable composite scaffolds were constructed, consisting of a titanium mesh cage of NBBM or a mixture of NBBM/autogenous bone particles (AB), which were preloaded with 660 μg recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or 4 μg recombinant human vascular endothelial growth factor165 (rhVEGF165). The scaffolds were implanted in bilateral latissimus dorsi muscles in eight pigs to construct in vivo bioreactors. Sequential fluorescence labeling was then applied to trace bone formation at the early stage. The implants were retrieved 12 weeks later. The undecalcified sections were observed in turn under the fluorescence microscope and light microscope to investigate early stage osteogenesis and histology. Moreover, new bone density (BD) was measured with histomorphometry. Compared with rhBMP-7-delivered NBBM scaffolds, rhVEGF165/rhBMP-7-delivered NBBM scaffolds were with more intense intra-scaffold osteogenesis at the early stage and the ultimate sizeable bone grafts of microstructurally more lamellae and trabeculae, and quantitatively higher BD (31.93% vs. 22.37%, p<0.01). This study demonstrated that as for the endocultivation of a large bone graft with bioactive factor-based in vivo bioreactor technique, dual delivery of rhVEGF165/rhBMP-7 has synergic effects on improving early stage bone formation and subsequently bone quality and quantity of the bone grafts.