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Institute
Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands
(2009)
The applications of quantum dots (QDs) in two-photon (2P) excitation applications demand reliable data about their 2P absorption (2PA) cross sections (sigma(2PA)). In the present study, sigma(2PA) values have been determined for a series of commercial colloidal CdSe/ZnS QDs and CdSeTe/ZnS QDs in aqueous media. For the first time for these QDs, the sigma(2PA) values have been determined over a wide spectral range, that is, between 720 and 900 nm, and are compared to the extinction coefficient (epsilon) values obtained under one-photon (1P) excitation. Furthermore, we present a QD in combination with an organic dye in a biotin-streptavidin Forster resonance energy transfer bioassay under 1P and 2P excitation. The results for the bioassay under 2P excitation are compared to those obtained under 1P excitation. The results demonstrate that in the case of the 2P excitation, higher sensitivity can be achieved because of an improved signal-to-noise ratio.
Total protein concentration (TPC) is a key parameter in many biochemical experiments and its quantification is often necessary for isolation, separation, and analysis of proteins. A sensitive and rapid nanobead-based TPC quantification assay based on Forster Resonance Energy Transfer (FRET) has been developed. A new, highly luminescent Tb(III) complex has been synthesised and applied as donor in this FRET assay with an organic dye (Cy5) as acceptor. FRET-induced changes in luminescence have been investigated both at donor and acceptor emission wavelength using time-resolved luminescence spectroscopy with time-gated detection. In the assay, the Tb(III) complex and fine-tuned polyglycidyl methacrylate (PGMA) nanobeads ensure that an improvement in sensitivity and background reduction is achieved. Using 40 nm large PGMA nanobeads loaded with the Tb(III) complex, it is possible to determine TPC down to 50 ng mL(-1) in just 10 minutes. Through specific assay components the sensitivity has been improved when compared to existing nanobead-based assays and to currently known commercial methods. Additionally, the assay is relatively insensitive to the presence of contaminants, such as non-ionic detergents commonly found in biological samples. Due to no need for any centrifugal steps, this mix-and-measure bioassay can easily be implemented into routine TPC quantification protocols in biochemical laboratories.
Calcium (Ca2+) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca2+ concentration ([Ca2+](i)) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca2+](i) is the use of fluorescent Ca2+ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca2+ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca2+-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca2+-free and Ca2+-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca2+-free and Ca2+-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca2+ sensitivity is reduced. Nevertheless, quantitative [Ca2+](i) recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.
pH sensing in living cells represents one of the most prominent topics in biochemistry and physiology. In this study we performed one-photon and two-photon time-domain fluorescence lifetime imaging with a laser-scanning microscope using the time-correlated single-photon counting technique for imaging intracellular pH levels. The suitability of different commercial fluorescence dyes for lifetime-based pH sensing is discussed on the basis of in vitro as well of in situ measurements. Although the tested dyes are suitable for intensity-based ratiometric measurements, for lifetime- based techniques in the time-domain so far only BCECF seems to meet the requirements of reliable intracellular pH recordings in living cells.
In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
Fluoroionophores of fluorophore-spacer-receptor format were prepared for detection of PdCl2 by fluorescence enhancement. The fluorophore probes 1-13 consist of a fluorophore group, in alkyl spacer and a dithiomaleonitrile PdCl2 receptor. First, varying the length of the alkylene spacer (compounds 1-3) revealed, dominant through-space pathway for oxidative photoinduced electron transfer (PET) in CH2-bridged dithiomaleonitrile fluoroionophores. Second. fluorescent probes 4-9 containing two anthracene or pyrene fragments connected through CH2 bridges to the dithiomaleonitrile unit were synthesized. Modulation of the oxidation potential (E-Ox) through electron-withdrawing or -donating groups on the anthracene moiety regulates file thermodynamic driving force for oxidative PET (Delta G(PET)) in bis(anthrylmethylthio)maleonitriles and therefore the fluorescence quantum yields (Phi(f)), too. The new concept was confirmed and transferred to pyrenyl ligands, and fluorescence enhancements (FE) greater than 3.2 in the presence of PdCl2 were achieved by 7 and 8 (FE=5.4 and 5.2). Finally, for comparison, monofluorophore ligands 10-13 were synthesized.
Fluoroionophores of fluorophore-spacer-receptor format were prepared for detection of PdCl2 by fluorescence enhancement. The fluorescent probes 1-13 consist of a fluorophore group, an alkyl spacer and a dithiomaleonitrile PdCl2 receptor. First, varying the length of the alkylene spacer (compounds 1-3) revealed a dominant through-space pathway for oxidative photoinduced electron transfer (PET) in CH2-bridged dithiomaleonitrile fluoroionophores. Second, fluorescent probes 4-9 containing two anthracene or pyrene fragments connected through CH2 bridges to the dithiomaleonitrile unit were synthesized. Modulation of the oxidation potential (EOx) through electron-withdrawing or -donating groups on the anthracene moiety regulates the thermodynamic driving force for oxidative PET (GPET) in bis(anthrylmethylthio)maleonitriles and therefore the fluorescence quantum yields (f), too. The new concept was confirmed and transferred to pyrenyl ligands, and fluorescence enhancements (FE) greater than 3.2 in the presence of PdCl2 were achieved by 7 and 8 (FE=5.4 and 5.2). Finally, for comparison, monofluorophore ligands 10-13 were synthesized.
Suppression of the near-infrared OH night-sky lines with fibre Bragg gratings - first results
(2012)
The background noise between 1 and 1.8 ?mu m in ground-based instruments is dominated by atmospheric emission from hydroxyl molecules. We have built and commissioned a new instrument, the Gemini Near-infrared OH Suppression Integral Field Unit (IFU) System (GNOSIS), which suppresses 103 OH doublets between 1.47 and 1.7?mu m by a factor of 1000 with a resolving power of 10?000. We present the first results from the commissioning of GNOSIS using the IRIS2 spectrograph at the Anglo-Australian Telescope. We present measurements of sensitivity, background and throughput. The combined throughput of the GNOSIS fore-optics, grating unit and relay optics is 36?per cent, but this could be improved to 46?per cent with a more optimal design. We measure strong suppression of the OH lines, confirming that OH suppression with fibre Bragg gratings will be a powerful technology for low-resolution spectroscopy. The integrated OH suppressed background between 1.5 and 1.7 mu m is reduced by a factor of 9 compared to a control spectrum using the same system without suppression. The potential of low-resolution OH-suppressed spectroscopy is illustrated with example observations of Seyfert galaxies and a low-mass star. The GNOSIS background is dominated by detector dark current below 1.67 mu m and by thermal emission above 1.67 mu m. After subtracting these, we detect an unidentified residual interline component of 860 +/- 210 photons s-1 m-2?arcsec-2?mu m-1, comparable to previous measurements. This component is equally bright in the suppressed and control spectra. We have investigated the possible source of the interline component, but were unable to discriminate between a possible instrumental artefact and intrinsic atmospheric emission. Resolving the source of this emission is crucial for the design of fully optimized OH suppression spectrographs. The next-generation OH suppression spectrograph will be focused on resolving the source of the interline component, taking advantage of better optimization for a fibre Bragg grating feed incorporating refinements of design based on our findings from GNOSIS. We quantify the necessary improvements for an optimal OH suppressing fibre spectrograph design.
The pressure dependence of sheath gas assisted electrospray ionization (ESI) was investigated based on two complementary experimental setups, namely an ESI-ion mobility (IM) spectrometer and an ESI capillary - Faraday plate setup housed in an optically accessible vacuum chamber. The ESI-IM spectrometer is capable of working in the pressure range between 300 and 1000 mbar. Another aim was the assessment of the analytical capabilities of a subambient pressure ESI-IM spectrometer. The pressure dependence of ESI was characterized by imaging the electrospray and recording current-voltage (I-U) curves. Qualitatively different behavior was observed in both setups. While the current rises continuously with the voltage in the capillary-plate setup, a sharp increase of the current was measured in the IM spectrometer above a pressure-dependent threshold voltage. The different character can be attributed to the detection of different species in both experiments. In the capillary-plate experiment, a multitude of charged species are detected while only desolvated ions attribute to the IM spectrometer signal. This finding demonstrates the utility of IM spectrometry for the characterization of ESI, since in contrast to the capillary-plate setup, the release of ions from the electrospray droplets can be observed. The I-U curves change significantly with pressure. An important result is the reduction of the maximum current with decreasing pressure. The connected loss of ionization efficiency can be compensated by a more efficient transfer of ions in the IM spectrometer at increased E/N. Thus, similar limits of detection could be obtained at 500 mbar and 1 bar.
A new ion mobility (IM) spectrometer, enabling mobility measurements in the pressure range between 5 and 500 mbar and in the reduced field strength range E/N of 5-90 Td, was developed and characterized. Reduced mobility (K-0) values were studied under low E/N (constant value) as well as high E/N (deviation from low field K-0) for a series of molecular ions in nitrogen. Infrared matrix-assisted laser desorption ionization (IR-MALDI) was used in two configurations: a source working at atmospheric pressure (AP) and, for the first time, an IR-MALDI source working with a liquid (aqueous) matrix at sub-ambient/reduced pressure (RP). The influence of RP on IR-MALDI was examined and new insights into the dispersion process were gained. This enabled the optimization of the IM spectrometer for best analytical performance. While ion desolvation is less efficient at RP, the transport of ions is more efficient, leading to intensity enhancement and an increased number of oligomer ions. When deciding between AP and RP IR-MALDI, a trade-off between intensity and resolving power has to be considered. Here, the low field mobility of peptide ions was first measured and compared with reference values from ESI-IM spectrometry (at AP) as well as collision cross sections obtained from molecular dynamics simulations. The second application was the determination of the reduced mobility of various substituted ammonium ions as a function of E/N in nitrogen. The mobility is constant up to a threshold at high E/N. Beyond this threshold, mobility increases were observed. This behavior can be explained by the loss of hydrated water molecules.
Electrospray ionization-ion mobility spectrometry was employed for the determination of collision cross sections (CCS) of 25 synthetically produced peptides in the mass range between 540-3310 Da. The experimental measurement of the CCS is complemented by their calculation applying two different methods. One prediction method is the intrinsic size parameter (ISP) method developed by the Clemmer group. The second new method is based on the evaluation of molecular dynamics (MD) simulation trajectories as a whole, resulting in a single, averaged collision cross-section value for a given peptide in the gas phase. A high temperature MD simulation is run in order to scan through the whole conformational space. The lower temperature conformational distribution is obtained through thermodynamic reweighting. In the first part, various correlations, e.g. CCS vs. mass and inverse mobility vs. m/z correlations, are presented. Differences in CCS between peptides are also discussed in terms of their respective mass and m/z differences, as well as their respective structures. In the second part, measured and calculated CCS are compared. The agreement between the prediction results and the experimental values is in the same range for both calculation methods. While the calculation effort of the ISP method is much lower, the MD method comprises several tools providing deeper insights into the conformations of peptides. Advantages and limitations of both methods are discussed. Based on the separation of two pairs of linear and cyclic peptides of virtually the same mass, the influence of the structure on the cross sections is discussed. The shift in cross section differences and peak shape after transition from the linear to the cyclic peptide can be well understood by applying different MD tools, e.g. the root-mean-square deviation (RMSD) and the root mean square fluctuation (RMSF). (C) 2018 Elsevier B.V. All rights reserved.
Spot variation fluorescence correlation spectroscopy (SV-FCS) is a variant of the FCS techniques which may give useful information about the structural organisation of the medium in which the diffusion takes place. We show that the same results can be obtained by post-processing the photon count data from ordinary FCS measurements. By using this method, one obtains the fluorescence autocorrelation functions for sizes of confocal volume, which are effectively smaller than that of the initial FCS measurement. The photon counts of the initial experiment are first transformed into smooth intensity trace using kernel smoothing method or to a piecewise-continuous intensity trace using binning and then a non-linear transformation is applied to this trace. The result of this transformation mimics the photon count rate in an experiment performed with a smaller confocal volume. The applicability of the method is established in extensive numerical simulations and directly supported in in-vitro experiments. The procedure is then applied to the diffusion of AlexaFluor647-labeled streptavidin in living cells.
Precision agriculture (PA) strongly relies on spatially differentiated sensor information. Handheld instruments based on laser-induced breakdown spectroscopy (LIBS) are a promising sensor technique for the in-field determination of various soil parameters. In this work, the potential of handheld LIBS for the determination of the total mass fractions of the major nutrients Ca, K, Mg, N, P and the trace nutrients Mn, Fe was evaluated. Additionally, other soil parameters, such as humus content, soil pH value and plant available P content, were determined. Since the quantification of nutrients by LIBS depends strongly on the soil matrix, various multivariate regression methods were used for calibration and prediction. These include partial least squares regression (PLSR), least absolute shrinkage and selection operator regression (Lasso), and Gaussian process regression (GPR). The best prediction results were obtained for Ca, K, Mg and Fe. The coefficients of determination obtained for other nutrients were smaller. This is due to much lower concentrations in the case of Mn, while the low number of lines and very weak intensities are the reason for the deviation of N and P. Soil parameters that are not directly related to one element, such as pH, could also be predicted. Lasso and GPR yielded slightly better results than PLSR. Additionally, several methods of data pretreatment were investigated.
Precision agriculture (PA) strongly relies on spatially differentiated sensor information. Handheld instruments based on laser-induced breakdown spectroscopy (LIBS) are a promising sensor technique for the in-field determination of various soil parameters. In this work, the potential of handheld LIBS for the determination of the total mass fractions of the major nutrients Ca, K, Mg, N, P and the trace nutrients Mn, Fe was evaluated. Additionally, other soil parameters, such as humus content, soil pH value and plant available P content, were determined. Since the quantification of nutrients by LIBS depends strongly on the soil matrix, various multivariate regression methods were used for calibration and prediction. These include partial least squares regression (PLSR), least absolute shrinkage and selection operator regression (Lasso), and Gaussian process regression (GPR). The best prediction results were obtained for Ca, K, Mg and Fe. The coefficients of determination obtained for other nutrients were smaller. This is due to much lower concentrations in the case of Mn, while the low number of lines and very weak intensities are the reason for the deviation of N and P. Soil parameters that are not directly related to one element, such as pH, could also be predicted. Lasso and GPR yielded slightly better results than PLSR. Additionally, several methods of data pretreatment were investigated.