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Eingeladener Vortrag
- nein (3)
Matrix-assisted laser desorption/ionization-timeof-flight (MALDI-TOF) imaging of polystyrenes with various molecular masses was applied to study spatial molecular mass distribution of polymers in sample spots prepared by the 'dried droplet' method. When different solvents and target surfaces were examined, a segregation of single homologous polymers was observed depending upon the evaporation rate of the solvent. For the observed Patterns left by the evaporating droplet, a hypothesis is offered taking into account different hydrodynamic interactions and diffusion.
The results illustrate that spot preparation using the conventionally 'dried droplet' method is prone to artifacts and should be avoided for reliable and reproducible MALDI mass spectrometry experiments with regards to the Determination of molecular masses and mass distributions.
A new setup for fast spatially resolved measurements of elemental trace amounts under total reflection conditions using a new colour X-ray camera is described. Samples prepared on conventional total reflection X-ray fluorescence (TXRF) reflectors were measured at BESSY II synchrotron. A spatial resolution of 50 × 50 µm² was obtained, while the required time for the investigation of a 10 × 10 mm² sample is 30 seconds. The set-up is up to 350 times faster than conventional X-ray fluorescence systems for elemental traces. The major components of the X-ray camera are polycapillary optics and a pn-CCD chip with an active area of 13 × 13 mm². This area is divided into 264 × 264 pixels of 48 × 48 µm². A full X-ray spectrum with a resolution of 152 eV @ 5.9 keV and a chip temperature of 246 K is recorded for each pixel. The chip has a read-out rate of 400 Hz.
The Radon transform is tested as a method for reconstruction of the emissivity distribution of asymmetric laser induced plasmas. Two types of experiments were carried out. First, the plasma asymmetry is introduced via focusing the laser by a cylindrical lens to create plasma plumes elongated along the symmetry axis of the lens. Second, an asymmetric power distribution across the laser beam is created by reflecting the latter from a damaged mirror. Various effects on the quality of the plasma emissivity reconstructed by the Radon tomography method are investigated. The understanding of these effects appears to be essential to design a proper experimental setup to study LIBS plasmas by the Radon tomography method. It is demonstrated that the Radon tomography can successfully be used for experimental studies of asymmetric LIBS plasmas.
Calibration of two commercially available glow discharge double focusing mass spectrometers, the VG 9000 and Element GD, is described using synthetic pin standards pressed from solution doped copper and zinc matrices. A special pressing die was developed for this purpose and optimal results were obtained with the highest possible pressures, i.e., 95 kN·cm- 2. This calibration approach permits the determination of trace element mass fractions down to µg·kg- 1 with small uncertainties and additionally provides traceability of the GD-MS results in the most direct manner to the SI (International System of Units). Results were validated by concurrent measurements of a number of compact copper and zinc certified reference materials. The impact of the sample pin cross-section (circular or square) was investigated with the use of a new pin-sample holder system for the Element GD. The pin-sample holder was designed by the manufacturer for pin-samples having circular cross-section; however, samples with square pin cross-section were also shown to provide acceptable results. Relative Sensitivity Factors for some 50 analytes in copper (VG 9000, Element GD) and zinc matrices (VG 9000) are presented. The field of applicability of GD-MS may be considerably extended via analysis of pin geometry samples based on their ease of preparation, especially with respect to the accuracy and traceability of the results and the enhanced number of analytes which can be reliably calibrated using such samples.
A new laser ablation (LA)-ICP-MS method for single cell and cell nucleus imaging was developed. Therein, iodine was employed as an elemental dye for fibroblast cells and for thin tissue sections. At an incubation time of 60 s, iodine is located mainly within the cell nuclei. This effect was illustrated in fibroblast cells, and iodine signal within the cell nucleus was as high as 5 × 104 cps at 4 µm laser spot size. The surrounding cytoplasm was iodinated as well, but to a lesser extent. The spatial resolution attained was sufficient to detect even smaller cell nuclei within a liver biopsy tissue. Furthermore, iodine was successfully employed for biomolecule labeling and we demonstrated that iodine signal increased with increasing thickness of a palatine tonsil tissue. Thus, the use of iodine as an internal standard to correct for tissue inhomogeneities in LA-ICP-MS was investigated for the simultaneous detection of two tumor markers (Her 2 and CK 7) in breast cancer tissue. Additionally, lanthanide background resulting from glass ablation can be corrected for by Eu standardization.
A laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 µm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 µm) were achieved, demonstrating that LA–ICP–MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.
We optimized multiplexed immunohistochemistry (IHC) on breast cancer tissue. Up to 20 tumor markers are routinely evaluated for one patient, and thus, a common analysis results in a series of time consuming staining procedures. As an alternative, we used lanthanides for labeling of primary antibodies, which are applied in IHC. Laser ablation (LA) ICPMS was elaborated as a detection tool for multiplexed IHC of tissue sections. In this study, we optimized sample preparation steps and LA ICPMS parameters to achieve a sufficient signal-to-background ratio. The results prove the high selectivity of applied antibodies, which was sustained after labeling. Up to three tumor markers (Her 2, CK 7, and MUC 1) were detected simultaneously in a single multiplex analysis of a 5 µm thin breast cancer tissue at a laser spot size of 200 µm. Furthermore, the LA ICPMS results indicate a significantly higher expression level of MUC 1 compared to Her 2 and CK 7, which was not obvious from the conventionally stained tissue sections.
Encapsulation of hydrophobic dyes in polystyrene micro- and nanoparticles via swelling procedures
(2011)
Aiming at the derivation of a generalized
procedure for the straightforward preparation of particles
fluorescing in the visible and near-infrared (NIR) spectral
region, different swelling procedures for the loading of the
hydrophobic polarity-probe Nile Red into nano- and
micrometer sized polystyrene particles were studied and
compared with respect to the optical properties of the
resulting particles. The effect of the amount of incorporated
dye on the spectroscopic properties of the particles was
investigated for differently sized beads with different
surface chemistries, i.e., non-functionalized, aminomodified
and PEG-grafted surfaces. Moreover, photostability
and leaking studies were performed. The main criterion
for the optimization of the dye loading procedures was a
high and thermally and photochemically stable fluorescence
output of the particles for the future application of these
systems as fluorescent labels.
The accurate knowledge of the properties of polycapillary X-ray semi-lenses has a significant influence on the quantitative results in a widespread field of applications involving microfocus X-ray beams. A routine procedure for the characterisation of a polycapillary X-ray semi-lens with a scanning electron microscope (SEM) having attached an energy dispersive spectrometer (EDS) is presented in this paper. A key issue of the procedure consists of fitting the semi-lens in front of the EDS for spectra acquisition. Relevant semi-lens parameters such as focal distance, full width at half maximum (FWHM) of the acceptance area, and transmission are determined in parallelising mode of the semi-lens. Special attention has been paid to the calculation of the transmission.