THE EFFECT OF LABELING INTENSITY, ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY, ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.</p

(LASER-EXCITED) SHPOLSKII FLUOROMETRY - APPLICATIONS IN MARINE ENVIRONMENTAL-ANALYSIS OF POLYNUCLEAR AROMATIC-HYDROCARBONS

Shpol'skii spectroscopy is a technique that can be employed to obtain highly resolved spectral signatures of molecules. The high-resolution spectra are obtained by using n-alkanes as solvents that form neat crystalline matrices at low temperatures. The specificity and sensitivity of the Shpol'skii method can be further improved by application of selective laser excitation. Summarizing, Shpol'skii spectra show the highly specific fingerprinting properties of IR spectra, but at the same time have the inherently high sensitivity of fluorescence techniques. In this paper attention will be given to principles and general experimental aspects of the Shpol'skii technique. Furthermore, some applications in environmental analysis will be discussed in more detail: (1) the qualitative determination of polycyclic aromatic hydrocarbons (PAH) in suspended matter collected in estuarine water; (2) the quantitative determination of PAH in NRCC sediment reference material SES-1; and (3) the application of laser-excited Shpol'skii spectroscopy for the qualitative determination of PAH in crude extracts of biotic samples. It is shown that the Shpol'skii technique can be used successfully as an independent reference method for both qualitative and quantitative checks of conventionally used determination methods. Because of the high selectivity of the low-temperature method, PAH can be quantitatively determined without any sample clean-up, even in extremely complex biotic samples

SHPOLSKII FLUOROMETRY AS AN INDEPENDENT IDENTIFICATION METHOD TO UPGRADE ROUTINE HPLC ANALYSIS OF POLYCYCLIC AROMATIC-HYDROCARBONS

High Performance Liquid Chromatography combined with fluorescence detection is routinely used in the Dutch Water Quality Survey to determine the Polycyclic Aromatic Hydrocarbons (PAH) contents of marine sediment samples. In this paper Shpol'skii spectroscopy is utilized to ascertain the identity and the purity of the peaks in the chromatogram by collection of several eluting fractions and subsequent spectroscopic analysis. The low temperature Shpol'skii technique provides high-resolution fluorescence spectra of PAHs that can serve as fingerprints. Thus, information concerning peak purity was obtained and the number of components identified was roughly doubled