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Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytornetry 1011-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.Key terms: Fluorescent biolabeling probes, fluorescent cyanine dyes, succinimidyl ester biolabeling probes, twocolor immunofluorescent imaging, longwavelength fluorescent probes, flow cytometry, chromophore equivalent weight Earlier papers from this laboratory (4,111 have described the preparation of two series of cyanine and merocyanine dyes which are useful as fluorescent labeling reagents for biological investigations. Functional groups utilized in these reagents for covalent linking to biomolecules have been the iodoacetamido group, for sulfhydryl tagging, and the isothiocyanato moiety, for attachment to primary or secondary amino groups. The incorporation of carboxylic acid groups into the basic cyanine structure was expected to permit fluorescent labeling through the use of derived active esters. The synthesis of (2,3,3-trimethyl-3-H-indol-5-yl)-acetic acid and derived intermediates suitable for the construction of highly fluorescent carboxyl-containing indocyanine dyes has been described recently (12).Here we describe the preparation of the dyes themselves, the procedures used for converting the carboxylic acid groups into active ester functions (esters of N-hydroxysuccinimide) which are reactive toward proteins, and procedures for attaching these dyes to various proteins. Advantages which characterize reagents in this group include ease of preparation, higher activity toward protein substrates in aqueous media under mild conditions, and the high extinction coefficients and quantum yields required for intense fluorescence. As with the other dyes treated in this series of papers, the fluorescence emission maxima are to be found from the long-wavelength portion of the visible range into the near infrared, well separated from the shorterwavelength autofluorescence which is characteristic of many biological specimens and which presents a difficulty in working with such fluorophores as fluorescein.

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Generally, it is recognized that inocula of Septoria tritici present on the basal leaves of winter wheat crops are spread towards the top of the canopy by splashy rainfall. This mechanism of inoculum dispersal is commonly accepted to be a key limit on disease progression. Therefore, attempts to forecast epidemics of S. tritici often quantify rainfall by some means, but largely ignore measurement of pathogen and host variables. In the present study, we show that new wheat leaves emerge initially at a height below established leaves that can contain sporulating lesions of S. tritici. This presents the possibility of horizontal inoculum transfer, even without splashy rainfall. The extent and duration of overlap between emergent and established leaves was found to differ considerably with cultivar and sowing date. Nitrogen application had little effect on overlap, because differences in crop phenology, e.g. leaf area and nodal length, were relative. However, estimates of raindrop penetration to the base of crop canopies suggested that vertical movement of inoculum is affected by nitrogen application. Crops receiving more nitrogen are denser, and therefore less rainfall reaches the base of the canopy. The interactions between crop and pathogen development are discussed with reference to the implications for predicting disease risk. In particular, cultivar traits that promote disease escape are quantified.

Rivers with high biological productivity typically show substantial increases in pH and dissolved oxygen (DO) concentration during the day and decreases at night, in response to changes in the relative rates of aquatic photosynthesis and respiration. These changes, coupled with temperature variations, may impart diel (24-h) fluctuations in the concentration of trace metals, nutrients, and other chemical species. A better understanding of diel processes in rivers is needed and will lead to improved methods of data collection for both monitoring and research purposes. Previous studies have used stable isotopes of dissolved oxygen (DO) and dissolved inorganic carbon (DIC) as tracers of geochemical and biological processes in streams, lakes, and marine systems. Although seasonal variation in 6180 of DO in rivers and lakes has been documented, no study has investigated diel changes in this parameter. Here, we demonstrate large (up to 13%o) cycles in delta18O-DO for two late summer sampling periods in the Big Hole River of southwest Montana and illustrate that these changes are correlated to variations in the DO concentration, the C-isotopic composition of DIC, and the primary productivity of the system. The magnitude of the diel cycle in delta18O-DO was greater in August versus September because of the longer photoperiod and warmer water temperatures. This study provides another biogeochemical tool for investigating the O2 and C budgets in rivers and may also be applicable to lake and groundwater systems.