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Notes: Abstract Confocal fluorescence microscopy enables visualisation and quantitation of fluorescent probes at high resolution deep within intact tissues, with minimal disturbance both of cell–cell interactions and the mechanical, ionic and physiological effects of the extracellular matrix. We illustrate the principles of multiple-parameter 3-D (x,y,z) imaging using reconstruction of nuclear channels in mammalian cells. Repeated sampling in time generates 4-D (x,y,z,t) images which can be used to follow dynamic changes, such as blue-light-dependent chloroplast re-orientation, in intact tissues. Quantitative measurements from multi-dimensional images require calibration of the spatial dimensions of the image and the fluorescence intensity response. This must be determined throughout the volume, which must be sampled to correct for geometric distortion as well as photometric errors arising from the complete optical system, including the specimen. The effects of specimen calibration are illustrated for morphological analysis of stomatal closing responses to abscisic acid in Commelina from 4-D images. Calibrated 4-D imaging allows direct volume measurements and we have followed volume regulation of chondrocytes in cartilage explants during osmotic perturbation. In intact cartilage, unlike in isolated cells, the chondrocytes exhibit volume regulatory mechanisms. In other cases, the fluorescence intensity of the probe may be related to a physiological parameter of interest and changes in its distribution within the cell. Optical sectioning permits discrimination of signal in separate compartments within the cell and can be used to follow transport events between different organelles. We illustrate 3-D (x,y,t) measurements of vacuolar glutathione conjugate pump activity in intact roots of Arabidopsis by following the sequestration of a fluorescent conjugate between glutathione and monochlorobimane. Dynamic measurements of protein localisation are now possible following the introduction of chimeric fusion proteins with green fluorescent protein (GFP) from Aequoria victoria. We have analysed the disposition of heterochromatin in nuclei of living Schizosaccharomyces pombe cells expressing a chimeric construct between Swi6 and GFP. Heterochromatin dynamics can be followed throughout mitosis in 4-D (x,y,z,t) images. Statistical analysis of the fluorescence histograms from each nucleus over time provides quantitative support for aggregation and dispersion of Swi6-GFP clusters during mitosis, rather than dissociation of Swi6 from the heterochromatin. A wide range of single-wavelength and ratio probes are available for imaging different ion activities. We compare 3-D (x,y,t) measurements of ion activities made using single-wavelength (Fluo-3 for calcium) and ratio (BCECF for pH) measurements, using stomatal responses in Vicia faba to peptides from the auxin-binding protein of maize and tip growth in pollen tubes of Lilium longiflorum as examples. Ratioing techniques have many advantages for quantitative fluorescence measurements and we conclude with a discussion of techniques to develop ratioing of single-wavelength probes against alternative references, such as DNA, protein or cell wall material.

Notes: It is known that acetylcholinesterase is secreted by the dopaminergic neurons of the substantia nigra and has a subsequent action independent of cholinergic transmission. Although non-cholinergic actions of this protein have been demonstrated, the subsequent fate of acetylcholinesterase is unknown. One possibility is that acetylcholinesterase is taken up following secretion into the extracellular space. This hypothesis has been tested in vivo, in both conscious and anaesthetized guinea-pigs. Exogenous acetylcholinesterase (2–20 pM) was infused via a push-pull cannula implanted into either the substantia nigra or the surrounding extranigral regions; the amount subsequently recovered in the perfusate was then compared with control values. Only when the push-pull cannulae were implanted in the substantia nigra was there a marked decrease in the amount of acetylcholinesterase recovered; this selective retention was abolished when the perfusion medium was cooled to 4°C or when the experiment was performed post mortem. Direct visualization of immunocytochemically identified nigral dopaminergic cells revealed co-localized deposits of labelled, exogenous acetylcholinesterase. Moreover, when exogenous acetylcholinesterase was boiled to prevent detection by the assay system and to eliminate any classical enzymatic action, an enhancement in perfusate levels of endogenous acetylcholinesterase was observed from nigral but not from extranigral sites, indicating that endogenous and exogenous acetylcholinesterases were in competition. These results suggest that, within the substantia nigra, secreted acetylcholinesterase may be subject to a temperature- and energy-dependent uptake mechanism.

Notes: Abstract A gene fusion plasmid was constructed by inserting the structural gene of the firefly luciferase into a vector which allows expression of a functional recombinant scFv. The fusion protein which is expressed in the periplasm of bacteria, retains dual activity and functions in bioluminescent immunoassays. Such a genetic approach offers many advantages over chemical cross-linking of antibodies to colorimetric enzymes and may be adaptable to clinical immuno-assays and high through-put drug screening assays.