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Ensuring domestic safety from terrorist attack is a daunting challenge because of the wide array of chemical agents that must be screened. A panel of purified fluorescent protein isoforms (FPs) was screened for the ability to detect various explosives, explosive simulants, and toxic agents. In addition to their commonly used visible excitation wavelengths, essentially all FPs can be excited by UV light at 280 nm. Ultraviolet illumination excites electrons in endogenous tryptophan (W) residues, which then relax by Forster Resonance Energy Transfer (FRET) to the chromophore of the FP, and thus the FPs emit with their typical visible spectra. Taking advantage of the fact that tryptophan excitation can be quenched by numerous agents, including nitroaromatics like TNT and nitramines like RDX, it is demonstrated that quenching of visible fluorescence from UV illumination of FPs can be used as the basis for detecting these explosives and explosive degradation products. This work provides the foundation for production of an array of genetically-modified FPs for in vitro biosensors capable of rapid, simultaneous, sensitive and selective detection of a wide range of explosive or toxic agents.

The lateral diffusion of 100 nm fluorescent latex microspheres (FS) bound to either N-biotinyl-phosphatidyl-ethanolamine or the glycosylphosphatidylinositol-linked protein Thy1 were monitored in the plasmalemma of primary rat fibroblasts by single particle tracking of FS centroids from digital fluorescence micrographs. A silicon intensified target camera was found to be superior to slow scan cooled CCD and intensified interline transfer CCD cameras for monitoring lateral diffusion of rapidly moving FS with nanometer level precision. To estimate the maximum tracking precision, a 4 sec-sequence comprising 120 images of FS fixed to a cover glass was obtained. The mean distance of the centroids from the origin was 7.5 +/- 0.4 nm, and no centroids were beyond 16 nm from the origin. The SIT camera was then used to track FS attached to lipids and Thy1 molecules on the surface of fibroblasts. The lateral diffusion of lipid-bound FS was unconstrained, and the ensemble averaged diffusion coefficient was 0.80 x 10(-9) cm2/sec. Thy1-bound FS existed in two mobility populations, both of which demonstrated constrained mobility. The rapidly moving population, comprising 61% of the total, had an ensemble diffusion coefficient of 6.1 x 10(-10) cm2/sec, and appeared to be restricted to domains with a mean length of about 700 nm. The slowly moving population, comprising about 39% of the total, had a diffusion coefficient of 5.7 x 10(-12) cm2/sec. These results demonstrate that nanovid can be extended to the realm of fluorescence microscopy and support previous studies indicating that while the lateral mobilities of at least some lipids are not constrained to small domains by barriers to lateral diffusion in the fibroblast plasmalemma, a peripheral membrane protein which is bound only by a lipid anchor can be prevented from diffusing freely.

Publisher Summary This chapter discusses imaging ion channel dynamics in living neurons by fluorescence microscopy. The distribution of ion channels and receptors over the neuronal surface is important for the receipt of incoming synaptic inputs and for the integration of these inputs. Most voltage-gated and ligand-gated ion channels have nonhomogeneous distributions in the neuronal membrane; many are restricted to dendritic, axonal, or somatic domains and are further localized within these domains to regions such as dendritic spines, nodes of Ranvier, or synaptic junctions. Determining where and how ion channels are distributed and maintained is important for a variety of reasons. Ion channels in growth cones have a role in neurite outgrowth mechanisms; they are obligatory for synaptic transmission and are required for amplification of neurotransmitter signals in the postsynaptic membrane. Changes in ion-channel distributions are an important aspect in development and plasticity. Several neurological disorders, including multiple sclerosis, are characterized by a change in ion-channel distribution, which is partially responsible for the clinical manifestations of the disease. Fluorescence techniques are invaluable in the study of ion-channel and receptor distributions and dynamics. Fluorescence microscopy has extremely high sensitivity, the lower limit being the ability to detect a single fluorophore under optimal conditions. Fluorescence measurements are specified by the wavelengths of emission and excitation.

Publisher Summary Acetylcholine (ACh) is stored by synaptic vesicles at about 100-fold higher concentration than present in the cytoplasm. As vesicular ACh is free in solution, energy input is required to establish the gradient of concentration. This is accomplished in a process having at least two macromolecular components that reside in the vesicular membrane. The first component is a proton-pumping ATPase known as a V-type ATPase. It pumps protons into the synaptic vesicle, thus acidifying the vesicle core to about pH 5.4. The enzyme is found in the vacuolar-type intracellular organelles, and it is evolutionally related to the mitochondria1 ATPase. The second component is the ACh transporter (AChT). It exchanges one or more vesicular protons for cytoplasmic ACh, thus accomplishing secondary active transport of ACh. The ACh exhibits a transport Michaelis dissociation constant (KM) of about 0.3 mM, which is less than the 4 mM that can be estimated for the concentration of ACh in the cytoplasm. Proton-exchanging transporters appear to account for vesicular storage of all of the classical neurotransmitters. As a class, these transporters are poorly characterized. A structure-activity study has demonstrated that the potency of vesamicol analogues, as assessed by inhibition of active transport, is highly dependent upon the structure of the analogue. This chapter reports the results of the recent ligand-interaction experiments that were directed toward making a choice between models 1 and 2, and toward the identification of the macromolecular structures of the AChT and VR.

The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.

The distribution of protein and lipid in the plasmalemma of some, and perhaps all, cells is non-homogeneous. Microdomains with compositions that are not representative of the bulk membrane exist in many cell types. This phenomenon is obligatory for spatially segregating certain functions performed by the plasma membrane. Obvious examples are concentration of neurotransmitter receptors at the synaptic gap, and voltage-dependent sodium ion channels at the nodes of Ranvier in myelinated nerve. Evidence has also emerged suggesting that lipids are capable of segregating into non homogeneous domains. A potential consequence of microdomain formation would be altered lateral mobility of components within the bilayer.Recently, nanovid microscopy was used to demonstrate that N-(fluoresceinthiocarbamoyl)- phosphatidyl ethanolamine (FITC-PE) was not constrained by diffusion barriers in fibroblasts. Colloidal gold probes conjugated to antiFITC-PE antibody were observed using DIC optics to undergo Brownian motion on the membrane surface. This study concentrated on the lamellar and super-nuclear regions of the cell membrane.

The distribution of ion channels and receptors over the neuronal surface is important for the receipt of incoming synaptic inputs and for the integration of these inputs. Most voltage-gated and ligand-gated ion channels have non-homogeneous distributions in the neuronal membrane, many being restricted to either dendritic, axonal or somatic domains and further localized within these domains to regions such as dendritic spines, nodes of Ranvier or synaptic junctions (1-3). For example voltage-dependent calcium channels are localized and immobilized on dendrites (4), while voltage-dependent sodium channel are localized on axon hillocks (5) and nodes of Ranvier. Determining where and how ion channels are distributed and maintained is important for a variety of reasons. Ion channels in growth cones have a role in neurite outgrowth mechanisms (6), they are obligatory for synaptic transmission and they are required for amplification of neurotransmitter signals in the post synaptic membrane (7). Changes in ion channel distributions are an important aspect in development and plasticity (8,9).