We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides three-dimensional distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L 2,3 edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.

We describe a method for correcting plural inelastic scattering effects in elemental maps that are acquired in the energy filtering transmission electron microscope (EFTEM) using just two energy windows, one above and one below a core edge in the electron energy loss spectrum (EELS). The technique is demonstrated for mapping low concentrations of phosphorus in biological samples. First, the single-scattering EELS distributions are obtained from specimens of pure carbon and plastic embedding material. Then, spectra are calculated for different specimen thicknesses t, expressed in units of the inelastic mean free path lambda. In this way, standard curves are generated for the ratio k0 of post-edge to pre-edge intensities at the phosphorus L2,3 excitation energy, as a function of relative specimen thickness t/lambda. Thickness effects in a two-window phosphorus map are corrected by successive acquisition of zero-loss and unfiltered images, from which it is possible to determine a t/lambda image and hence a background k0-ratio image. Knowledge of the thickness-dependent k0-ratio at each pixel thus enables a more accurate determination of the phosphorus distribution in the specimen. Systematic and statistical errors are calculated as a function of specimen thickness, and elemental maps are quantified in terms of the number of phosphorus atoms per pixel. Further analysis of the k0-curve shows that the EFTEM can be used to obtain reliable two-window phosphorus maps from specimens that are considerably thicker than previously possible.

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3 ′ ,5 ′ -bisphosphate derivatives act as selective P2Y 1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y 1 and A 1 and A 3 ARs.Modulation of adenosine receptors (P1) and nucleotide (P2) receptors by selective agonists and antagonists (1,2) has the potential for the treatment of wide range of diseases, including those of the cardiovascular, inflammatory, and central nervous systems. There are four subtypes of adenosine receptors (A 1 , A 2A , A 2B , and A 3 ), all of which are G protein-coupled receptors (GPCRs) generally coupled to adenylate cyclase. Extracellular nucleotides, principally ATP, ADP, UTP, and UDP, act through two families of membrane-bound P2 receptors: P2Y subtypes, GPCRs which are activated by both adenine and uracil nucleotides and generally coupled to phospholipase C; and P2X subtypes, ligand-gated ion channels which are activated principally by adenine nucleotides (2). As many as seven subtypes have been cloned within each family. Agonists of adenosine and P2 receptors are almost exclusively nucleosides and nucleotides, respectively, while antagonists of these receptors are structurally more diverse (1). In comparison to the adenosine receptors, much less is known about the specific effects of P2 receptors, largely due to the lack of selective ligands.We are currently designing and synthesizing novel ligands for both adenosine and P2 receptors. Recent methods utilized in these investigations include: conformationally constraining the ribose, or ribose-like, moiety of nucleosides and nucleotides to freeze a conformation that may provide favorable affinity and/or selectivity at P1 and P2 receptors (3,4); modifying known receptor antagonists (5-7); use of a template approach based on the