We present a depth-resolved Image Mapping Spectrometer (IMS) which is capable of acquiring 4D (x, y, z, λ) datacubes. Optical sectioning is implemented by structured illumination. The device's spectral imaging performance is demonstrated in a multispectral microsphere and mouse kidney tissue fluorescence imaging experiment. We also compare quantitatively the depth-resolved IMS with a hyperspectral confocal microscope (HCM) in a standard fluorescent bead imaging experiment. The comparison results show that despite the use of a light source with four orders of magnitude lower intensity in the IMS than that in the HCM, the image signal-to-noise ratio acquired by the IMS is 2.6 times higher than that achieved by the equivalent confocal approach. OCIS codes: (110.4234) Multispectral and hyperspectral imaging; (180.2520) Fluorescence microscopy; (170.6280) Spectroscopy, fluorescence and luminescence.

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M 2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M 2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M 2 receptor to enhanced green fluorescent protein-M 2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n ‫؍‬ 2, 0.495 ؎ 0.019; n ‫؍‬ 4, 0.202 ؎ 0.010; n ‫؍‬ 6, 0.128 ؎ 0.006; n ‫؍‬ 8, 0.093 ؎ 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 -0.24, identifying the M 2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.Much evidence now indicates that G protein-coupled receptors can exist as oligomers (1, 2), a development that has implications for all aspects of GPCR 4 -mediated signaling. Among the many questions prompted by the emergence of such structures is that of oligomeric size. Although commonly referred to as dimers, oligomers of GPCRs have been detected most often by means of coimmunoprecipitation or resonance energy transfer (3). As typically applied, neither technique can distinguish dimers from larger oligomers. The latter have been identified on the basis of their electrophoretic mobility (reviewed in Ref. 4), but the composition of the bands may be unclear, and the size under the conditions of electrophoresis may have little in common with that in the membrane. Larger oligomers also have been identified by approaches in which detection requires the colocalization of three or four proteins, each bearing a different tag (5-11), but such procedures place only a lower limit on the possible size of the array.There have been comparatively few attempts to examine the oligomeric status of a GPCR in a more quantitative and explicit manner. Measurements of BRET at different ratios of acceptor to donor have pointed to dimers of the melatonin receptor (12), the ␤ 1 -and ␤ 2 -adrenergic receptors (13), the M 1 , M 2 , and M 3 muscarinic receptors (14), and the neurotensin receptor (15)....

The M 2 muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)-and FLAGtagged human M 2 receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[ 3 H]methyl scopolamine dissociates from the orthosteric site (k obsd ). The predominantly monomeric status was confirmed by coimmunoprecipitation and by crosslinking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease k obsd by 4.5-and 9.1-fold, respectively; the corresponding estimates of affinity (log K L ) are Ϫ2.55 Ϯ 0.13 and Ϫ2.29 Ϯ 0.14. Gallamine and the allosteric ligand obidoxime decreased k obsd by more than 100-fold (log K L ϭ Ϫ4.12 Ϯ 0.04) and by only 1.1-fold (log K L ϭ Ϫ1.73 Ϯ 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M 2 receptor, albeit at comparatively high concentrations.

Chirality and activity relationships are paramount to pharmaceutical design and synthesis. Generally, point chirality (enantiomeric R and S configurations) is emphasized most in molecules and drugs; however, axis chirality (present when structures adopt conformations with asymmetrical distributions of electron density) must also be considered as a stereogenic unit of interest. Together, stereogenic units and optical isomerism describe the chiral disposition of electron density about nuclei. In this essence, different components of chirality are always present in molecular systems. In assessing the different chiral parameters of pharmaceuticals, the cardiovascular drug carvedilol serves as an ideal example because both enantiomers produce different physiological effects. In the current study, R-and S-4-(2-hydroxypropoxy)carbazol (carvedilol fragment A), along with prochiral and chiral analogues, are studied to investigate the chiral components of carvedilol. Further, the effects of substituent variation about a stereocenter are investigated and discussed using conformational energy as a surrogate of structure (based on the fact that energy is a function of molecular spatial orientation) to determine the energetic equivalency of prochiral and chiral structures. Multidimensional conformational analysis (MDCA) was performed on selected structures using restricted Hartree-Fock (RHF) and density functional theory (DFT with the Becke 3LYP hybrid exchange-correlation functional) molecular orbital computations to elucidate the structural and energetic basis of chirality. The analogues had the following prochiral and chiral structures: R-CH 2 -OH, [R] and [S] R-CHMe-OH, and R-CMe 2 -OH, with substituent R being either MeCH 2 -or ArCH 2 -, where Ar is the carbazole moiety. Potential energy curves (PECs) of torsional angles χ 1 , χ 2 , χ 3 , and χ 10 for R-and S-4-(2-hydroxypropoxy)carbazol verified that all torsional angles are indeed enantiomeric. Correspondingly, the potential energy hypersurfaces (PEHSs) of R-and S-4-(2hydroxypropoxy)carbazol were also enantiomeric, as illustrated with optimizations of conformational minima; converged minima occurred in equivalent point chiral and axis chiral pairs. Similarly to R-and S-4-(2hydroxypropoxy)carbazol, achiral and chiral analogues analyzed by MDCA displayed axis chirality while chiral structures displayed both axis and point chirality. As such, the presence of point and axis chirality in molecular systems allows predictions to be made concerning the orientations of viable conformations of a respective PEHS. Further, the data indicate that chirality induced by an asymmetric distribution of electron density (axis chirality) is always present whenever a structure adopts asymmetric conformations. Like enantiomers of point chirality, axis chiral conformers also occur in pairs. Potential energy surfaces (PESs) were generated about the prochiral and chiral centers for all structures at the RHF/3-21G level of theory and used to test the equivalency of conformational energy between s...

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Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.