Membrane potential around single molecules has been measured by using the nonlinear optical phenomenon of second harmonic generation. This advance results from the interaction between a highly dipolar molecule with a selectively directed highly polarizable 1-nm gold particle. With this approach, a second harmonic signal, which is enhanced by the nanoparticle, is detected from a volume of nanometric dimensions. This present work clearly shows that functional cellular imaging around single molecules is possible by selectively directing an antibody with a 1-nm gold label to a specific membrane protein. The results of this work open the way for three-dimensional, high resolution functional imaging of membrane electrophysiology in cells and cellular networks.Recently, it has been shown that the nonlinear optical phenomenon of second harmonic generation (SHG) is a sensitive monitor of cellular membrane potential (1, 2). To observe SHG with sensitivities comparable to linear optical phenomena, tightly focused pulsed laser light with high peak power is required. As reviewed by Nie and Zare (3) single molecules were successfully observed in linear optical imaging as early as 1981 and most recently, with great success, by using confocal microscopy to limit the out-of-focus light that severely degrades the signal to noise in linear optical imaging. However, SHG, either in an imaging or a nonimaging mode, has not been accomplished with single molecule sensitivity.SHG arises from the second term of the expansion of the molecular electron polarizability shown below and thus is called a second-order nonlinear optical phenomenon.where P is the polarization, E is the applied optical electric field, and (n) are the nth order optical susceptibilities. In the recent past, the third term of this equation has generated a lot of interest in biological imaging because it is responsible for the nonlinear optical phenomenon of two-photon fluorescence (TPF) (4-8). The use of nonlinear optics results in naturally high x, y, and z spatial resolution, and, in general with nonlinear optical microscopy, photodamage and photobleaching are reduced because the out-of-focus light does not result in excitation. However, for the particular case of SHG, no photochemistry occurs even in the focal plane because the signal, stimulated by nonresonant radiation, does not involve an excited state with a finite lifetime. Optical effects arising from the first term (linear processes) and the third terms of this equation have no fundamental restriction on the symmetric distribution of the dye molecules that give rise to these phenomena in the cells. Second harmonic generation, on the other hand, has a requirement that symmetrically distributed chromophores will not contribute to the observed signal. This symmetry restriction results in a considerable advantage to the nonlinear optical imaging of cell membranes and their membrane potential because only those molecules that are asymmetrically distributed in the cell membrane contribute to the observe...

Single-molecule studies of the conformations of the intact ␤2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the ␤2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.G protein-coupled receptors (GPCRs) represent one of the largest families of integral membrane proteins, and GPCRs are responsible for most transmembrane signal transduction by hormones and neurotransmitters, as well as for the senses of vision, smell, and taste. GPCRs are characterized by seven transmembrane (TM) domains with an extracellular N terminus and a cytoplasmic C terminus (1). For many GPCRs, such as the ␤ 2 adrenergic receptor (␤ 2 AR), small molecular weight ligands bind to sites within the hydrophobic core formed by the TM ␣-helices. The ␤ 2 AR ( Fig. 1) belongs to the major subfamily of GPCRs that includes rhodopsin, for which a high-resolution crystal structure is now available (2). The binding of agonist ligands to a receptor induces conformational changes that facilitate interactions between the receptor and its cognate G protein.We have developed a method for site-specifically labeling Cys-265 in the ␤ 2 AR with fluorescein-5-maleimide (FM; ref. 3) to yield what we call FM␤ 2 AR. Cys-265 is adjacent to a G protein-coupling domain at the end of TM6 (see Fig. 1). In ensemble measurements, we demonstrated previously that FM bound to Cys-265 sensitively reports conformational changes, with agonists inducing a decrease in the fluorescence intensity of FM␤ 2 AR in proportion to the biological efficacy of the agonist. By using exogenous quenchers localized to different environments, we determined that during agonist-induced activation, the domain adjacent to Cys-265 rotates and͞or tilts to a more buried environment, which is closer to both the surface of the micellar compa...

The regulation of plant hydraulic conductance and gas conductance involves a number of different morphological, physiological and molecular mechanisms working in harmony. At the molecular level, aquaporins play a key role in the transport of water, as well as CO₂, through cell membranes. Yet, their tissue-related function, which controls whole-plant gas exchange and water relations, is less understood. In this study, we examined the tissue-specific effects of the stress-induced tobacco Aquaporin1 (NtAQP1), which functions as both a water and CO₂ channel, on whole-plant behavior. In tobacco and tomato plants, constitutive overexpression of NtAQP1 increased net photosynthesis (A(N)), mesophyll CO₂ conductance (g(m)) and stomatal conductance (g(s)) and, under stress, increased root hydraulic conductivity (L(pr)) as well. Our results revealed that NtAQP1 that is specifically expressed in the mesophyll tissue plays an important role in increasing both A(N) and g(m). Moreover, targeting NtAQP1 expression to the cells of the vascular envelope significantly improved the plants' stress response. Surprisingly, NtAQP1 expression in the guard cells did not have a significant effect under any of the tested conditions. The tissue-specific involvement of NtAQP1 in hydraulic and gas conductance via the interaction between the vasculature and the stomata is discussed.

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