RESUMO
Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional objects in a two-dimensional dataset. SW microscopy is performed with high-magnification, high-numerical aperture objective lenses, and while this results in high-resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low-magnification and high-numerical aperture. With this method, we produce SW images within a field of view of 4.4 mm × 3.0 mm that can readily accommodate over 16,000 cells in a single dataset. We demonstrate the method using both single-wavelength excitation and the multi-wavelength SW method TartanSW. We show application of the method for imaging of fixed and living cells specimens, with the first application of SW imaging to study cells under flow conditions.
RESUMO
Some neurotransmitter-gated ion channels are very much more sensitive to general anesthetics than others, even when they are genetically and structurally related. The most striking example of this is the extreme sensitivity of heteromeric neuronal nicotinic acetylcholine receptors to inhalational general anesthetics compared with the marked insensitivity of the closely related homomeric neuronal nicotinic receptors. Here we investigate the role of the alpha subunit in determining the anesthetic sensitivity of these receptors by using alpha(3)/alpha(7) chimeric subunits that are able to form functional homomeric receptors. By comparing the sensitivities of a number of chimeras to the inhalational agent halothane we show that the short (13 amino acids) putative extracellular loop connecting the second and third transmembrane segments is a critical determinant of anesthetic sensitivity. In addition, using site-directed mutagenesis, we show that two particular amino acids in this loop play a dominant role. When mutations are made in this loop, there is a good correlation between increasing anesthetic sensitivity and decreasing acetylcholine sensitivity. We conclude that this extracellular loop probably does not participate directly in anesthetic binding, but rather determines receptor sensitivity indirectly by playing a critical role in transducing anesthetic binding into an effect on channel gating.