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The optical stack is composed of four layers deposited by magnetron sputtering on stainless steel substrates.
The spectral optical constants of the single layers were calculated from the reflectance and transmittance measurements and used to design the optical stack.
Images are presented as z-projections through the entire optical stack unless otherwise noted.
The MGL content of these randomly selected terminals was subsequently examined in a dual-color 3D projection throughout the entire optical stack in NIS-Elements.
To test this idea, intensity values for all four neurochemical markers were quantified from unaltered single images collected from the center of each optical stack and compared between the two genotypes (Fig. 8).
Boutons labeled with a terminal type-specific marker were selected in a single-channel image obtained from the center of the optical stack (approximately 5 μm from the surface of the section), and analysed by Adobe Photoshop CS5.
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Such variances include the spatial resolution between metal aperture walls, color filters used, and optical stacking thickness.
Optical stacks of 1-ìm thick sections were obtained through putatively double-labeled cells.
The combination of such effective fluorophore delivery with confocal laser scanning microscopy allows high quality acquisition of optical stacks and consequent 3D reconstruction.
The micrometer resolution in conjunction with the high number of optical stacks required for reconstruction of an islet over time introduces a considerable breathing artifact.
Terminals were selected from the center of unaltered deconvolved optical stacks, and the optical section in which the MGL-positive puncta were largest and most intense was identified.
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