An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescencefrom the specimen, which is normally placed in contact with a first surface reflector. The resulting excitationSW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular tothe optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart.Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminatedregions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescencemicroscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs withdifferent axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased thesampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateralresolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with imagesof the membrane and cytoskeleton of living and fixed cells.