Supplementary Figure 2: Light-sheet characterization.
From: 3D high- and super-resolution imaging using single-objective SPIM
(a) Top, light sheet imaged in the xy plane of a fluorescent solution obtained by scanning a Gaussian beam along the mirror axis. Bottom, single Gaussian beam at different defocusing strengths illustrating the ability to localize the thinnest portion of the light sheet at a variable distance from the reflecting mirror. The red rectangles represent the position of the mirror where the excitation beam is reflected. (b) Top, spatial characterization of the Gaussian excitation laser beam reflected on the 45° mirror (red rectangle). Bottom, Gaussian beam cross-section (red dots) and Gaussian fits (black lines) at the three positions (i, ii and iii) represented in the top panel along the light propagation. The cross-sections show perfect Gaussian profiles, as expected. (c) Beam profile along the light propagation (red dots) measured from the width of the Gaussian fitting represented in b all along the propagation axis of the Gaussian beam. The black line represents the fit with the equation of propagation of a Gaussian beam (Online Methods). The dotted lines represent the position of the beam cross-section represented in the lower panels of b. (d) Light-sheet field of view (two times the Rayleigh length) and thickness (FWHM) for different objectives (100×/1.4-NA, 60×/1.2-NA, 40×/0.75-NA, 20×/0.5-NA, 10×/0.3-NA) and beam diameters at the back focal plane of the objective. The black line represents the theoretical relation between the field of view and the thickness of a light sheet created by the focalization of a Gaussian beam at 561 nm (Online Methods).
