Figure 1. Schematic representation of the SPIM illumination geometry (1A).

IML-SPIM optical system adapted for two-photon photoactivation (1B). M mirrors, DM dichroic mirrors, BE beam expander, CL cylindrical lens, TL tube lens, F bandpass filter. Either a sample or a mirror for alignment can be alternatively inserted into the holder mounted on the motorised stages. Direct observation of the light-sheet illumination intensity distribution created by the cylindrical lens is achieved by inserting a plane mirror into the sample holder, oriented at 45 degrees with respect to the illumination axis, acquiring the reflected light on the EMCCD camera. Illumination light-sheet distributions with a comparable thickness were chosen for single-photon and two-photon excitation (1C insets). The illumination intensity (fig. 1C insets) can be described by an elliptical gaussian-lorentzian distribution and its thickness can be obtained by performing a fitting procedure: examples of sample curves and the relative fits are shown in fig. 1C for single-photon excitation (R²≈0.99) and two-photon excitation (R²≈0.98)). Scale Bar = 50 µm. Objective: Leica HCX APO L U–V–I 20×.