Fig. 1 Scheme of the Selective Plane Illumination Microscope (A) adapted from [5]. Figure B and fig. C show the adaptation of the system for the experimental measurements. With the cylindrical lens in its standard position, a light sheet is produced in the focus of the objective lens, and a homogeneous fluorescent image of the phantom sample is produced on the CCD Camera (B). Rotating the cylindrical lens by 90 degrees, the axial section of the intensity excitation distribution appears on the CCD sensor.

Fig. 2 Experiments performed on various phantom samples mimicking the different optical properties of biological tissues (5, 10, 30, 50 mm⁻¹ respectively), considering the intensity excitation profile in the direction of propagation. Schematic representation (A-C) of the peak shift (green dot) of the excitation intensity distribution for single photon excitation (blue) and two-photon (red) excitation configurations. The arrows underline how the peak tends to shift back from the ideal case. While keeping constant the travel within the sample in the detection path (150μm) and varying both illumination depth and the scattering properties of the phantom sample, we were able to measure a significant shift for the single photon excitation configuration with respect to the two-photon configuration. D shows an example of the axial section of the measured intensity excitation distributions for the 1P and 2P excitation lightsheets for a phantom sample with 5 mm⁻¹ scattering coefficient at 400 μm illumination depth. E shows the relative peak position of the uniform region with respect to the supposed focus (in a non-scattering sample) for single photon excitation at different illumination depths (400,600 and 800μm deep within the sample) while increasing the scattering coefficient of the phantom sample. 2PE allows the shift of the uniform region to be reduced. Excitation Wavelengths: λ = 488 nm (1P) and λ = 790 nm (2P) Intensity used in 1P experiments: I = 0.13 kW/cm²; Intensity used in 2P experiments: I = 19.78 kW/cm². Detection Objective: Leica 20x, NA 0.5.

Fig. 3 Images of different homogeneously scattering phantom samples with fluorescent beads (diameter = 0.17 μm) by means of single photon and two-photon excitation SPIM. Increasing the scattering coefficient within the sample, it is possible to appreciate how signal to noise ratio in 2PE-SPIM degrades much less in comparison to the single photon case. All images are acquired at 600 μm illumination depth within the samples. Scale bar is 5 μm. Detection objective Leica HCX APO L U-V-I 12 40X, NA 0.8.Additional magnification introduced:2,5X to get a total magnification of 100X. Intensity used in single photon experiments: I = 0.11 kW/cm²; intensity used in 2P experiments: I = 19.23 kW/cm²

Fig. 4 Z-stack of mammary epithelial acini (zstep 1 µm) has been acquired (see Suppl. Media 1). Only representative planes within the cell spheroids (spaced 3μm) are shown (A). Scale bar: 10 µm . Maximum intensity projection of the entire volume (B). Excitation wavelength λ = 750nm, I = 54.94 KW/cm². Objective lens: HCX APO L U-V-I 40x/0.8 WATER.