## 6 Excitation of Single/Multiple Fluorescent Beads in 2D/3D  
To estimate the resolution of the setup in a more realistic environment, we used fluorescent beads of different color to mimic single cells in future optogenetic in vivo experiments. We used polystyrene particles (refractive index 1.59) by Spherotec with the dyes \"Nile Red\" (maximum absorption at 505 nm, maximum emission at 555 nm, \(\varnothing\) 5.0-7.9 \(\upmu\)m) and \"Purple\" (maximum absorption at 595 nm, maximum emission at 615 nm, \(\varnothing\) 2.5-4.5 \(\upmu\)m) in aqueous solution and placed 0.5 \(\upmu\)L per layer between the microscope cover slides with a thickness of 170 \(\upmu\)m. For multi-layer measurements, we stacked multiple slides. The layers with beads were observed in transmission using an inverted Microscope (Nikon Ti Eclipse) with 10\(\times\) magnification and an  
Figure 4: **Left**: Distribution of average focus intensity normalized to camera acquisition time over number of generated foci as a log–log plot. Data are further normalized to the maximum at \(n=1\). A wavelength-dependent drop in SNR is visible for both colors starting at around \(n=20\) for 589 nm and \(n=60\) for 488 nm, which might be attributed to the differences in spatial frequencies necessary for displaying foci in a fixed field of view. **Right**: Increase of the mean focus diameter over the number of foci. additional telescope for demagnifying the resulting beam to fit a camera (IDS UI-3042 SE-M). Fluorescence intensities were calculated by fitting Gaussian functions to fluorescent particle recordings, using the mean intensity of all camera pixels for distances smaller than two times the fitted standard deviation. Since laser power and illumination time were adjusted to use the full dynamic range of the camera, we normalized to both, followed by a normalization to the maximum observed value in each measurement. We tested the capability of the setup to excite several beads in one layer simultaneously, as depicted in Figure 5 for the blue laser, by illuminating 5 to 50 fluorescent beads, adjusting the laser power to fully use the dynamic range of our camera. As can be seen in panels (a) and (b), there is only negligible cross-talk between the illuminated beads and neighboring particles, which shows that our setup at 4x magnification is capable of exciting single particles. As panel (c) shows, most of the illuminated particles, shown in red superimposed on the wide-field image of all particles within the field of view shown in blue, are closely packed with other particles. The halos around the particles can be attributed to the emitted fluorescent light being scattered by surrounding unilluminated particles and the high refractive index difference of the particles compared to water. As described in the previous section, by illuminating a number m of fluorescent beads with a holographical illumination setup, the available light energy should split evenly between the fluorescent beads, resulting in a \(m^{-1}\) behavior of the fluorescent light emitted by each particle. Normalizing for the used laser power, panel (d) shows the measured fluorescence intensity over the number \(m\) of illuminated particles as a double-logarithmic plot for both Nile Red and Purple beads. Linear fits reveal a \(m^{-1.37}\) and \(m^{-1.24}\) dependency for 488 nm and 589 nm illumination, respectively. The much higher standard deviations of the measured intensities for the 589 nm illumination are the result of each focus illuminating several neighboring particles simultaneously, since the diameters of the purple beads are on average only half as big as the diffraction limit.