## 4 Discussion  
Our setup can provide multiple foci with a diameter down to about 8 \(\upmu\)m. This does not meet the diffraction limit, a fact that we mainly attribute to spherical aberrations, and, in the case of multiple spots, to not meeting the sampling theorem of the superposed binary Fresnel zone plates. Using the same focal length, this problem can partly be solved either by using a smaller pixel pitch or by using more pixels and a higher magnification. Nevertheless, the focus size is well below the size of neuron soma and therefore suited for single cell stimulation. Employing two-photon excitation using femtosecond lasers is quite common as of today and provides higher lateral and axial resolution. The object of our future investigations, however, will be nearly two-dimensional neural networks that do not require ultrafast lasers. The power density in a single focus can be as high as 1.5 W/mm\({}^{2}\). Regarding single cell stimulation, it can be estimated from the data presented in [40] that a minimum PBR \(r_{min}\) of about 20 should be observed to ensure minimal unwanted optical stimulation. Using the fit function to our measured data of multiple focus generation, the maximum number of simultaneously stimulatable cells in future experiments can be estimated as \(n=\sqrt[14]{\frac{8200}{r_{min}-5.9}}\sim 100\), at the full laser diode power of 60 mW. We showed that the ferroelectric modulator is able to switch modulation patterns at a rate of up to 1.7 kHz, which allows for single cell stimulation times down to less than 600 \(\upmu\)s. More importantly, multiple foci can be generated with delays that are down to this timescale. It has to be noted that temporal modulation can be done using the modulator only without the need for modulated light sources, which stresses the need for a fast modulator. Furthermore, a fast switching between wavelengths for the alternating stimulation and inhibition of neural ability becomes feasible. Regarding speed alone, DMD/MEMS-based modulators could be faster. However, using amplitude modulation such systems offer poor light efficiency, as discussed in [32] as well as a lower diffraction efficiency when compared to binary phase modulation, as demonstrated here in accordance with [34]. Our simulations indicate that for the generation of randomly positioned foci, an about six times higher PBR can be achieved using our system as compared to the amplitude modulation of DMD-based approaches. This is of higher importance for our future experiments employing simultaneous cell stimulation than multi-kHz stimulation speed. In our first experiments, we compared common full field optogenetic stimulation with single-cell optical stimulation and could observe a localized highly correlated and even increased response to the single-cell stimulation. Spike sorting of the respective electrode's spike train shows we are able to stimulate a single neuron. Figure 10: Spike sorting of the spike train of electrode 32 during full field stimulation resulted in two clusters (**a**,**b**). Holographic single-cell stimulation revealed only a single cluster (**c**). Future iterations of the setup could be used e.g., for subcellular stimulation by several means: According to Equation (4), either the magnification \(m\) or the pixel pitch \(\Delta\) of the modulator could be decreased. Since the latter mainly depends on technological development, the former is the immediate option. However, the resulting reduction of the working distance has to be taken into account. So far, experiments showing the effects of stimulating several individual input channels, i.e., dendrites, of neurons have only been done using patch-clamp techniques, which opened a new view on neurons as multi-threshold signal switches. Investigations along the same lines with more individually controlled inputs might offer new views on neural network connections and signal pathways, as well as into the forming of memory.