## 7 Conclusions  
The technique presented in this paper allows for the simultaneous holographic shaping of light of different wavelengths for advanced optogenetic experiments. In the presented configuration, experiments using simultaneous stimulation by ChR2 (Channelrhodopsin 2) [2] or ChRimson [44] and inhibition by eNpHR [45], Jaws [46] or BiPOLES [47] are possible. Compared to micro-LED- or OLED-based approaches, a high lateral spatial resolution with high intensities can easily be reached by the flexible use of laser light. Other than DMD or generalized phase contrast methods, system aberrations can be controlled with the same spatial light modulator. We have shown that, using an iterative optimization scheme, the lateral spatial resolution can be pushed to less than 1.3 times the diffraction limit. However, a compromise between spatial resolution and available field of view has to be made. Diffraction-limited performance of the system is essential when sub-cellular resolution is required, for example, for probing responses to the stimulation of individual processes of single neurons. Splitting the laser beams to up to 100 individual foci using computer-generated holograms and illuminating up to 50 fluorescent beads with sizes below that of single cells, we have shown that the optical system is capable of illuminating multiple individual cells at once with high contrast, where the addressing of 20 to about 50 individual cells appears feasible. It can be expected that, using common liquid crystal on silicon SLMs, higher diffraction efficiencies and therefore higher contrast and more simultaneous foci can be achieved, the binary phase modulators used in this paper, however, offer much higher temporal resolution, and therefore higher flexibility in the temporal design of experiments. Testing on fluorescent beads, we have shown that the axial resolution with a 4 \(\times\) 0.1 \(NA\) microscope objective is only suited for addressing individual cells in well-engineered low-density three-dimensional samples with about 300 \(\upmu\)m distance between axially collinearly located cells, as can be expected for single-photon holographic illumination approaches. Under these restrictions, foci can be freely placed in a sample in three dimensions. Here, multi-photon stimulation systems [19; 26] have a clear advantage. For future experiments, the setup can be adapted to employ femtosecond light sources for two-photon stimulation experiments. In conclusion, the setup presented is a dedicated computational adaptive instrument, addressing, in particular, in vitro experiments for neural network analysis or arrhythmia control of cardiomyocytes, where highly flexible optogenetic stimulation and inhibition are crucial.  
F.S.: experimental setup. T.H.: conduction of experiments. F.S. and T.H.: data evaluation. F.S. and L.B.: writing of manuscript. V.B. and J.W.C.: manuscript revision and supervision. All authors have read and agreed to the published version of the manuscript.  
Funding by the German Research Foundation (Deutsche Forschungsgemeinschaft (DFG), grants CZ 55/39-1 and BU 2974/4-1) is greatly acknowledged.  
Not applicable.  
Data Availability Statement: Data supporting the findings of this study are available from the corresponding author upon reasonable request.  
The authors thank [PERSON] and [PERSON] from the Center for Regenerative Therapies (CRTD), TU Dresden, and [PERSON] from Universitats-Augenklinik Bonn, University of Bonn, for valuable discussions.  
The authors declare no conflict of interest.