(a) Summary of the optical setup designed to generate 12 μm (Full Width Half Maximum), temporally focused, Gaussian, Generalised Phase Contrast (GPC) and holographic (CGH) spots. The setup was equipped with three lasers, two of them delivering nJ-pulse energies at 80 MHz (Coherent Discovery, 1 W, 80 MHz, 100 fs tuned to 920, 940 or 1030 nm; Spark Alcor, 4 W, 80 MHz, 100 fs, 920 nm) and the third a custom Optical Parametric Amplifier (OPA) pumped by an amplified fibre laser, with fixed wavelength output (Amplitude Satsuma Niji, 0.5–0.6 W, 250 kHz, 100 fs, 940 nm). Fluorescence signals were acquired using an sCMOS camera. The microscope was equipped for electrophysiology patch-clamp recordings. (b) Lateral and axial cross sections of two-photon excited fluorescence generated with Gaussian (yellow), GPC (blue) and GCH (red) beams, as indicated in the legend. Scale bars represent 10 μm. (c) Lateral and axial profiles of two-photon excited fluorescence generated with each excitation modality, and the corresponding system response, demonstrating single-cell resolution.