Multi-photon holographic patterned illumination using temporal focusing (TF) pulses provides 10 μm multi-spots with an axial confinement of 5 μm, enabling parallel photo-stimulation of multi-target cells in three dimensions. However, interference fringes and speckles degrade the uniformity of the spots, causing spatio-temporal fluctuations in photoactivation. Here, we demonstrate fringe- and speckle-free holographic patterned illumination using multifocal time-multiplexed (TM) TF pulses even when the multi-spots overlap spatially. The uniform patterned illumination enabled the expansion of the field-of-view of TM-TF microscopy by more than fivefold and enhanced the axial resolution to 1.3 μm.
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