Arbitrary Two-Dimensional Multiphoton Excitation Patterns with Temporally Focused Digital Holograms

Multiphoton excitation has recently found application in the fields of bioimaging, uncaging and lithography. In order to fully exploit the advantages of nonlinear excitation, in particular the axial resolution due to nonlinearity, most systems to date operate with point or multipoint excitation, while scanning either the laser beam or the sample to generate the illumination pattern. Here we combine the recently introduced technique of scanningless multiphoton excitation by temporal focusing with recent advances in digital holography to generate arbitrarily shaped, depth resolved, two-dimensional excitation patterns completely without scanning. This is of particular importance in applications requiring uniform excitation of large areas over short time scales, such as neuronal activation by multiphoton uncaging of neurotransmitters. We present an experimental and theoretical analysis of the effect of spatial patterning on the depth resolution achieved in temporal focusing microscopy. It is shown that the depth resolution for holographic excitation is somewhat worse than that achieved for uniform illumination. This is also accompanied by the appearance of a speckle pattern at the temporal focal plane. The origin of the two effects, as well as means to overcome them, are discussed.