Multi-photon attenuation-compensated light-sheet fluorescence microscopy
Madhu Veettikazhy, Jonathan Nylk, Federico Gasparoli, Adrià Escobet-Montalbán, Anders Kragh Hansen, Dominik Marti, Peter Eskil Andersen, and Kishan Dholakia
Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-of-view in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging. Here we show this promising approach may be implemented in multi-photon (two-photon) light-sheet fluorescence microscopy and, furthermore, be achieved in a facile manner utilizing a graded neutral density filter, circumventing the need for complex beam shaping apparatus. A “gold standard” system utilizing a spatial light modulator for beam shaping is used to benchmark our implementation. The approach will open up enhanced depth penetration in light-sheet imaging to a wide range of end users.