Multi-photon attenuation-compensated light-sheet fluorescence microscopy

Abstract

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 (twophoton) 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.

Challenges and advances in optical 3D mesoscale imaging

“…In light-sheet microscopy, both the illumination light path as well as the detection light path can be affected by scattering and absorption, with penetration of the light-sheet being a concern for the illumination, and the imaging depth relative to the detection lens affecting the emitted light traveling through the sample. Although much effort has been spent in obtaining a greater degree of penetration for light-sheet microscopy, 86,87 to date, this effort has been focused on obtaining a signal via 'attenuation compensation' rather than correcting for inhomogeneity in the sample per se. The presence of differentially attenuating/scattering structures in a mesoscale sample casts 'shadows', leading to concealed regions in the sample or distortion of the expected signal strength at the detector.…”