Planar Airy beam light-sheet for two-photon microscopy

Abstract

We demonstrate the first planar Airy light-sheet microscope. Fluorescence light-sheet microscopy has become the method of choice to study large biological samples with cellular or sub-cellular resolution. The propagation-invariant Airy beam enables a tenfold increase in field-of-view with single-photon excitation; however, the characteristic asymmetry of the light-sheet limits its potential for multi-photon excitation. Here we show how a planar light-sheet can be formed from the curved propagation-invariant Airy beam. The resulting symmetric light sheet excites two-photon fluorescence uniformly across an extended field-of-view without the need for deconvolution. We demonstrate the method for rapid two-photon imaging of large volumes of neuronal tissue.

Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

“…We anticipate the benefit demonstrated in this work can be obtained for any implementation of 2P-SPIM, including using Bessel [10,27,29] or Airy [31,32] beams instead of Gaussian beams, as long as the laser peak intensity remains lower than in the point-scanning case. The nonlinear photodamage threshold involved in 2P-SPIM using different beam shapes would however require further investigations.…”

“…In multiphoton light-sheet microscopy, adjusting the laser pulse frequency remains an attractive yet unexplored strategy to balance signal and photoperturbation. Indeed, most multiphoton light-sheet microscopes described so far [4,[10][11][12][13][25][26][27][28][29][30][31][32] were implemented using a mode-locked Ti:sapphire laser at f ∼80 Mhz, which is the most commonly used laser source in multitphoton microscopy. However, this choice is questionable and may not take full advantage of light-sheet illumination and its orthogonal geometry.…”

Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

“…We have also demonstrated that rotation to a standard Airy beam profile gives a modest increase in the useable field of view, with no loss of resolution. Creation of an Airy beam profile in a light sheet microscope is straightforward to implement, either by the positioning and rotation of a cubic phase mask 17,52 or cylindrical lens 19,22 , or by design of SLM pattern 55 . Experimentalists using an Airy beam light sheet microscope should strongly consider use of the symmetric rotated Airy beam (SRA) beam profile rather than the standard Airy beam profile to realise the increase in useable FOV and resolution uniformity afforded by this beam type.…”

“…Analysis of the empirical data showed that the FOV was extended slightly (4%) using the SRA profile, whilst it was reduced slightly (7%) in the NSRA profile, when compared to the Airy profile (Fig 1f; Airy, 130 µm; SRA 136 µm; NSRA 121 µm). Larger improvements to effective FOV are expected when using different combinations of objective and Airy beam profile, with an improvement of up to 33% demonstrated 52 .…”

“…For developing a deconvolution-free SHG-LSM method we tested two alternate orientations: with the secondary lobes placed symmetrically above and below the imaging plane, termed Symmetrical Rotated Airy (Fig 1b; SRA), or with all secondary lobes being either above or below the imaging plane, termed Non-Symmetric Rotated Airy (Fig 1b, NSRA). A recent publication by Hosny et al uses the term Planar Airy Light Sheet which is the same case as our Symmetrical Rotated Airy 51 . In this work, we distinguish the two beam types by their symmetry about the illumination plane.…”