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. 2019 Aug 20;13:77. doi: 10.3389/fnana.2019.00077

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Copyright © 2019 Rakotoson, Delhomme, Djian, Deeg, Brunstein, Seebacher, Uhl, Ricard and Oheim.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Principle and performance of the On-axis 2-photon virtual light-sheet generation in vivo imaging system (OASIS). (A) Simplified optical path of our custom 2P microscope. Red: infrared (IR) excitation, green: fluorescence. Ti:Sapph, fs-pulsed IR laser; DS, DeepSee module (pulse stretcher); BE, beam expander; π-shaper, optical element that converts the Gaussian beam into a top hat profile; M, mirrors; dic, dichroic mirrors; SD, spinning disk; TL, tube lens; CDE, corrective distance element; OL, objective lens; ISD, image-splitting device; sCMOS, camera. Inset ❶: (simplified) multi-spot excitation pattern and epi-collection of the generated fluorescence through the same objective. Inset ❷: detail of microlens/pinhole/dichroic coating arrangement. Note the offset between the excitation (exc.) and fluorescence foci (fluo.) at the level of the disk, produced by the CDE. (B) Measured depth penetration in turbid samples. Log-plot of 2P-excited fluorescence from a green-fluorescent Chroma test slide, topped either with water (0%) or increasing concentrations of milk (a model for the multi-scale scatterers present in tissue), for the OASIS (red dots) and a ZEISS LSM710. Confocal pinhole diameters were 1 and 2 Airy, as indicated. (C) OASIS prototype, note the compact size and space available around the objective, inset. VCD, voice coil z-drive; BFC, bright-field camera. (D) Assessment of OASIS lateral resolution. Equatorial section through an autofluorescent thorny pollen grain. Scale bar, 10 μm. Inset: magnified view of an in-focus spine and intensity profiles across the lines shown; Scale bar, 2 μm. (E) OASIS z- resolution. Axial-intensity profile measured from a z-stack of images acquired from a green fluorescent Chroma slide (solid line), and its derivative dF/dz (dashed). The 10%–90% intensity range was taken as axial optical sectioning capacity Δz.