Spinning disk confocal modules are “core facility friendly”; they insert conveniently between a commercial microscope base and camera, improve image quality and add no significant drawbacks. In contrast, high numerical aperture (NA) light sheet microscopy often requires radical sample modification, substantial user re-training and fully customized hardware. We present a “core-facility friendly” light-sheet: a “black box” that inserts between a commercial microscope base and camera, greatly reducing photo-toxicity without degrading image quality or breaking compatibility with existing sample preparation.
In 2008, based on the ingenious ‘Remote-Refocus’ of E.J. Botcherby and T. Wilson [Botcherby 2007] , C. Dunsby [Dunsby 2008] invented a brilliant single-objective light sheet technique called 'Oblique Plane Microscopy' (OPM) that bypassed many typical light sheet drawbacks. However his theoretical NA of 0.74 for a water immersion objective was significantly lower than the 1.33 limit for an aqueous sample. In 2018 B. Yang and B. Huang [Yang 2018] made a clever modification to OPM to achieve an NA of 1.06 and incorporated the elegant galvo-based volumetric scanning method invented by M.B. Bouchard and E.M.C. Hillman [Bouchard 2015].
Building on this work, we present a simple, robust and modular light sheet design with an NA in the 1.2-1.3 range, and discuss the key concepts and considerations for high NA single objective light sheet.
[Botcherby 2007] An optical technique for remote focusing in microscopy; E.J. Botcherby, R. Juškaitis, M.J. Booth and T. Wilson; Optics Communications, vol 281(4), p880-887, (2007) https://doi.org/10.1016/j.optcom.2007.10.007
[Dunsby 2008] Optically sectioned imaging by oblique plane microscopy; C. Dunsby; Optics Express, vol 16(25), p20306-20316, (2008) https://doi.org/10.1364/OE.16.020306
[Bouchard 2015] Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms; M.B. Bouchard, V. Voleti, C.S. Mendes, C. Lacefield, W. B. Grueber, R.S. Mann, R.M. Bruno and E.M.C. Hillman; Nature Photonics, vol 9, p113-119, (2015) https://doi.org/10.1038/nphoton.2014.323
[Yang 2018] High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy; B. Yang, Y. Wang, S. Feng, V. Pessino, N. Stuurman and B. Huang; bioRxiv, (2018) https://doi.org/10.1101/273359