Fast optical sectioning for widefield fluorescence mesoscopy with the mesolens based on HiLo microscopy

Schniete, Jan and Franssen, Aimee and Dempster, John and Bushell, Trevor J and Amos, William Bradshaw and McConnell, Gail (2018) Fast optical sectioning for widefield fluorescence mesoscopy with the mesolens based on HiLo microscopy. Scientific Reports, 8. ISSN 2045-2322

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    Abstract

    We present here a fast optical sectioning method for mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm x 3 mm x 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets. Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8±0.2 μm and raw acquisition speed of 1 minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4 mm with lateral resolution of 0.7 μm and axial resolution of 7 μm. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.