RT Journal Article
SR Electronic
T1 Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 374884
DO 10.1101/374884
A1 Jan Schniete
A1 Aimee Franssen
A1 John Dempster
A1 Trevor Bushell
A1 William Bradshaw Amos
A1 Gail McConnell
YR 2018
UL http://biorxiv.org/content/early/2018/07/23/374884.abstract
AB We present here a fast optical sectioning method for optical mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm × 3 mm × 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.