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 × 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.

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 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.

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Rapid imaging of large biological tissue specimens such as ultrathick sections of mouse brain cannot easily be performed with a standard microscope. Optical mesoscopy offers a solution, but thus far imaging has been too slow to be useful for routine use. We have developed two different illuminators for light-sheet mesoscopy with the Mesolens and we demonstrate their use in high-speed optical mesoscale imaging of large tissue specimens. The first light-sheet approach uses Gaussian optics and is straightforward to implement. It provides excellent lateral resolution and high-speed imaging, but the axial resolution is poor. The second light-sheet is a more complex Airy light-sheet that provides sub-cellular resolution in three dimensions that is comparable in quality to point-scanning confocal mesoscopy, but the light-sheet method of illuminating the specimen reduces the imaging time by a factor of 14. This creates new possibilities for high-content, higher-throughput optical bioimaging at the mesoscale.

We demonstrate a femtosecond single pass Raman generator based on an YVO crystal pumped by a high energy fiber laser at a wavelength of 1064 nm and a repetition rate of 1 MHz. The Raman generator shifts the pump wavelength to 1175 nm, in a broad band spectrum, making it suitable for multi-photon microscopy. We use the Raman generator for third harmonic generation imaging of live plant specimens as well as for two-photon fluorescence imaging of red fluorescent protein (RFP) expressing HeLa cells. We demonstrate that the photo-damage to a live specimen is low