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 × 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.
Highlights
Widefield techniques capable of performing optical sectioning are highly sought after in biological imaging to reduce photodamage and photobleaching as well as increasing contrast at high acquisition speed
The scaling in At this setting our MATLAB an optical section thickness of 6.8 ± 0.2 μm was measured by evaluating the average FWHM of Gaussian fits to horizontal intensity line plots through the processed image of a tilted fluorescent layer discussed in the Materials and Methods section
We have shown here a fast widefield optical sectioning method for the Mesolens using HiLo microscopy capable of section thickness of 6.8 ± 0.2 μm over the full field of view (FOV) of 4.4 mm, on par with confocal laser scanning microscopy (CLSM) on the Mesolens which can generate sections of 5 μm thickness by imaging a thin fluorescent layer which was set at a tilt
Summary
Widefield techniques capable of performing optical sectioning are highly sought after in biological imaging to reduce photodamage and photobleaching as well as increasing contrast (reduction of out-of-focus blur) at high acquisition speed. The only method to cover such a large FOV with a thin sheet would be to scan the light sheet and acquire multiple images or use a rolling shutter as presented in recent work[12,13,14] This approach would be similar to stitching and tiling on standard microscope lenses but with the FOV remaining unchanged and the illumination moving through several positions in the FOV. A related method called HiLo microscopy (“Hi” and “Lo” representing the high and low spatial frequency components, not to be confused with HILO, highly inclined and laminated optical sheet17) has been developed recently[18] which makes use of the optical sectioning capability of SIM without any super-resolution content. We elected to write our own script to perform the computational side of HiLo microscopy in MATLAB and compared its performance to an ImageJ plugin which was written by the developers of HiLo microscopy
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