Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

Area of Science:

• Biomedical Imaging
• Microscopy Techniques
• Optical Sectioning

Background:

• Mesoscopy requires high-resolution imaging of large biological samples.
• Existing optical sectioning methods can be slow and limited in field of view.
• Confocal laser scanning microscopy (CLSM) offers good resolution but is time-consuming for large volumes.

Purpose of the Study:

• To present a fast optical sectioning method for mesoscopy based on HiLo microscopy.
• To enable rapid, subcellular resolution imaging of large specimens.
• To overcome limitations of speed and field of view in current mesoscopic imaging.

Main Methods:

• Utilized HiLo microscopy principles for optical sectioning.
• Employed Mesolens with a high pixel-number camera and sensor-shifting for a 259.5 Megapixel image.
• Developed custom software for HiLo processing of large datasets.
• Performed widefield epifluorescence imaging.

Main Results:

• Achieved imaging of specimens up to 4.4 × 3 × 3 mm³ in under 17 hours (estimated for 1000 images).
• Obtained sectioning strength comparable to CLSM, with sections as thin as 6.8 ± 0.2 µm.
• Demonstrated a raw acquisition speed of 1 minute per slice, up to 30 times faster than CLSM.
• Achieved lateral resolution of 0.7 µm and axial resolution of 7 µm over a 4.4 mm field of view.

Conclusions:

• The HiLo mesoscopy method provides a fast and effective solution for optical sectioning of large specimens.
• This technique offers subcellular resolution throughout large volumes, surpassing traditional methods in speed.
• Successfully applied to image fixed hippocampal neuronal specimens and zebrafish larvae, demonstrating its utility.