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Mirror-assisted light-sheet microscopy: a simple upgrade to enable bi-directional sample excitation.

Asaph Zylbertal1, Isaac H Bianco1

  • 1University College London, Department of Neuroscience, Physiology & Pharmacology, London, United Kingdom.

Neurophotonics
|August 8, 2024
PubMed
Summary

Mirror-assisted light-sheet microscopy (mLSM) overcomes tissue occlusion in biological samples. This cost-effective upgrade enables uniform excitation and cellular-resolution imaging of previously inaccessible regions, enhancing light-sheet microscopy utility.

Keywords:
calcium imaginglight-sheet microscopyselective plane illumination microscopyvolumetric imagingzebrafish

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Area of Science:

  • Biomedical Imaging
  • Microscopy Techniques
  • Neuroscience

Background:

  • Light-sheet microscopy offers optical sectioning but struggles with opaque tissues.
  • Unidirectional illumination in light-sheet microscopy can lead to uneven sample excitation due to occlusions, as seen in larval zebrafish brain imaging.

Purpose of the Study:

  • To introduce mirror-assisted light-sheet microscopy (mLSM) as a method to improve sample illumination.
  • To overcome limitations of unidirectional illumination in light-sheet microscopy for imaging occluded biological structures.

Main Methods:

  • Developed mLSM by incorporating a mirrored micro-prism into existing light-sheet setups.
  • Utilized a scanned laser beam reflected off the prism to create a second excitation path.
  • Employed online tuning of scanning and laser power for uniform dual illumination.

Main Results:

  • mLSM successfully imaged zebrafish brain regions previously inaccessible due to occlusion.
  • Achieved cellular-resolution imaging in areas illuminated by both direct and reflected light sheets.
  • Demonstrated adjustable imaging quality by translating the excitation objective.

Conclusions:

  • mLSM is a simple, inexpensive, and effective upgrade for light-sheet microscopy systems.
  • This technique enhances data acquisition from biological samples by improving excitation uniformity.
  • mLSM facilitates more comprehensive imaging of complex biological structures without interfering with sample behavior.