Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

Area of Science:

• Biomedical imaging
• Optical microscopy
• Live-cell imaging

Background:

• Confocal laser scanning microscopy provides optical sectioning but can be slow and limited in field of view.
• Need for advanced microscopy techniques that offer high-quality imaging with improved speed and versatility.

Purpose of the Study:

• Introduce and characterize HiLo microscopy, a novel wide-field imaging technique.
• Compare HiLo microscopy performance against standard confocal laser scanning microscopy.
• Demonstrate the applicability of HiLo microscopy for in vivo imaging.

Main Methods:

• HiLo microscopy combines two raw images: one with speckle illumination and another with uniform illumination.
• Numerical fusion of images allows adjustable optical section thickness using a single parameter.
• Performance evaluated based on sectioning strength and imaging quality, including 3-D imaging of GFP-labeled mouse brain hippocampus.

Main Results:

• HiLo microscopy achieves real-time, optically sectioned images comparable in quality to confocal microscopy.
• 3-D imaging of mouse brain hippocampus shows comparable quality between HiLo and confocal methods.
• HiLo microscopy enables faster imaging rates and larger fields of view than confocal microscopy.

Conclusions:

• HiLo microscopy is a simple, robust, and versatile technique for high-quality optical sectioning.
• It offers advantages in speed and field of view over traditional confocal microscopy.
• Demonstrated successful in vivo imaging in planaria, C. elegans, and zebrafish highlights its broad utility.