Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture

Area of Science:

• Microscopy
• Biophotonics
• Optical Imaging

Background:

• Light-sheet microscopy faces trade-offs between field of view, resolution, and detection efficiency.
• High-NA objectives offer resolution but have limited depth-of-field, hindering efficient signal capture in thick samples.
• Existing methods struggle to balance imaging depth and resolution in light-sheet microscopy.

Purpose of the Study:

• To develop an improved light-sheet microscopy strategy, ExD-SPIM, to overcome the depth-of-field limitation of high-NA objectives.
• To match the detection depth-of-field to the illumination light sheet thickness for enhanced fluorescence collection.
• To improve signal-to-noise ratio and volumetric imaging capabilities in light-sheet microscopy.

Main Methods:

• Introduced ExD-SPIM (extended depth-of-field selective-plane illumination microscopy).
• Utilized a phase mask to axially extend the depth-of-field of high-NA detection objectives.
• Validated through numerical simulations, imaging of bead phantoms, and live animal imaging.

Main Results:

• ExD-SPIM successfully extended the depth-of-field of high-NA objectives while preserving lateral resolution.
• Increased total fluorescence collection, reduced background, and improved signal-to-noise ratio (SNR).
• Achieved over threefold SNR increase compared to conventional low-NA light-sheet imaging and reduced photobleaching.

Conclusions:

• ExD-SPIM effectively addresses the depth-of-field limitation in high-NA light-sheet microscopy.
• The technique significantly enhances imaging performance, offering improved SNR and reduced photobleaching.
• ExD-SPIM enables more sensitive volumetric imaging, as demonstrated by increased neuron detection in whole-brain activity imaging.