Uniform Illumination Module

Standard light-sheet microscopes use lasers with a Gaussian beam profile to form the light sheet. Gaussian beams are divergent in nature, and, as a result, their use causes axial blurring and reduces z resolution at the edge of large fields of view (FOVs), i.e., FOVs that are larger than the Rayleigh length of the Gaussion beam.

Our uniform illumination module is designed to overcome the limitations of standard Gaussian beams. With this module:

• A tightly focused Gaussian beam is swept at a high frequency along the illumination axis;
• Integration over time and space produces an elongated, uniform, thin light-sheet, which provides uniform axial resolution over large FOVs; and
• Reduced out-of-focus by using a scanned light sheet.

This module enables the visualization of fine details deep inside the specimen that are hardly resolvable without uniform illumination.

▲ Profile of Gaussian beam generates non-uniform resolution (top). Uniform illumination beam generates a homogeneous illumination profile (bottom).
 

▲ Image of cleared mouse brain section showing cortical motor neurons. Left image acquired without uniform illumination module. Neuron somata at the edge of the FOV are not separable due to axial blurring. Right image recorded with the uniform illumination module. Neuron somata are clearly separable due to uniform and enhanced axial resolution.