Uniform Illumination Module

Typical digital scanning light-sheet microscopes use a Gaussian beam profile for their excitation beam. However, as a Gaussian beam is convergent at the start, ideal in the middle, and divergent at the end, axial blurring and reduced z-resolution at the edge of large fields of view (FOVs) can occur. These limitations of Gaussian beam profiles are overcome with Bruker's uniform illumination module.

How Does the Uniform Illumination Module Work?

A tunable acoustic gradient lens (or TAG lens) achieves fast axial scanning with superior light-sheet control, beam uniformity, and consequently uniform PSFs across the FOV.

With the uniform illumination module:

• A focused Gaussian beam moves along the illumination axis at ~70 kHz.
• Integration of the illumination beam over time and space produces an elongated, uniform light sheet.
• Out-of-focus information is reduced using a scanned light sheet in combination with line mode.

This module is available as an optional add-on for the MuVi SPIM and TruLive3D Imager and it comes as a built-in module for the LCS SPIM.

The uniform illumination module is particularly well-suited for high signal-to-noise ratio samples. By providing consistent lighting across the entire FOV, the module ensures that the signal-to-noise ratio remains stable throughout the imaging process. This is crucial for capturing accurate and reliable data, especially when dealing with high-contrast samples where subtle details are essential. 

▲ The relationship of resolution and FOV. (1) High NA objectives allow a high resolution across a small FOV. (2) Low NA objectives allow a low resolution across a large FOV. (3) The uniform illumination module uses fast axial scanning to enable a high resolution across a large FOV.
 

The uniform illumination module allows for clear visualization of details within specimens that would otherwise be difficult to distinguish without consistent illumination and an extended FOV. Otherwise, the undistinguishable features on the edges of the FOV are equally well resolved as those closer to the middle of the FOV. One such application is imaging large, cleared samples, such as a cleared mouse brain section displaying cortical motor neurons. 

Without uniform illumination, neuronal somata at the edge of the FOV suffer from axial blurring, making them indistinguishable. Capturing them with the uniform illumination module enhances the axial resolution at the edges, which allows for a clear separation of neuronal somata throughout the FOV.

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