New Technique Improves Capture of Multifocus Microscopy Images

Boston University researchers have developed a straightforward and fast way to acquire information from different depths with standard microscopy. The new technique can be added to most existing systems and is easy to replicate, making it accessible to other researchers.

Standard camera-based microscopy systems acquire sharp images at a single focal plane. Although researchers have tried various strategies to simultaneously acquire images with different focal depths, these approaches typically require multiple cameras or use a specialized diffractive optical element to perform image splitting with a single camera. Both strategies are complex, and a diffractive optical element can be difficult to manufacture.

"We used a z-splitter prism that can be assembled entirely from off-the-shelf components and is easily applied to a variety of imaging modalities such as fluorescence, phase-contrast or darkfield imaging," explained Sheng Xiao, first author on the paper published in Optica today. The z-splitter prism divides detected light to simultaneously produce several images in a single camera frame. Each image is focused at a different depth in the sample. Using a high-speed camera with a large sensor area and high pixel count allowed the researchers to distribute multiple high-resolution images on the same sensor without any overlap.

The multifocal images acquired with the new technique make it possible to estimate the out-of-focus background from the sample much more accurately than can be done with a single image. The researchers used this information to develop an improved 3D deblurring algorithm that eliminates the out-of-focus background light that is often a problem when using widefield microscopy.

The team demonstrated their new technique with commonly used microscopy modalities, including fluorescence, phase-contrast, and darkfield imaging.