A New Way to Capture High Quality 3D Images of Live Cells and Organisms

By Hilary Katulak and Katherine Gianni

Optical microscopy has been an indispensable tool for studying complex biological systems. The most common technique to record images with a microscope is with a digital camera, which is cost effective, light efficient, low noise, simple to use and involves no moving parts. However, this method is often hampered by problems of speed and complexity when performing 3D volumetric imaging.

Researchers from Boston University’s Biomicroscopy Lab have uncovered an innovative and simple solution. Detailed in a paper published in Optica, the researchers, led by biomedical engineering professor Jerome Mertz, outline a flexible and versatile platform for high speed, high contrast, large field-of-view 3D imaging. The platform makes use of a simple z-splitter implemented with standard widefield microscopy which can be added to existing systems and is easy to replicate — making it an accessible and attractive tool for biological and biomedical research.

Capturing multifocus images

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.

The researchers used their multifocus technique to capture phase-contrast microscopy images of the feeding behavior of tiny rotifers. The video shows nine different focal planes. Video courtesy of Sheng Xiao, Boston University.

“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,” said Sheng Xiao, a lead member of the BU research team.

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.

Researchers developed a new multifocus technique that uses a z-splitter prism (right) to split detected light in a standard microscope. This simultaneously produces several images, each focused to a different depth in the sample, in a single camera frame. Photo courtesy of Sheng Xiao, Boston University.

“Our extended volume 3D deblurring algorithm suppresses far-out-of-focus background from sources beyond the imaging volume,” said Xiao. “This improves both the image contrast and signal-to-noise ratio, making it particularly beneficial in fluorescence imaging applications involving thick samples.”

Demonstrated versatility

The researchers demonstrated the new technique with commonly used microscopy modalities, including fluorescence, phase-contrast and darkfield imaging. They captured large field-of-view 3D images encompassing hundreds of neurons or entire freely moving organisms as well as high-speed 3D images of a rotifer cilia, which beat every hundredth of a second. This showed how the approach provides the flexibility to prioritize a large field-of-view or high speed.

The researchers used their new technique to capture fluorescent microscopy images of freely swimming worms known as C. elegans. The video shows nine different imaging depths, or focal planes. Video courtesy of Sheng Xiao, Boston University.

To demonstrate the capabilities of the extended volume 3D deblurring algorithm, the researchers imaged various thick samples, including the brain of a living mouse. They observed significant contrast and signal-to-noise ratio improvements compared to both raw multifocus images and more traditional 3D deblurring algorithms.

“Optical microscopy has been an indispensable tool for studying 3D complex biological systems and processes,” said Xiao.

“Our new multifocus technique allows live cells and organisms to be observed at high speeds and with high contrast.”

This new imaging system has broad application, from helping neuroscientists better understand brain function and cure neurological diseases, to aiding researchers conducting behavioral studies of small animals.

Moving forward, the researchers are now working on expanding the technique so that it will work with even more imaging modalities.

Read the full paper here.

Learn about the Biomicroscopy Lab: http://sites.bu.edu/biomicroscopy/.