High Speed Imaging of Living Cells

A new multifocus technique uses a z-splitter prism 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. (Source: S. Xiao, Boston U.)

Researchers have developed a simple method for simul­taneously acquiring images at different depths with a standard micro­scope. The new technique can be applied to a variety of micro­scopy methods, making it useful for a wide range of biological and bio­medical imaging appli­cations. “Optical microscopy has been an indis­pensable tool for studying 3D complex biological systems and processes,” said Sheng Xiao, a member of the research team from Boston University. “Our new multi­focus technique allows live cells and organisms to be observed at high speeds and with high contrast.”

The researchers led by Jerome Mertz developed a new straight­forward and fast way to acquire information from different depths with standard micro­scopy. The new approach can be simply 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,” said Xiao. 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. “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.”

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. 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. The researchers are now working on expanding the technique so that it will work with even more imaging moda­lities. (Source: OSA)

Reference: S. Xiao et al.: High-contrast multifocus microscopy with a single camera and z-splitter prism, Optica 7, 1477 (2020); DOI: 10.1364/OPTICA.404678

Link: Neurophotonics Center, Boston University, Boston, USA

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