U. ILLINOIS (US) — A new advance in microscopy offers fast, detailed, 3D views of cells’ internal structures without the use of fluorescence or contrast agents.
In a paper published in the journal PLoS ONE, researchers at the University of Illinois who developed the technique report they were able to use it to visualize the E. coli bacteria with a combination of speed, scale, and resolution unparalleled for a label-free method.
The method is based on a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM) that was designed by Gabriel Popescu, a research at the University of Illinois’ Beckman Institute, as an add-on module to a commercial phase contrast microscope.
SLIM is extremely fast and sensitive at multiple scales (from 200 nm and up) but, as a linear optical system, its resolution is limited by diffraction.
By applying a novel deconvolution algorithm to retrieve sub-diffraction limited resolution information from the fields measured by SLIM, Popescu and his fellow researchers were able to render tomographic images with a resolution beyond SLIM’s diffraction limits.
They used the sparse reconstruction method to render 3D reconstructed images of E. coli cells, enabling label-free visualization of the specimens at sub-cellular scales.
Last year the researchers successfully demonstrated a new optical technique that provides 3D measures of complex fields called Spatial Light Interference Tomography (SLIT) on live neurons and photonic crystal structures.
In this project they developed a novel algorithm to further extend the three-dimensional capabilities by performing deconvolution on the measured 3D field, based on modeling the image using sparsity principles. This microscopy capability, called dSLIT, was used to visualize coiled sub-cellular structures in E. coli cells.
These structures have only been observed using specialized strains and plasmids and fluorescence techniques, and usually on non-living cells, the researchers say. These new methods provide a practical way for non-invasive study of such structures.
Mustafa Mir, first author on the paper and member of Popescu’s Quantitative Light Imaging Laboratory at Beckman, says studying and understanding the three-dimensional internal structure of living cells is essential for furthering our understanding of biological function.
“Visualizing them is extremely challenging due to their small size and transparent nature,” Mir says. “This new method, however, provides a way to take advantage of the intrinsic properties of these very small, transparent cells non-invasively and without the use of fluorescence techniques and contrast agents.
“Previous studies have thus used extrinsic contrast such as fluorescence and specialized strains in combination with complex superresolution techniques for such studies. This will allow biologists to study sub-cellular structures while minimally perturbing the cell from its natural state.”
The researchers wrote that the method addresses two major issues in cell microscopy: lack of contrast, due to the thin and optically transparent nature of cells, and diffraction limited resolution.
“Although several such structures have been previously identified, little is known about their function and behavior due to the practical difficulties involved in imaging them,” they conclude. “The results presented here indicate that dSLIT can be used to characterize and study such sub-cellular structure in a practical and non-invasive manner, opening the door for a more in depth understanding of the biology.”
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