Foreground: The black dots plot the brightness of a cluster of fluorescently tagged viruses inside a host cell that gets milled with a focused ion beam. The red line is the fitted curve used to guide the cut, ensuring the remaining ultrathin section contains the viruses of interest. Background: A fluorescence heat map from the final slice is overlaid on a grayscale cryogenic electron tomography image, where circular virus clusters line up with the brightest fluorescence. Credit: Anthony Sica/Peter Dahlberg/SLAC National Accelerator Laboratory

Taking images of tiny structures within cells is tricky business. One technique, cryogenic electron tomography (cryoET), shoots electrons through a frozen sample. The images formed by the electrons that emerge allow researchers to reconstruct the internal architecture of a cell in 3D with near-atomic resolution.

The thing is, this method doesn’t work if a sample is too thick. Electrons can’t penetrate through most cells, including human cells. Instead, researchers use a beam of ions to mill these beefier cells down to 200 nanometer-thick slices, but then another challenge arises: ensuring the structure of interest—a ribosome or chloroplast, say—is actually contained in this thin slice. It often takes multiple tries to capture the target object in an eroded sample.

New technique improves milling accuracy

Now, by combining light microscopy with ion beam milling, researchers at the Department of Energy’s SLAC National Accelerator Laboratory have identified an additional signal in fluorescent light that can guide the milling process. This technique improves the accuracy of optically guided milling by roughly an order of magnitude, which will allow cryoET to more easily target small, rare structures within cells, including invading viruses.

The paper is published in the journal Nature Communications.

"Our approach tells you where your object is inside that final thin cell section very accurately and with a high success rate," said Peter Dahlberg, an assistant professor at SLAC and Stanford University. "This improvement also greatly enhances efficiency, as you spend less time milling and missing your targets." Dahlberg developed this time-saving technique with his research associates Anthony Sica and Magda Zaoralová.

Credit: SLAC National Accelerator Laboratory

How the tri-coincident system works

The team used what’s called a tri-coincident system, which aligns the focal planes of three different instruments: a scanning electron microscope, an ion beam for cell milling and an optical microscope for observing tiny objects tagged with fluorescent chemicals. Unlike commercial systems, which do not have co-aligned instruments, this system allows researchers to monitor fluorescence while milling.

Although tri-coincident systems can’t normally resolve objects smaller than 200 nanometers due to the fundamental optical limitations of these instruments, Dahlberg and his team turned to interference, a phenomenon of light wave interactions, to overcome this limitation.

As an ion beam erodes the top of a cell, fluorescent light from the object of interest shines up from underneath. The sample’s top surface reflects these light waves, which then turn around to interfere with the incoming light—much like ripples on a pond that cross and create more complex patterns. In this case, as the surface of the sample is eroded, interference causes the fluorescent light from tagged objects to dim and brighten. Sica wrote software that can precisely locate the fluorescent object based on these patterns of dimming and brightening, allowing more accurate milling.

Applications and future directions

To test out the approach, the team imaged a 26 nanometer-wide virus infecting a human cell, demonstrating that this approach can target biological structures previously off limits with cryoET. The technique could be applied to other viral particles and tiny, transient structures involved in cell division, as well as other objects.

"I want to show the field that using this effect and this tri-coincident geometry is the right way to target something very small in cells in their native state," Dahlberg said.

Next, the team wants to incorporate advanced light microscopy methods into the tri-coincident system to further improve the optical image quality and take the most informative cryoET images possible.

"There’s a lot of research and development into milling, but I want to incorporate fancier, sophisticated fluorescence techniques," Sica said.

Publication details

Anthony V. Sica et al, Optical interference for the guidance of cryogenic focused ion beam milling beyond the axial diffraction limit, Nature Communications (2026). DOI: 10.1038/s41467-025-65548-8

Citation: Tightening the focus of subcellular snapshots: Combined approach yields better cell slices for cryoET imaging (2026, January 17) retrieved 17 January 2026 from https://phys.org/news/2026-01-tightening-focus-subcellular-snapshots-combined.html

This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.