New electron microscopy method opens window into structural studies of thicker, more complex biological samples

Scientists at the Rosalind Franklin Institute have demonstrated a novel way to recover high-resolution structural details of biological objects using transmission electron microscopy – a technique that could reveal new details from thicker, more complex specimens than before.

The approach, called electron Fourier ptychography (eFP), reconstructs key properties of the wave of electrons that exit a sample as a beam passes through, allowing visualisation of structure with exceptional clarity. The proof-of-principle study, led by Dr Jingjing Zhao and published in Scientific Reports, shows that eFP can achieve high-resolution imaging at ultra-low fluence – crucial for studying delicate biological materials that are easily damaged by the electron beam.

“Our motivation was to apply a technique that can solve current imaging issues and meet future requirements for imaging thicker biological specimens,” explains Dr Zhao, Postdoctoral Scientist at the Franklin.

In conventional electron microscopy, detectors record only the intensity of transmitted electrons, not their phase – a property relating to the timing of the wave’s oscillations which contains vital structural information. Recovering that phase is a significant challenge, with a traditional ptychographic approach – one that scans a convergent beam across the sample – requiring specialised hardware and complex data collection.

Electron Fourier ptychography takes a different route in which the specimen is illuminated with slightly tilted beams and a series of corresponding images is recorded. A computational algorithm then combines these images to recover the complex electron wave leaving the sample – in effect, reconstructing the structural information.

In Jingjing and colleagues’ study, the method achieved atomic-level detail on gold particles using a high electron fluence, and sub-nanometre resolution on protein crystals under cryogenic conditions at ultra-low fluence. Importantly, it required no modifications to the microscope – only a change in how data is collected and processed. This means other researchers could adopt the technique without needing expensive new equipment.

One of the main motivations for developing eFP, Jingjing explains, is its potential to recover useful data from thicker biological specimens, which are currently difficult to interpret with conventional cryo-electron microscopy imaging. For thin biological samples, such as those below 200 nanometres, scientists generally assume that image contrast is mainly determined by small phase differences – tiny shifts in how the electron waves pass through the sample which reveal structural details. In thicker samples, however, the contrast becomes more complex – a mixture of both electron phase shifts and changes in amplitude.

Electron Fourier ptychography could help to separate these two contributions, allowing researchers to study the structures of thicker specimens. Jingjing says: “With conventional cryo-electron microscopy imaging, when the sample is thick, it’s hard to separate the phase and amplitude signals. This method can help us decouple them, opening the way to imaging thicker and more complex biological objects in the future.”

Beyond thickness, the technique also offers the potential for three-dimensional imaging. By extending the method from 2D to 3D, researchers could reconstruct volumetric information from large biological samples, such as small organelles or sections of cells, at unprecedented resolution.

“We’ve proved the concept in 2D,” says Jingjing. “The next step is to move into 3D and integrate this with existing workflows for cryo-electron microscopy and cryo-electron tomography. We want to push the boundary of electron microscopy for imaging thick biological samples. The Franklin is uniquely equipped to enable this by providing access to advanced instruments specifically designed for experimentation and the development of new methods.”

The Franklin’s forthcoming chromatic aberration-corrected electron microscope – the first of its kind in the UK – will further enhance what eFP can achieve, allowing even sharper and more accurate reconstructions.

Jingjing sees electron Fourier ptychography as a bridge between materials science and life science, bringing proven computational imaging methods from one field into another. “This is an advanced imaging method to help us understand biological structures and, through them, life itself,” she says. “Structural studies are the foundation for understanding how life works at the molecular level, which is crucial in learning more about health and disease. With electron Fourier ptychography, we’re opening a new window into that world.”