Seeing the intricate structures inside biological tissue in three dimensions is essential for understanding how cells are organized and how they function. But looking at those structures at the nanoscale is challenging. Conventional microscopy methods can provide high-resolution images, yet they often require samples to be cut into thin sections. X-ray imaging offers a different approach: It can penetrate relatively large samples and produce three-dimensional views without physically slicing those samples apart. At synchrotron facilities, highly focused and coherent X-ray beams can push this technique into the nanoscale.
One such technique is X-ray nano-holotomography, a form of three-dimensional phase-contrast imaging. Rather than simply measuring how much X-ray radiation passes through a sample, the technique also detects subtle phase shifts in the X-rays caused by the material. The technique is full-field, capturing the entire field of view in a single measurement rather than through sequential scanning, which makes it significantly faster than scanning-based methods. Images are collected at multiple distances from the sample, allowing researchers to recover information about the phase of the X-ray wave and reconstruct the sample’s three-dimensional refractive index. Hard X-rays, with their ability to penetrate materials and biological tissue, make the technique particularly useful for nondestructive nanoscale imaging. Modern synchrotron beamlines can achieve spatial resolutions below 50 nanometers, with data acquisition times of less than one hour.
However, achieving high resolution also makes the imaging process more sensitive to imperfections. The X-ray beam used for nano-holotomography is not perfectly uniform. Small errors in the position of the sample can also affect the measurements. These effects become particularly important when imaging features only tens of nanometers across. Traditionally, researchers address these problems in separate stages: First align the images, then retrieve the phase information, and finally reconstruct the three-dimensional object. While this approach works well for many experiments, errors introduced during one stage can carry into the next.
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Image: A new joint nano-holotomography reconstruction technique, based on the bilinear Hessian method and multi-node, multi-GPU acceleration, enables processing of large 3D brain volumes (one slice is shown) and reveals synapses (orange), mitochondria (cyan), endoplasmic reticulum (pink), and chromatin (green) in a neuronal nucleus.