Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy

TLDR

Microscopy has advanced biology, yet optical methods require labeling and cryo‑electron microscopy is limited to thin specimens; cryogenic X‑ray diffraction microscopy promises sub‑10‑nm 3‑D imaging of whole cells. The authors aimed to demonstrate quantitative 3‑D imaging of an entire, unstained cell at 50–60 nm resolution using X‑ray diffraction microscopy. They employed X‑ray diffraction microscopy to reconstruct a whole yeast spore cell, achieving 50–60 nm resolution. The reconstruction revealed the 3‑D morphology of organelles—including cell wall, vacuole, ER, mitochondria, granules, nucleus, and nucleolus—and a protruding structure suggestive of germination, showing that X‑ray diffraction microscopy enables quantitative 3‑D imaging of thick biological specimens at nanometer scale.

Abstract

Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3–5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50–60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5–10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.