Toward Compositional Contrast by Cryo-STEM

Electron microscopy (EM) is the most versatile tool for studying matter from subatomic to visible scales, but high vacuum and charged irradiation demand careful stabilization of sensitive specimens, especially biological samples composed of light elements in water. Early heavy metal staining enabled contrast and earned the 1974 Nobel Prize for foundational cell biology. A decade later, cryogenic fixation and vitrification preserved native hydrated states at below −150 °C, enabling high-resolution cryo-EM while contending with ongoing radiation damage. Although cryo-EM preserves morphology at near-atomic to atomic resolutions, a relatively untapped advantage is preservation of native composition without added stains. Conventional analytical spectroscopies (EELS/EDS) have low interaction cross sections incompatible with the low-dose requirements of cryo-EM, and conventional cryo-TEM phase contrast complicates quantitative interpretation of pixel intensities, especially at low spatial frequencies. Inspired by soft X-ray cryo-tomography in the water window, which yields quantitative, composition-based absorption maps, this Account asks how quantitative compositional contrast can be transferred to cryo-electron microscopy. Scanning transmission EM (STEM) can provide compositional contrast via incoherent elastic scattering sensitive to atomic number Z. In amorphous materials where coherent effects are absent, dark-field signals quantitatively measure scattered flux integrated over detector angles. Learning to interpret such signals in cryo-STEM opens a new dimension for cryo-EM and tomography of biological specimens. This Account outlines the principles, demonstrates cryo-STEM and cryo-STEM tomography (CSTET) on biological cells and macromolecules, and discusses prospects for further development.