Spectral images of cryo-preserved macromolecular complexes are collected in the STEM-EELS mode. In addition to two spatial dimensions ( x and y ), these contain an energy-loss dimension ( E ). From these images, the zero-loss portion is extracted to form elastic bright-field (EBF) images, serving as reference images, from which particle positions and reconstruction poses can be determined by refining a reference reconstruction. Then, E images are extracted for all energy bins along the energy-loss spectrum. The particle coordinates and reconstruction poses determined from the reference are then applied to each of these imagesindicated here at one exemplary energy lossuch that a volume can be reconstructed for every energy loss, generating a 4D data set with three spatial dimensions and one energy-loss dimension. In addition to analysis of the reconstructions, localized spectra can be generated by plotting the values of specific voxels or voxel regions as a function of energy loss.
