In this paper, for the first time it is shown how in-line holography in Transmission Electron Microscopy (TEM) enables the study of radiation-sensitive nanoparticles of organic and inorganic materials providing high-contrast holograms of single nanoparticles, while illuminating specimens with a density of current as low as 1–2 e−Å−2s−1. This provides a powerful method for true single-particle atomic resolution imaging and opens up new perspectives for the study of soft matter in biology and materials science. The approach is not limited to a particular class of TEM specimens, such as homogenous samples or samples specially designed for a particular TEM experiment, but has better application in the study of those specimens with differences in shape, chemical composition, crystallography, and orientation, which cannot be currently addressed at atomic resolution.