Advanced Environmental Scanning Electron Microscopy Reveals 3D Organization of Condensed Mitotic Chromosomes in Their Native State

Abstract The challenge of in-situ handling and high-resolution, low-dose imaging of intact, sensitive and moist samples in their native state at the nanometer scale or live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM uses machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator in an in-house modified electron microscope equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. An invisible thin film of ionic liquid is used to increase the thermal and electrical conductivity of the samples and minimize damage from free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not directly observed before by any microscopic technique.