Progress in Electron Energy Loss Spectroscopic Imaging and Analysing Biological Specimens with a Field Emission Scanning Transmission Electron Microscope

A major problem raised in electron microscopy imaging and analytical studies on biological sections is the conflict between high dose requirements for getting useful signal/noise values on the different channels of information and low dose requirements for beam damage reduction. Among various solutions, our approach consists in developing a strategy which optimize the signal extraction associated to the scattering process suffered by any primary electron. The basic design of the Scanning Transmission Electron Microscope (STEM) is well adapted because it is possible, for each probe position on the specimen, to filter and simultaneously record different fractions of the transmitted beam, discriminated in scattering angle with a set of variable apertures and in energy loss with an EELS spectrometer. Electron energy loss spectroscopy constitutes then a basic component of the microscope as pointed out as early as 20 years ago by Crewe (1966). It can be used fruitfully either as a contrast-enhancement device or as a real analytical tool for chemical microanalysis, depending on the recorded fraction of the EELS spectrum. In a previous publication (Colliex et al. (1984)), we had described unconventional modes for STEM imaging of biological structures. The present paper intends to update it, covering two specific aspects: novel imaging methods and analytical microscopy. The basic methodology has not changed noticeably but the concomitant progress in detection design and in data read-out and processing have enlarged the field of applications and improved the limits of detection by at least an order of magnitude.