Analysis of Cellular Organelles and Macromolecular Assemblies by Electron Energy Loss Spectroscopy

Abstract

Recent advances in electron energy loss spectroscopy (EELS) have significantly extended the range of applications for biological microanalysis. For example, EELS can now detect physiological concentrations of the important element calcium in rapidly frozen cells with a sensitivity greater than that achievable by energy-dispersive x-ray spectroscopy (EDXS). It can also detect small numbers of phosphorus atoms bound to macromolecular assemblies, and measure water distributions in frozen hydrated tissue. Here we discuss some of these developments in the context of detection limits and mapping techniques in the scanning transmission electron microscope (STEM) and energy-filtering transmission electron microscope (EFTEM).The useful information about elemental composition in EELS of biological specimens generally resides in weak core-edge signals corresponding to atomic concentrations in the 10−5−10−3(1–100 mmol/kg dry weight) range. For example, the Ca L2,3signal/background ratio is typically only 10−3and it is necessary to measure differences in signal that are only 104 of the background. Changes in low-loss fine structure corresponding to varying chemical composition are also very subtle; for example, detection of a 3% change in water content requires reliable measurement of a 0.1 eV shift in the low-loss intensity maximum. To extract such information requires efficient parallel detection of the energy loss spectrum and a high-brightness source to provide a sufficient number of incident electrons. The dedicated STEM is particularly well-suited for analyzing low concentrations of biological elements. If desired, the probe current can be reduced into the picoampere range for low-dose, high-resolution imaging prior to elemental analysis. The STEM’S field-emission source can then be used to deliver a current approaching 10 nA into a ~10 nm diameter probe. High electron flux conditions are ideal for spectrum-imaging applications where adequate counting statistics must be achieved within a limited pixel dwell time. The cold field-emission source of the STEM has the additional advantage of providing electrons with a narrow energy spread of <0.5 eV which is important in fine structure studies.