The thickness determination of organic crystals under low dose conditions using electron energy loss spectroscopy

In the 3-dimensional (3-D) reconstruction of protein crystals with variable thicknesses the electron images and diffraction patterns can only be merged if the crystal thickness is known. Measurement of the thickness using the ratio of the number of inelastically scattered electrons to the number of electrons in the zero loss peak can be accomplished with parallel electron energy loss spectrometry (PEELS). A theoretical analysis of the accuracy of the technique on paraffin crystals of different thicknesses is presented. Our experimental studies with paraffin crystals show the feasibility of measuring a single layer of 47 A with good accuracy under low dose and low temperature conditions. A simple experimental apparatus is proposed to obtain thicknesses from small regions of unstained protein crystals prior to collecting the 3-D data sets from the unexposed area of the same crystal.