Energy filtered STEM imaging of thick biological sections

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Abstract
Energy filtered imaging of thick biological specimens was analysed using a dedicated STEM fitted with an energy loss spectrometer and interfaced with a sophisticated data collection set-up. All images were digital, thus permitting a quantitative analysis of the data. We also present a mathematical explanation of the data, which is useful in predicting the quality of thick specimen images formed with energy filtered filtered electrons. It is known that increasing specimen thickness leads to a decrease of the zero energy loss intensity and an increase in higher (multiply scattered) energy loss electrons. We show that contrast decreases gradually with increased energy loss but, most important, the signal to noise ratio is maximal at an energy loss position slightly below the intensity maximum. This is the optimal position for imaging thick specimens. Moreover our studies confirm that the following parameters have similar effects on the energy loss spectra: (1) increased thickness (t); (2) higher average Z number elements (or lower mean free path Ai); and (3) lower primary voltage (V0).