Preparation of biological tissue sections for correlative ion, electron, and light microscopy

In order to correctly interpret the chemical images obtained using ion microscopy (IM), it is useful to correlate them with the information provided by conventional light microscopy (LM), secondary electron imaging (SEI), backscattered electron imaging (BEI), and electron probe microanalysis (EPMA). Accordingly, we have devised a technique of specimen preparation which allows for the application of several different microanalytical techniques to a single histologic section mounted on the same substrate. Sections are cut onto polyester plastic coverslips (devoid of peaks for any element with atomic number > 9 using EPMA) and studied by LM. After a light rotary coating with carbon (to prevent charging), the section can then be examined by SEI, BEI, and EPMA. Specific areas can be marked for IM study either with an objective‐mounted pin tissue microlocater, or by placing small pieces of metal foil, cut in specific geometric shapes, over features of interest. After sputter‐coating the sample with platinum, metal‐free shadows are visible using a low‐power reflected light microscope available on a typical IM sample chamber as a guide for ion beam placement. The conductive coatings also minimize specimen charging during IM. Post‐IM light microscopy, SEI, and BEI are used to confirm the location of specific areas probed in the IM experiments and to provide information on differential ion‐sputtering artifacts and tissue contaminants. This new correlative technique should permit better understanding of the images obtained with these diverse instruments.