In situ elemental analysis and visualization in cryofixed nervous tissues: X‐ray microanalytical investigations of embryological and mature brain, inner ear, photoreceptors, muscle and muscle spindles. Comparison of preparation methods for analysis and visualization at cellular and subcellular levels

Abstract

Summary For meaningful X‐ray microanalysis (XRMA) in biology and medicine, the development of preparative and quantitative methods has been necessary. The methods need to preserve close to in vivo distribution of diffusible ions with at the same time reasonable morphological preservation of the tissue. Analyses at low and middle microanalytical resolution are useful at the initial stages of an investigation or when data from large populations of samples have to be acquired. Cryomicrotomy, which makes it possible for the single cells within semi‐thin and thick cryosections examined by X‐ray microanalysis to be further characterized histochemi‐cally (enzyme and substrate content), has been adopted for several pathophysiological studies. The method is particularly suitable for the analysis of complex morphological tissues with many cell types as in the brain or sensory organs of the internal ear. For microanalysis at the subcellular level, we developed a preparative procedure based on the frozen fixed preparation which is freeze‐dried in vacuo at −80°C and then at the same temperature, without breaking the vacuum, impregnated with a low‐temperature Lowicryl‐type resin. The resin is polymerized by u.v. light. This method prevents redistribution of the ions in the tissue and retains the antigenicity of the tissue. A considerable number of cells can be analysed simultaneously and the elemental composition in different cell compartments can be compared due to the similar analytical conditions within the section. An alternative to thin plastic sections of freeze‐dried material is thin cryosections cut at −150°C and analysed at low temperatures. Although some methodological problems still exist in preparation of cryosections, this type of section is potentially the most useful in analysis of diffusible ions, especially calcium which in most biological systems is present in very low concentrations. New preparative techniques for XRMA brought severe problems in visualization of the specimens prepared by cryomethods. Charging, low contrast, mass loss and contamination, which are often negligible in conventional electron microscopy, have still to be solved in XRMA of cryoprepared specimens. However, the methods of semi‐thin and thick cryosectioning and low‐temperature embedding were successfully used for analysis of cells and organelles and for the study of fluids in restricted biological spaces such as the inner ear, muscle spindles and ventricles of the brain in rats. Accordingly, examinations which were impossible by micropuncture and ion selective techniques could be carried out by XRMA. New data on the developing embryological organs and maturation of biological fluids in small biological cavities were obtained.