J. Phys. IV France 104 (2003) @EDP Sciences, Les Ulis DOI : 10. 1051/jp4 : 200300096 345 Soft X-ray contact microscopy and transmission electron microscopy : Comparative study of biological samples T. Limongi', L. Palladino1 5, E. Bernieri2, G. Tomassetti', L. Reale', F. Flora3, P. Cesare4, C. Ercole4, P. Aimola4 and A. M. Ragnelli4 1 Physics Department, L'Aquila University, Italy 2 INFN, National Laboratory of Frascati, Italy 3 ENEA, Frascati, Italy 4 Basic and Applied Biology Department, L'Aquila University, Italy 5 Prof. Libero Palladino, Physics Department, L'Aquila University, 67010 Coppito, L'Aquila, Italy Abstract. Isolated cellular organelles (mitochondria, chloroplasts) and cultured bacteria were analysed both by soft X-ray contact microscopy (SXCM), and by transmission electron microscopy (TEM) after négative staining. For each sample, a comparison was performed betweenimages obtainedwith either technique,with the aim of facilitating the interprétation of SXCM images.The validity and the limits of this comparative approach are discussed. 1. INTRODUCTION Soft X-ray Contact Microscopy (SXCM) was developed for the observation of living biological Systems in their physiological state, at a resolution superior to that of light microscopy, but the interprétation of the images produced by this technique is not always easy, both for the examination of intact single cells and for the study of isolated cellular organelles, which is currently limited to chloroplasts [1]. Searching for a simple way to interpret SXCM images, we decided to try a comparative method : we compared images of the same biological samples obtained with SXCM, and with Transmission Electron Microscopy (TEM) after négative staining, which does not require any treatment of chemical fixation, so that similar observation conditions are achieved. 2. MATERIALS AND METHODS 2. 1 Biological samples préparation Chloroplasts were isolated from fresh spinach leaves by differential centrifugation. The pellets were suspended in 0.35 M NaCI. The chloroplast préparations were tested in order to assess their photosynthetic ability by means of the Hill reaction activity. Mitochondria were isolated from bovine heart and liver by differential centrifugation and prepared for the observations either directly, or after further purification on Sucrose density gradient. The mitochondrial pellets were resuspended in 0. 148 M NaCI. The mitochondrial preparations were tested to verify their functional activity, by determining the oxidation rate and the control of respiration by phosphate accepter. Only the préparations having an accepter control ratio higher than 3.5 were used. The f/ï/'ocfgr sp. strain used was isolated and cultured by our microbiology laboratory. Bacterial cells were harvested by centrifugation and the pellets suspended in 0.9% NaCI. 2.2 Transmission electron microscopy observations Samples of mitochondria, chloroplasts and bacteria were examined after négative staining with the drop method [2] that requires less than one minute to be accomplished. The staining solution was Ammonium