Scanning transmission ion microscopy mass measurements for quantitative trace element analysis within biological samples and validation using atomic force microscopy thickness measurements B Guillaume Deve `s a, * , Touria Cohen-Bouhacina b , Richard Ortega a a Laboratoire de chimie nucle ´aire analytique et bioenvironnementale, UMR 5084, CNRS-Universite ´ de Bordeaux 1, BP 120 Chemin du solarium, F33175 Gradignan cedex, France b Centre de Physique Mole ´culaire Optique et Hertzienne, Universite ´ de Bordeaux 1, 351, cours de la Libe ´ration, F33405 Talence cedex, France Received 5 October 2003; accepted 2 April 2004 Available online 11 September 2004 Abstract We used the nuclear microprobe techniques, micro-PIXE (particle-induced X-ray emission), micro-RBS (Rutherford backscattering spectrometry) and scanning transmission ion microscopy (STIM) in order to perform the characterization of trace element content and spatial distribution within biological samples (dehydrated cultured cells, tissues). The normalization of PIXE results was usually expressed in terms of sample dry mass as determined by micro-RBS recorded simultaneously to micro-PIXE. However, the main limit of RBS mass measurement is the sample mass loss occurring during irradiation and which could be up to 30% of the initial sample mass. We present here a new methodology for PIXE normalization and quantitative analysis of trace element within biological samples based on dry mass measurement performed by mean of STIM. The validation of STIM cell mass measurements was obtained in comparison with AFM sample thickness measurements. Results indicated the reliability of STIM mass measurement performed on biological samples and suggested that STIM should be performed for PIXE normalization. Further information deriving from direct confrontation of AFM and STIM analysis could as well be obtained, like in situ measurements of cell specific gravity within cells compartment (nucleolus and cytoplasm). D 2004 Elsevier B.V. All rights reserved. Keywords: STIM; PIXE; AFM; Quantitative analysis; Specific gravity 1. Introduction From the main components of organic molecules (H, C, O and N) to the mineral elements (Na, Mg, P, S, Cl, K, Ca) constituting the substance of all living matter and the essential trace elements (F, Mn, Fe, Cu, Zn, Se, I), around 20 chemical elements are involved to build the entire life [1]. Their distribution within cells and cellular compart- ments as well as their regulation are strictly governed by the DNA. Abnormal distribution or unregulated incorpo- ration of these trace elements can alter cellular metabolism and may lead to cell death. In the same way, any exogenous element, such as toxic heavy metals, could lead to cellular dysfunction. In the post genomic era, where one searches for a complete understanding of cell functioning, the ability to map at cellular level the distribution of chemical elements is then of primary importance. Few analytical techniques are powerful enough to reach that aim because both spatial resolution in the micrometer range and good sensitivity are required. Relevant literature [2] can easily be found about the most used techniques: 0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2004.04.015 $ This paper was presented at the International Congress on X-Ray Optics and Microanalysis (ICXOM XVII), held in Chamonix, Mont Blanc, France, 22-26 September 2003, and is published in the special issue of Spectrochimica Acta Part B, dedicated to that conference. * Corresponding author. Tel.: +33 557 120 903; fax: +33 557 120 900. E-mail address: deves@cenbg.in2p3.fr (G. Deve `s). Spectrochimica Acta Part B 59 (2004) 1733 – 1738 www.elsevier.com/locate/sab