Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots Silvia Corezzi a, * ,1 , Lorena Urbanelli b,1 , Peter Cloetens c , Carla Emiliani b , Lukas Helfen c,d , Sylvain Bohic c,e , Fausto Elisei f , Daniele Fioretto a,g a Dipartimento di Fisica, Università di Perugia, 06123 Perugia, Italy b Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Perugia, 06123 Perugia, Italy c European Synchrotron Radiation Facility, F-38000 Grenoble, France d ISS/ANKA, Forschungszentrum Karlsruhe, D-76021 Karlsruhe, Germany e INSERM U-836 (Team 6), Rayonnement Synchrotron et Recherche Médicale, Institut des Neurosciences Grenoble (GIN), Université Joseph Fourier UMR-S 836, F-38042 Grenoble, France f Dipartmento di Chimica, Università di Perugia, 06123, Perugia, Italy g Research Center SOFT, CNR–INFM, Università di Roma ‘‘La Sapienza,” 00185 Roma, Italy article info Article history: Received 7 November 2008 Available online 5 February 2009 Keywords: Synchrotron-based X-ray fluorescence Quantum dots Chemical imaging abstract Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of ‘‘artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S- XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD–secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and b-tubulin, a protein associated with cytoskeleton microtubules. We set up sam- ples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal speci- ficity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion. Ó 2009 Elsevier Inc. All rights reserved. During recent years, the field of molecular diagnostics has wit- nessed an explosion of interest in the use of nanomaterials in assays for protein markers for many diseases. Intense research has been fueled by the need for practical, robust, and highly sensitive and selective detection agents that can address the deficiencies of con- ventional technologies. The number and uses of bionanoconjugates are growing daily in biomedical applications such as imaging tech- niques, subcellular component identification, and intracellular traf- ficking of molecules. Diagnostics, therapeutics, and basic science all are beginning to benefit from the use of nanoscale materials such as metal and metal oxide nanoparticles, nanowires, and quantum dots (QDs) 2 [1–4]. Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for the mapping of elemental distributions at a subcellu- lar level. Sub-micron spatial resolutions can be achieved in two- dimensional scans by an appropriate focusing of the incident high energy X-ray beam produced by a third-generation synchrotron source [5]. S-XRF is the only available technique for quantitative elemental imaging of whole cells due to the high spatial resolution and to the large penetration depth of hard X-rays, with 10 to 30 lm-thick samples able to be measured without the need for being sectioned. S-XRF has a very high sensitivity for most elements of biological interest, and the detection limit for trace elements has been estimated to be of few thousand atoms within the irradiated section of the sample [6]. Recent examples of elemental imaging of cells include microbes [7], mouse fibroblast cells [8], phagosomes of infected macrophages [9], human lung cells [10], melanoma cells [11], nerve cells of brain tissue [12], microvascular endothelial 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.01.044 * Corresponding author. Fax: +39-075-44666. E-mail address: corezzi@fisica.unipg.it (S. Corezzi). 1 To be equally considered as first authors. 2 Abbreviations used: QD, quantum dot; S-XRF, synchrotron-based X-ray fluorescence; HER2, human epidermal growth factor receptor 2; IgG, immunoglobulin G; D-PBS, Dulbecco’s phosphate-buffered saline; RT, room temperature; PFA, paraformaldehyde; BSA, bovine serum albumin; Si 3 N 4 , silicon nitride. Analytical Biochemistry 388 (2009) 33–39 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio