Experimental ATR device for real-time FTIR imaging of living cells using brilliant synchrotron radiation sources Cestelli-Guidi Mariangela a , Yao Seydou b , Sali Diego c , Castano Sabine b , Marcelli Augusto a , Cyril Petibois b, ⁎ a INFN - Laboratori Nazionali di Frascati, Via E. Fermi 40, 00044 Frascati (Roma), Italy b Université de Bordeaux, CNRS UMR 5248, IPB. Allée de Saint Hillaire, F33600 Pessac, France c Bruker Optics S.r.l. Viale V. Lancetti 43, 20158 Milano, Italy abstract article info Available online 9 December 2011 Keywords: FTIR imaging Live cell Evanescent wave Surface analysis Spectroscopy In this contribution we present the design of an original Attenuated Total Reflection (ATR)-based device designed for an IR microscope coupled to a FPA detector and optimized for in-vivo cell imaging. The optical element has been designed to perform real time experiments of cell biochemical processes. The device in- cludes a manually removable Ge-crystal that guarantees an ease manipulation during the cell culture and a large flat surface to support the cell growth and the required change of the culture wells. This layout will allow performing sequential ATR IR imaging with the crystal immersed in the culture wells, minimizing con- tributions due to water vapors in the optical system. Using existing brilliant synchrotron radiation sources this ATR device may collect images at the surface of the Ge crystal at a sub-cellular spatial resolution with a penetration depth of the evanescent wave inside the sample of ~500 nm within few seconds. A brief sum- mary of the cellular components that should be detected with such optical device is also presented. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Imaging techniques allowing to analyze live cells in real-time are still limited and not yet optimized (Blow, 2008). The significance of real-time imaging for cell analyzes ranges between picoseconds for structural properties of molecules (ex. folding/unfolding of proteins) and minutes to hours for cell motility, growth, division…etc. The signif- icance of in vivo (or in vitro) imaging deals with the interaction of cells and imaging technology, i.e., the effects of lasers, waves, and other probes on cell homeostasis. In general, taking benefit of the high axial resolution of the modern microscopes, enhancing the spatial lateral res- olution to image smaller objects inside a cell requires just more brilliant sources capable to collect data with enough signal-to-noise ratio (SNR) (Petibois et al., 2009). The drawback in this case is the large heat (and the dose when using ionizing radiation such as X-ray or UV radiation) released to the cell that also means a large interaction with the cellular components. Moreover the convergence of both in vivo and real-time requirements implies that only a limited number of imaging techniques can be applied on the same cells for studying their biochemistry or physiology in normal biological conditions (Stephens and Allan, 2003). The standard approach in the field is UV-confocal fluorescence microscopy, which is widely used due to its capability to image the distribution of a given fluorescent probe inside a cell. The transfection method with GFP (green fluorescent protein) now allows imaging the distribution of a specific molecule without significant cell modification or manipulation, i.e., satisfying the condition of the “normal” biology of a sample (Heim et al., 1995). Although powerful, also the UV- confocal microscopy is a limited technique. Indeed, although the catalog of the existing fluorescent probes is large they do not cover all scientific demands. Fluorescent probe lifetime for dynamic/kinetic imaging is limited and also the compatibility of multiple information with simulta- neous multiple probes is required (Niino et al., 2009). At present, the limitation between fluorescent probes, up to 3 or 4 at the same time, is probably the most important drawback of this technique. The molec- ular and cell biology fields are now looking to different approaches or techniques for the study of cellular processes. There is also a need for quantitative studies, which require to access to the geometrical param- eters of the cell such as size, thickness and volume. Nowadays, the best tomographic imaging should allow differentiating intracellular and ex- tracellular compartments and volumes. This would open the way to quantitative imaging of sub-cellular parameters. The future demand will be coupling chemical/molecular and topographic/morphological techniques to provide accurate quantitative information of the sample. Some chemical imaging techniques may provide global or multi- parametric information combining the advantage of being non- damaging, a condition particularly important for biosamples. These techniques take advantage of the low energy level and non-ionizing character of the visible-IR radiation. This is the case for Fourier- transform infrared (FTIR) spectroscopy, which has been setup as a microscopy technique with fast spatially resolved image acquisition Biotechnology Advances 31 (2013) 402–407 Abbreviations: ATR, attenuated total reflectance; FPA, focal plane array; FTIR, Fourier- transform InfraRed; IRE, internal reflection element; NA, numerical aperture; SNR, signal- to-noise ratio; SR, synchrotron radiation; TIR, total internal reflection. ⁎ Corresponding author. Tel.: + 33 540006848; fax: + 33 540005200. E-mail address: c.petibois@cbmn.u-bordeaux.fr (C. Petibois). 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.11.009 Contents lists available at SciVerse ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv