SOLEIL Users’ Meeting 2012
Four accounts

In January 2012 we met Sylvain Clède, winner of the Best Student Poster at the 2012 SOLEIL Users Meeting, so he could tell us about the research described in the award-winning poster. These same results have just been published in the journal Chemical Communications, so this is an opportunity to (re) discover Sylvain’s answers to our questions.

Research group working on this topic at ENS.
Prof. Clotilde Policar is on the 1^st row, 2^nd on the left and Sylvain Clède is on the 2^nd row, 2^nd on the left.

After graduating in science (in Pau) and two years of preparatory classes in physics and chemistry (Toulouse), I was enrolled in 2006 for a Senior Diploma in molecular physical chemistry, jointly accredited by Cachan ENS and Paris Sud. I graduated with a B.Sc. in 2007 and a M.Sc. in physical chemistry in 2008. Following my admission to Cachan ENS in 2008, I prepared for and obtained the Chemistry teaching diploma (called “agrégation”) in 2009.
I then obtained a Masters in Biophysics at Paris 6. My training took place under the direction of Clotilde Policar and François Lambert, in the Laboratory of the BioMolecules (UMR 7203 – UPMC – ENS) in the Chemistry Department at ENS Paris. I started a PhD the following year with the same group while working as a teacher in the ENS (I do my thesis and contribute to the teaching in the school).

My project focuses on the synthesis and intracellular detection of transition metal complexes, specially metal carbonyl complexes of the M(CO)₃ type. We recently showed that such compounds could be mapped in cellulo using their infrared signature.

One example of the mappings obtained by infrared spectromicroscopy on the SMIS beamline is shown in Figure 1. MDA-MB-231 cells (breast cancer) were incubated with P89, a metal carbonyl analogue of tamoxifen (molecule developed in the Friedel laboratory, ENSCP). The maps were generated by measuring and plotting the integral of specific infrared bands (hot spots were obtained). P89 was thus detected by one of its absorption bands at 1925 cm^-1 (image b). The nucleus presents different infrared signatures, including the absorption of amides (amide I band at 1650 cm^-1) reflecting a high concentration of DNA folding proteins, histones (image c). When both maps were superimposed (image d, overlay in yellow) we were able to deduce the localization of P89 in the nucleus. A correlative study with fluorescence microscopy (using DAPI to label the nucleus) backed this observation. 

 
Figure 1. Two MDA-MB-231 breast cancer cells (cells 1 and 2) incubated with a metal-carbonyl tamoxifen analog P89 (10 µM, 1 h, 37 °C, 5% CO₂). (a) Bright field image (scale bar 10 µm). IR mappings recorded at the SMIS beamline: (b) P89-band hot spot (1925 cm^—1, green); (c) amide I-band hot spot (1650 cm^—1, red); (d) P89-band hot spot (green), amide I-band hot spot (red), overlay (yellow). Pixel size: 6 x 6 µm².

Given the diversity of problems encountered in biology, it may be useful to have "multimodal" sensors, detectable in complementary ways. There are examples in the literature of molecules combining fluorescence and MRI or fluorescence and radio emissions. The majority of these objects are of high molecular weight and each modality is assured by a different fragment. We are looking to synthesize probes the multimodality of which is based on a single molecular core (we suggest the acronym SCoMPI for "Single Core Multimodal Probe for Imaging") to cause the least disturbance to the biological system under study.
At the moment we are interested in bimodal probes, both infrared active and luminescent. Fluorescence microscopy is a technique widely adopted by biologists and a wide range of organelle markers is available. Infrared spectroscopy, in turn, involves radiation that is much less damaging to cells and tissues and allows the use of endogenous signals to detect certain organelles (such as the absorption of phosphates and amides to identify the nucleus).
The poster presents the physicochemical characteristics of one of these probes and its potential as a bimodal marker for subcellular imaging.
Its infrared spectral signatures and fluorescence emissions are outside the intrinsic responses of the cell, allowing an unambiguous detection of the compound in cellulo.

From an imaging point of view, a first objective was to show that the information provided by the two spectroscopies was coherent, that the infrared and luminescence maps could be superimposed, giving the same cellular distribution for the probe. We provided evidence of perinuclear localization using both methods, showing the relevance of SCoMPI for bioimaging. Colocalization studies with fluorescence markers showed that the probe was localized to the Golgi apparatus.
The results, for which the poster provides a summary, have just been published in the journal Chemical Communications

Clède S, et al, A Rhenium tris-Carbonyl Derivative as a Single Core Multimodal Probe for Imaging (SCoMPI) Combining Infrared and Luminescent Properties, Chem. Commun. 2012, 48,7729-7731

We had access to two beamlines, each presenting the radiation range required to study our compounds in cellulo using spectromicroscopy: SMIS for infrared and DISCO for UV-visible. We needed spectral information at a subcellular resolution, provided by these two beamlines.

Working on two beamlines (SMIS and DISCO) during sessions programmed close together was crucial: we were able to compare the signals from identical cells and thus validate our methodology and our compounds. This proximity in time and space was a real advantage for us. We also greatly benefited from the strong collaboration between the two groups that operate these beamlines.

We are working on bimodal probes as markers of biologically active molecules.
 

Publication :
Clède S, et al, Synchrotron radiation FTIR detection of a metal-carbonyl tamoxifen analog. Correlation with luminescence microscopy to study its subcellular distribution, Biotechnol. Adv. 2012