Simple FISH-based evaluation of spermatic nuclear architecture shows an abnormal chromosomal organization in balanced chromosomal rearrangement carriers.

INTRODUCTION: Interphasic DNA has a constant three-dimensional conformation, which is particularly striking for spermatic DNA, with distinct chromosomal territories and a constant chromosomal conformation. We hypothesized that this organization is fragile, and that an excess or a lack of chromosomal segments could hinder the genomic structure as a whole. METHODS: Five human male chromosomal translocation carriers and five controls were included. Spermatic DNA spatial organization was studied, in both balanced and unbalanced spermatozoa, with two-dimensional fluorescent in situ hybridization (FISH) via analysis of chromosomes not implicated in the cases' translocations, compared to that of normal controls. Two parameters were studied: the distance between the two telomeric ends of chromosome 1, and the area of the chromosomal territories of chromosomes 1 and 17. RESULTS: Sperm FISH analysis of rearrangement carriers revealed changes in the nuclear architecture compared to that of controls. Inter-telomeric distance and chromosomal territories areas were both significantly increased. DISCUSSION: We show that an excess or lack of chromosomal segments can hinder the normal spatial nuclear architecture in sperm. These results show that nuclear architecture is a fragile assembly, and that local chromosomal abnormalities may impact the nucleus as a whole. This suggests a potential avenue for selection of spermatozoa prior to in vitro fertilization, not only in rearrangement carriers but also in the infertile population at large. Furthermore, we suggest that 2D-FISH could possibly be a useful tool in assessing spermatic nuclear organization in a way to evaluate male fertility.

Monitoring the Kinetics of the Cellular Uptake of a Metal Carbonyl Conjugated with a Lipidic Moiety in Living Cells Using Synchrotron Infrared Spectromicroscopy.

Presented here is the exploitation of synchrotron infrared spectromicroscopy to evaluate the feasibility of monitoring the cellular uptake of rhenium-tris-carbonyl-tagged (Re(CO)3) lipophilic chains in living cells. To this aim, an in-house thermostated microfluidic device was used to limit water absorption while keeping cells alive. Indeed, cells showed a high survival rate in the microfluidic device over the course of the experiment, proving the short-term biocompatibility of the device. We recorded spectra of single, living, fully hydrated breast cancer MDA-MB231 cells and could follow the penetration of the rhenium complexes for up to 2 h. Despite the strong variations observed in the uptake kinetics between individual cells, the Re(CO)3 complex was traced inside the cells at low concentration and shown to enter them on the hour time scale by active transport.

Bimodal X-ray and Infrared Imaging of an Organometallic Derivative of Praziquantel in Schistosoma mansoni.

An organometallic derivative of praziquantel was studied directly in worms by using inductively coupled plasma-mass spectrometry (ICP-MS) for quantification and synchrotron-based imaging. X-ray fluorescence (XRF) and IR absorption spectromicroscopy were used for the first time in combination to directly locate this organometallic drug candidate in schistosomes. The detection of both CO (IR) and Cr (XRF) signatures proved that the Cr(CO)3 core remained intact in the worms. Images showed a preferential accumulation at the worm's tegument, consistent with a possible targeting of the calcium channel but not excluding other biological targets inside the worm.

A rhenium tris-carbonyl derivative as a model molecule for incorporation into phospholipid assemblies for skin applications.

A rhenium tris-carbonyl derivative (fac-[Re(CO)3Cl(2-(1-dodecyl-1H-1,2,3,triazol-4-yl)-pyridine)]) was incorporated into phospholipid assemblies, called bicosomes, and the penetration of this molecule into skin was monitored using Fourier-transform infrared microspectroscopy (FTIR). To evaluate the capacity of bicosomes to promote the penetration of this derivative, the skin penetration of the Re(CO)3 derivative dissolved in dimethyl sulfoxide (DMSO), a typical enhancer, was also studied. Dynamic light scattering results (DLS) showed an increase in the size of the bicosomes with the incorporation of the Re(CO)3 derivative, and the FTIR microspectroscopy showed that the Re(CO)3 derivative incorporated in bicosomes penetrated deeper into the skin than when dissolved in DMSO. When this molecule was applied on the skin using the bicosomes, 60% of the Re(CO)3 derivative was retained in the stratum corneum (SC) and 40% reached the epidermis (Epi). Otherwise, the application of this molecule via DMSO resulted in 95% of the Re(CO)3 derivative being in the SC and only 5% reaching the Epi. Using a Re(CO)3 derivative with a dodecyl-chain as a model molecule, it was possible to determine the distribution of molecules with similar physicochemical characteristics in the skin using bicosomes. This fact makes these nanostructures promising vehicles for the application of lipophilic molecules inside the skin.

An easy-to-detect nona-arginine peptide for epidermal targeting.

A correlative approach combining synchrotron radiation based IR microscopy and fluorescence microscopy enabled the successful detection and quantification of a nona-arginine peptide labelled with a Single Core Multimodal Probe for Imaging (SCoMPI) in skin biopsies. The topical penetration of the conjugate appeared to be time dependent and occurred most probably via the extracellular matrix.

Nuclear volume differences between balanced and unbalanced spermatozoa in chromosomal translocation carriers.

While chromosomal translocations are usually associated with a normal phenotype, they can still cause male infertility as well as recurrent miscarriages and fetal malformations related to their transmission in an unbalanced state. The distinction between balanced and unbalanced spermatozoa on morphological criteria is still unfeasible. However, we previously showed that: i) spermatozoa with an unbalanced content have a higher rate of DNA fragmentation; and ii) that density gradient centrifugation partially separates balanced from unbalanced sperm cells. We hypothesized that a chromosomal imbalance could alter the fine spermatic nuclear architecture and consequently the condensation of DNA, thus modifying normal sperm density. Spermatic nuclear volumes in four translocation carriers were analyzed using confocal microscopy. Secondarily, FISH analysis was used to establish the segregation mode of each spermatozoon. We found the average spermatic nuclei size to be higher among unbalanced spermatozoa in all patients but one. All the unbalanced modes were associated with larger nuclei in two patients, while this was the case for the 3:1 mode only in the other two, suggesting an abnormal condensation. This could be the first step in elaborating a procedure to completely eliminate unbalanced spermatozoa from semen prior to in vitro fertilization.

Metal-carbonyl units for vibrational and luminescence imaging: towards multimodality.

Metal-carbonyl complexes are attractive structures for bio-imaging. In addition to unique vibrational properties due to the CO moieties enabling IR and Raman cell imaging, the appropriate choice of ancillary ligands opens up the opportunity for luminescence detection. Through a classification by techniques, past and recent developments in the application of metal-carbonyl complexes for vibrational and luminescence bio-imaging are reviewed. Finally, their potential as bimodal IR and luminescent probes is addressed.

Luminescence modulations of rhenium tricarbonyl complexes induced by structural variations.

Octahedral d(6) low-spin Re(I) tricarbonyl complexes are of considerable interest as noninvasive imaging probes and have been deeply studied owing to their biological stability, low toxicity, large Stokes shifts, and long luminescence lifetimes. We reported recently the bimodal IR and luminescence imaging of a Re(I) tricarbonyl complex with a Pyta ligand (4-(2-pyridyl)-1,2,3-triazole) in cells and labeled such metal-carbonyl complexes SCoMPIs for single-core multimodal probes for imaging. Re(I) tricarbonyl complexes have unique photophysical properties allowing for their unequivocal detection in cells but also present some weaknesses such as a very low luminescence quantum yield in aqueous medium. Further optimizations would thus be desirable. We therefore developed new Re(I) tricarbonyl complexes prepared from different ancillary ligands. Complexes with benzothiadiazole-triazole ligands show interesting luminescent quantum yields in acetonitrile and may constitute valuable luminescent metal complexes in organic media. A series of complexes with bidentate 1-(2-quinolinyl)-1,2,3-triazole (Taquin) and 1-(2-pyridyl)-1,2,3-triazole (Tapy) ligands bearing various 4-substituted alkyl side chains has been designed and synthesized with efficient procedures. Their photophysical properties have been characterized in acetonitrile and in a H2O/DMSO (98/2) mixture and compared with those of the parent Quinta- and Pyta-based complexes. Tapy complexes bearing long alkyl chains show impressive enhancement of their luminescent properties relative to the parent Pyta complex. Theoretical calculations have been performed to further characterize this new class of rhenium tricarbonyl complexes. Preliminary cellular imaging studies in MDA-MB231 breast cancer cells reveal a strong increase in the luminescence signal in cells incubated with the Tapy complex substituted with a C12 alkyl chain. This study points out the interesting potential of the Tapy ligand in coordination chemistry, which has been so far underexploited.

Influence of the side-chain length on the cellular uptake and the cytotoxicity of rhenium triscarbonyl derivatives: a bimodal infrared and luminescence quantitative study.

Rhenium triscarbonyl complexes fac-[Re(CO)3 (N^N)] with appropriate ancillary N^N ligands are relevant for fluorescent bio-imaging. Recently, we have shown that [Re(CO)3 ] cores can also be efficiently mapped inside cells using their IR signature and that they can thus be used in a bimodal approach. To describe them we have coined the term SCoMPIs for single-core multimodal probes for imaging. In the context of the use of these SCoMPIs in bio-imaging, the questions of their cellular uptake and cytotoxicity are critical. We report here a series of compounds derived from the [Re(CO)3 Cl(pyta)] core (pyta=4-(2-pyridyl)-1,2,3-triazole). The pyta ligand is of interest because it can be easily functionalized. Aliphatic side chains (C4 , C8 , and C12 ) were appended to this core. A correlative study involving IR and luminescence was performed to monitor and quantify their cellular internalization. We studied the relationship between lipophilicity (log P(o/w)), cytotoxicity (IC50 ), and cellular uptake, and we showed that both uptake and cytotoxicity increase with the length of the side chain, with a higher uptake for the C12 derivative. This study stresses the distinction that has to be made between apparent toxicity, determined as an incubation concentration IC50 , and intrinsic toxicity. Indeed, the intrinsic toxicity of a compound can remain hidden if it is not cell permeable. Therefore it must be kept in mind that IC50 values are composite values, reflecting both cellular uptake and intrinsic toxicity.

Fourier transform infrared (FT-IR) spectromicroscopy to identify cell organelles: correlation with fluorescence staining in MCF-7 breast cancer cells.

Biomolecules display specific vibrational signatures in the infrared (IR) range, and organelles that concentrate these biomolecules can be identified by these IR signatures. Subcellular identification and location of cell organelles using IR signatures is attractive as it does not require the use of any specific trackers and is thus non-invasive and non-destructive. We show here that endogenous IR absorptions are relevant to detecting and imaging the nucleus, the cytoplasm, and the Golgi apparatus/endoplasmic reticulum in MCF-7 breast cancer cells, and we compare these results with our previous work on the HeLa cell line. We correlate maps of fixed and dried cells obtained by synchrotron radiation Fourier transform infrared (SR FT-IR) spectromicroscopy with epifluorescence images using fluorescent trackers for Golgi apparatus and nucleus, namely BODIPY TR C5-ceramide complexed to BSA and DAPI, respectively. Interestingly, the ratios of the IR bands CH2 : CH3 (both asymmetric and symmetric) and CO((ester)):amide I were shown to be reliable gauges of the lipidic character of a cellular compartment, the -CH2 and the CO((ester)) absorptions increasing with the presence of inner membranes like in the Golgi apparatus.

Toward optimal spatial and spectral quality in widefield infrared spectromicroscopy of IR labelled single cells.

Advancements in widefield infrared spectromicroscopy have recently been demonstrated following the commissioning of IRENI (InfraRed ENvironmental Imaging), a Fourier Transform infrared (FTIR) chemical imaging beamline at the Synchrotron Radiation Center. The present study demonstrates the effects of magnification, spatial oversampling, spectral pre-processing and deconvolution, focusing on the intracellular detection and distribution of an exogenous metal tris-carbonyl derivative 1 in a single MDA-MB-231 breast cancer cell. We demonstrate here that spatial oversampling for synchrotron-based infrared imaging is critical to obtain accurate diffraction-limited images at all wavelengths simultaneously. Resolution criteria and results from raw and deconvoluted images for two Schwarzschild objectives (36×, NA 0.5 and 74×, NA 0.65) are compared to each other and to prior reports for raster-scanned, confocal microscopes. The resolution of the imaging data can be improved by deconvolving the instrumental broadening that is determined with the measured PSFs, which is implemented with GPU programming architecture for fast hyperspectral processing. High definition, rapidly acquired, FTIR chemical images of respective spectral signatures of the cell 1 and shows that 1 is localized next to the phosphate- and Amide-rich regions, in agreement with previous infrared and luminescence studies. The infrared image contrast, localization and definition are improved after applying proven spectral pre-processing (principal component analysis based noise reduction and RMie scattering correction algorithms) to individual pixel spectra in the hyperspectral cube.

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques.

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

Synchrotron radiation FTIR detection of a metal-carbonyl tamoxifen analog. Correlation with luminescence microscopy to study its subcellular distribution.

1,1-Di(4-hydroxyphenyl)-2-cyrhetrenylbut-1-ene 1 is an organometallic conjugate where a [(Cp)Re(CO)(3)] unit is linked to a hydroxytamoxifen-like structure. Its subcellular nuclear distribution was previously observed in a single cell using the near-field technique AFMIR. We show here that synchrotron radiation FTIR spectromicroscopy (SR-FTIR-SM) enabled the mapping of 1 based on its IR-signature (characteristic bands in the 1850-2200 cm(-1) range) and pointed out the colocalization of 1 with an area of high amide density. Fluorescence microscopy using DAPI staining performed on the same cells confirmed that this area corresponds to the cell nucleus.

A rhenium tris-carbonyl derivative as a single core multimodal probe for imaging (SCoMPI) combining infrared and luminescent properties.

A rhenium tris-carbonyl derivative has been designed to couple infrared and luminescent detection in cells. Both spectroscopies are consistent with one another; they point out the reliability of the present SCoMPI (for Single Core Multimodal Probe for Imaging) for bimodal imaging and unambiguously indicate a localization at the Golgi apparatus in MDA-MB-231 breast cancer cells.