The search for predictive indicators of disease has largely focused on molecular markers. However, biophysical markers, which can integrate multiple pathways, may provide a more global picture of pathophysiology. The adequacy of the chosen, mostly combined, advanced imaging techniques is often determined by their ability for multi-scale follow-up of molecular processes. I will present through two studies the different level of information we have obtained on the pathophysiological processes of two muscular illnesses that are the amyotrophic lateral sclerosis (ALS) and the Duchenne muscular dystrophy (DMD). Early symptoms of ALS usually include muscle weakness or stiffness. Therefore, mechanical response of differentiated myotubes from primary cultures of mice expressing the ALS-causing SOD1 mutation was examined by atomic force microscopy. Simultaneous acquisition of topography and cell elasticity of ALS myotubes was performed by force mapping method, compared with healthy myotubes and supplemented with immunofluorescence and qRT-PCR studies. Wild type myotubes reveal a significant difference in elasticity between a narrow and a wide population, consistent with maturation occurring with higher actin expression. However, this is not true for SOD1 expressing myotubes, where a significant shift of thin population towards higher elastic modulus values was observed indicating that SOD1 mutation induces structural changes that occurs very early in muscle development.Duchenne muscular dystrophy (DMD) is a severe and lethal disease linked to mutations in the dystrophin gene. However, the link between the dystrophin deficiency and the contractile dysfunction of the cardiomyocytes is unclear. Second harmonic generation (SHG) microscopy was used to acquire images on a mouse model of DMD (mdx mice) and control alive cardiomyocytes at different ages and in different experimental conditions to study the cellular cytoskeleton and contractile apparatus organization. SHG makes functional imaging possible as without need of fluorescent staining, by measuring the SHG anisotropy important results are extracted on the myosin filament conformation and the organization of key sarcomeric cardiac proteins.

Using stem cells as anticancer drug carriers aims for targeting tumors and reducing the side effects of chemotherapy. Coupling the multipotential capacities of stem cells with the nanomedical properties of the drugs provides an attractive platform for cancer therapy. Raman microscopy with its high spatial resolution and sensitivity is used as a unique tool to trace drugs and cellular components without labeling. Drug uptake, transfer, and release by stem cells, in addition to the effect on tumor cells were studied by Confocal Raman Microscope.Moreover, to further understand the intra-cellular effect of paclitaxel drug exposure and stem cell treatment upon the stem and cancer cells, we have traced different enzymes and biomarkers intra-cellularly. By software-aided Raman analysis, we were able to detect and measure the enzymes: cytochrome c in the mitochondria and Matrix Metalloproteinase MMP-1, as cellular markers pre and post-treatment. This was performed by calculating the Pearson correlation coefficient for the selected reference spectrum to the spectral correlation matrix. We investigated the cellular viability by means of confocal Raman microscope, also verified by apoptosis and cytotoxic assays.Our results prove the promising use of dental pulp stem cells as cellular vehicles, and significant phenotypic change in cellular marker expression after treatment with paclitaxel. Biochemical imaging of intracellular drug uptake using Confocal Raman would help to have a better understanding of drugs mechanism of action and cellular behavior, including cellular interaction mechanisms.

Multiphoton microscopy (MPM) is a nonlinear high-resolution technique that can be applied to collagen studies such as collagen type I denaturation during caries process. The MPM lies in its simultaneous image creation based on the fluorescence signals produced by a multiphoton excitation (2-photon excited fluorescence [2PEF]) and a coherent nonlinear phenomenon, second harmonic generation (SHG). This study aims to investigate the structural modifications during the dentin caries process by monitoring the variations of the SHG and the SHG/2PEF ratio recorded by nonlinear optical spectroscopy (NLOS). The International Caries Detection and Assessment System (ICDAS) was used as a clinical reference in vitro.To this end, teeth for each ICDAS score were selected and longitudinal sections through the caries lesion were performed. SHG and 2PEF images and spectra were recorded for each ICDAS score (0 to 6).2PEF and SHG images highlighted the microarchitecture and optical properties of the collagen denaturation in dentin during the caries process. The SHG images of carious dentin demonstrated a destruction of the collagen matrix. The variation of intensity of the 2PEF, SHG and the ratio SHG/2PEF allowed the merge of ICDAS scores: ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6. The new groups tend to be similar to the International Caries Classification and Management System (ICCMS). The 2PEF and SHG intensities and the ratio SHG/2PEF are highly altered during the caries process. The ratio SHG/2PEF is a relevant parameter to monitor the caries evolution and it defined three stages: initial lesion (ICDAS 0/1), moderate lesion (ICDAS 2/3) and extensive lesion (ICDAS 4/5/6). The development of a clinical device based on the nonlinear optics will bring a valuable diagnostic tool for caries detection in an everyday practice.

Luminescent nanodiamonds (ND) are attractive tools for nanoscale biologic cellular imaging allowing both photoluminescence (PL) and magnetic resonance imaging [1]. Recent technological developments enable to fabricate bright NDs with high content of nitrogen-vacancy centres [2] that are anticipated to serve as a cell probes. In this work we present novel method of NDs detection in cellular environment. We demonstrate simultaneous visualization of NDs and of the non- labeled nucleus of living cells based on Raman and PL detection as a new tool for the localization of internalized nanoparticles.To this end, NDs of size ranging from ultra-small particles ~ 5 nm to 60 nm were used, prepared from Ib synthetic diamond. Cells used for this experiment were from mammalian breast cancer (MCF7). We report on accomplishing to successfully internalize ND particles in MCF7 cells. We show that ND internalization can be monitored by Raman imaging method using K-mean cluster analysis. Whilst standard Raman imaging methods of NDs make use of the sp3 diamond Raman signal, which limits their use to 100 nm size particles or bigger [3], here we employ Raman imaging in a novel way to detect small near-IR cellular probe. Changes of cells stiffness were detected by force measurement in atomic force microscopy after incubating cells with NDs, suggesting cell membrane hardening upon ND uptake.[1] L. Moore, M. Nesladek et al., Nanoscale (2014)[2] J. Havlik, M. Gulka, M. Nesladek et al., Nanoscale (2013) [3] C.-Y. Cheng et al., Applied Physics Letter (2007)

The Side effects of chemotherapy in cancer treatments are unavoidable due to the lack of anticancer drug’s specificity and it has a painful impact on patient quality of life. Over the past 30 years, increasing efforts is done to optimize chemotherapy dosing to reduce drug toxicity while increasing its efficacy. A new study proves stem cells can act as a drug reservoir and they will release anticancer drug in its original form in nearby area of cancer cells. Stem cells, due to their capacity to uptake drug, can control the drug toxicity. Dental Pulp Stem Cells DPSCs are able to uptake Paclitaxel PTX and could release it in the culture medium gradually. The conditioned culture medium (culture medium plus released PTX from DPSC) is transferred to the breast cancer cells MCF7. Visualizing the drug uptake intracellular could provide us mechanism of action of the drug. Confocal Raman microscopy as a noninvasive label free method is being used to trace drugs intracellular [1,2]. Applying Confocal Raman Microscopy, anticancer drug uptake by MCF7 is imaged. Surprisingly MCF7 - without any direct contact with PTX- showed drug uptake. It proves the stem cells carry and deliver anticancer drug without its modification. It could be a revolution in chemotherapy to avoid the side effects and increase the drug efficacy.[1] H. Salehi et al. Label-free detection of anticancer drug paclitaxel in living cells by Confocal Raman Microscopy. Applied physics letter. 2013, 102, 113701.[2] H. Salehi et al. Confocal Raman data analysis to comparison of apoptotic and non-apoptotic MCF-7 cells caused by anticancer drug paclitaxel. Journal of Biomedical Optics. 2013,18(5), 056010.

We investigated bone regeneration in vitro and in vivo, using porous silicon (pSi) microparticles coupled with nacre proteins. We prepared a composite powder formed by pSi particles loaded with phosphate, and nacre proteins carrying calcium. For in vitro experiments, we used primary culture of human Dental Pulp Stem Cells. Results showed efficiency for both cell adhesion, cell differentiation and CaPO4 formation. But the most interesting results were that this composite was highly efficient to regenerate bone in vivo, even without any additional stem cells grafting.