Stefano Managò

Non-invasive sex assessment in bovine semen by Raman spectroscopy

X- and Y-chromosome-bearing sperm cell sorting is of great interest, especially for animal production management systems and genetic improvement programs. Here, we demonstrate an optical method based on Raman spectroscopy to separate X- and Y-chromosome-bearing sperm cells, overcoming many of the limitations associated with current sex-sorting protocols. A priori Raman imaging of bull spermatozoa was utilized to select the sampling points (head-neck region), which were then used to discriminate cells based on a spectral classification model. Main variations of Raman peaks associated with the DNA content were observed together with a variation due to the sex membrane proteins. Next, we used principal component analysis to determine the efficiency of our device as a cell sorting method. The results (>90% accuracy) demonstrated that Raman spectroscopy is a powerful candidate for the development of a highly efficient, non-invasive, and non-destructive tool for sperm sexing.

Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia

Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.

Bioderived Three-Dimensional Hierarchical Nanostructures as Efficient Surface-Enhanced Raman Scattering Substrates for Cell Membrane Probing

In this work, we propose the use of complex, bioderived nanostructures as efficient surface-enhanced Raman scattering (SERS) substrates for chemical analysis of cellular membranes. These structures were directly obtained from a suitable gold metalization of the Pseudonitzchia multistriata diatom silica shell (the so called frustule), whose grating-like geometry provides large light coupling with external radiation, whereas its extruded, subwavelength lateral edge provides an excellent interaction with cells without steric hindrance. We carried out numerical simulations and experimental characterizations of the supported plasmonic resonances and optical near-field amplification. We thoroughly evaluated the SERS substrate enhancement factor as a function of the metalization parameters and finally applied the nanostrucures for discriminating cell membrane Raman signals. In particular, we considered two cases where the membrane composition plays a fundamental role in the assessment of several pathologies, that is, red blood cells and B-leukemia REH cells.

Combined Raman Spectroscopy and Digital Holographic Microscopy for Sperm Cell Quality Analysis

The diagnosis of male infertility is vastly complex. To date, morphology, motility, and concentration have been used as key parameters to establish the sperm normality and achieve pregnancy both in natural and in assisted fecundation. However, spermatozoa from infertile men could present a variety of alterations, such as DNA fragmentation, alterations of chromatin structure, and aneuploidy, which have been demonstrated to decrease reproductive capacity of men. Therefore, the ability to see detailed relationships between morphology and physiology in selected spermatozoa with submicrometric resolution in a nondestructive and noninvasive way and within a functional correlated context could be extremely important for the intracytoplasmic sperm injection procedure. In this review, we describe label-free optical spectroscopy and imaging techniques, based on the combination of Raman spectroscopy/imaging with holographic imaging, which are able to noninvasively measure the (bio)chemistry and morphology of sperm cells. We discuss the benefits and limitation of the proposed photonic techniques, with particular emphasis on applications in detection/characterization of sperm cell morphological defects and photodamage, and the identification/sorting of X- and Y-bearing bovine spermatozoa.

Raman detection and identification of normal and leukemic hematopoietic cells

The analysis of leukocytes of peripheral blood is a crucial step in hematologic exams commonly used for disease diagnosis and, typically, requires molecular labelling. In addition, only a detailed, laborious phenotypic analysis allows identifying the presence and stage of specific pathologies such as leukemia. Most of the biochemical information is lost in the routine blood tests. In the present study, we tackle 2 important issues of label-free biochemical identification and classification of leukocytes using Raman spectroscopy (RS). First, we demonstrate that leukocyte subpopulations of lymphocytes (B, T and NK cells), monocytes and granulocytes can be identified by the unsupervised statistical approach of principal component analysis and classified by linear discriminant analysis with approximately 99% of accuracy. Second, we apply the same procedure to identify and discriminate normal B cells and transformed MN60 lymphocyte leukemic cell lines. In addition, we demonstrate that RS can be efficiently used for monitoring the cell response to low-dose chemotherapy treatment, experimentally eliciting the sensitivity to a dose-dependent cell response, which is of fundamental importance to determine the efficacy of any treatment. These results largely expand established Raman-based research protocols for label-free analysis of white blood cells, leukemic cells and chemotherapy treatment follow-up.

Nanosphere Lithography on Fiber: Towards Engineered Lab-On-Fiber SERS Optrodes

In this paper we report on the engineering of repeatable surface enhanced Raman scattering (SERS) optical fiber sensor devices (optrodes), as realized through nanosphere lithography. The Lab-on-Fiber SERS optrode consists of polystyrene nanospheres in a close-packed arrays configuration covered by a thin film of gold on the optical fiber tip. The SERS surfaces were fabricated by using a nanosphere lithography approach that is already demonstrated as able to produce highly repeatable patterns on the fiber tip. In order to engineer and optimize the SERS probes, we first evaluated and compared the SERS performances in terms of Enhancement Factor (EF) pertaining to different patterns with different nanosphere diameters and gold thicknesses. To this aim, the EF of SERS surfaces with a pitch of 500, 750 and 1000 nm, and gold films of 20, 30 and 40 nm have been retrieved, adopting the SERS signal of a monolayer of biphenyl-4-thiol (BPT) as a reliable benchmark. The analysis allowed us to identify of the most promising SERS platform: for the samples with nanospheres diameter of 500 nm and gold thickness of 30 nm, we measured values of EF of 4 × 105, which is comparable with state-of-the-art SERS EF achievable with highly performing colloidal gold nanoparticles. The reproducibility of the SERS enhancement was thoroughly evaluated. In particular, the SERS intensity revealed intra-sample (i.e., between different spatial regions of a selected substrate) and inter-sample (i.e., between regions of different substrates) repeatability, with a relative standard deviation lower than 9 and 15%, respectively. Finally, in order to determine the most suitable optical fiber probe, in terms of excitation/collection efficiency and Raman background, we selected several commercially available optical fibers and tested them with a BPT solution used as benchmark. A fiber probe with a pure silica core of 200 µm diameter and high numerical aperture (i.e., 0.5) was found to be the most promising fiber platform, providing the best trade-off between high excitation/collection efficiency and low background. This work, thus, poses the basis for realizing reproducible and engineered Lab-on-Fiber SERS optrodes for in-situ trace detection directed toward highly advanced in vivo sensing.

Normal-State Optical Features Study of Nd123 and Gd1212 HTSC Materials for Photonics and Metamaterials Fabrication

YBCO-like Nd123 and Gd1212 superconductors in the normal state, in the visible and infrared regime, have been analyzed in this work. Superconductors, particularly high-temperature superconductors (HTSCs), hold indeed a great interest in the highly innovative research field of metamaterials and photonics with important applications. Indeed nanostructured devices suffer from increasing resistivity with frequency in metals, and superconducting materials provide a viable route to approach this problem. Moreover, HTSCs are plasmonic materials at nonzero temperature, and their features, not available in conventional materials, make them very attractive for metamaterials use. In particular, we investigate Nd123, for its high Tc, and Gd1212, for the coexistence, in its unitary cell, of magnetic order and superconductivity. Samples were extracted from top-seeded melt-textured growth bulk pellets and bars. Fourier transform infrared spectroscopy analysis, performed in the far-infrared, shows articulated peak structures with k <; kmin and a plateau for higher k, allowing for Nd123 and Gd1212 pseudogap measurement. Raman spectroscopy and microscopy provided a structural mapping of our Nd123 samples, allowing for distinguishing the superconducting matrix from nonsuperconducting phase inclusions (precipitates) and providing an instrument to discriminate among optical responses of the different compounds.

Diatomite nanovectors uptake in cancer cells: a Raman imaging study

In this work, we employed the Raman microscopy to study the internalization kinetics and spatial distribution of small interfering RNA (siRNA)-diatomite nanoparticles (DNPs) complex in human lung epidermoid carcinoma cell line (H1355) up to 72 h. Raman images are compared with confocal fluorescence microscopy results. The Raman analysis provides that the siRNA-DNPs are internalized and co-localized in lipid vesicles within 18 h, after that equilibrium is achieved.