FULL ARTICLE Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy Francesca R. Bertani 1 , Elisabetta Botti 2 , Luisa Ferrari 1 , Valentina Mussi 1 , Antonio Costanzo 2 , Marco D ' Alessandro 1 , Francesco Cilloco 1 , and Stefano Selci * ,1 1 CNR-ISC Istituto del Sistemi Complessi, Consiglio Nazionale delle Ricerche, Via fosso del Cavaliere, 100 00133 Rome, Italy 2 Dermatology Unit, NESMOS Department, Sapienza University of Rome, via di Grottarossa 1035, 00189 Rome, Italy Received 5 March 2015, revised 8 July 2015, accepted 5 August 2015 Published online 17 September 2015 Key words: hyperspectral microscopy, multivariate analysis, melanoma cells HaCaT cells co-culture. 1. Introduction The possibility of discriminating single cell and tissue properties without perturbation of their metabolic or differentiation status has enormous potential impact in diagnostic [1] and therapeutic fields [2]. The chance of gaining information from cells living and evolving in their environment opens a new way for cell therapy, regenerative medicine, personalized im- munotherapy and cancer treatment [3]. The defini- tion of methods for label free and non-invasive high resolution imaging represents one of the principal goals of several methodological and instrumental ap- proaches. In recent years, we are experiencing a sort of change of paradigm in imaging methods, similarly to what happened in life sciences. There, the change in genomic paradigm has meant the transition from the strict correspondence of one protein to only one function, to a more complex view of genetic and epi- genetic world. As regards the imaging methods we are going toward the " global analysis " of cell fea- * Corresponding author: e-mail: stefano.selci@isc.cnr.it , Phone: +39 06 4993 4167, Fax: +39 06 4548 8043 A novel hyperspectral confocal microscopy method to se- parate different cell populations in a co-culture model is presented here. The described methodological and instru- mental approach allows discrimination of different cell types using a non-invasive, label free method with good accuracy with a single cell resolution. In particular, mela- noma cells are discriminated from HaCaT cells by hyper- spectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluores- cence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity.

We present a procedure for the determination of the interaction potential from the knowledge of the radial pair distribution function. The method, realized inside an inverse Monte Carlo simulation scheme, is based on the application of the maximum entropy principle of information theory and the interaction potential emerges as the asymptotic expression of the transition probability. Results obtained for high density monoatomic fluids are very satisfactory and provide an accurate extraction of the potential, despite a modest computational effort.

The forward and backward scattering parts (phase II) of the new neutron spectrometer, TOSCA, replacing the old TFXA and TOSCA (phase I), have been successfully installed at the ISIS pulsed neutron source. The results show a significant enhancement in the counting rate due to the larger detector area. The improved resolution (to 1.5%-–3% of the energy transfer) as compared with the previous instruments (TXFA: 2.5%-–3.5%; TOSCA-I: 2%–-3.5%) has been achieved by increasing the primary flight path from 12 m to 17 m. A chopper has been added in order to avoid neutron frame overlap and to reduce the fast neutron background. Additional diffraction capability will be installed in the near future. An example of the high resolution that is routinely available is provided by the spectrum of potassium hydrogen phtalate at 20 K.

Inelastic neutron scattering experiments were performed at intermediate and high momentum transfer, up to 88.2 Å-1, to study the temperature dependence of single hydrogen mean kinetic energy in polycrystalline and liquid hydrogen sulphide (H2S), in the temperature range 16-206 K. Values of the hydrogen mean kinetic energy were extracted, within the impulse approximation, by fitting to the high momentum transfer data a model response function, obtained from a momentum distribution which is the orientational average of a multivariate Gaussian function. The extracted kinetic energies are compared with a harmonic model for the vibrational and roto-translational dynamics. The model makes use of the hydrogen-projected density of states worked out from intermediate momentum transfer data, as well as of optical frequencies determined from Raman and infrared (IR) spectroscopy. A fairly good agreement is obtained in the whole temperature range, while noticeably lower values for the kinetic energy are found if a single atom momentum distribution of isotropic Gaussian shape is assumed in the model response function.

The forward and backward scattering parts of a new neutron spectrometer, TOSCA-II, replacing the old TFXA and TOSCA-I, have been successfully installed at the ISIS pulsed neutron source. The results show a significant enhancement in the counting rate due to the larger detector area. The improved resolution (to 1.5÷3% of the energy transfer) as compared with the previous instruments (TXFA 2.5 ÷ 3.5%, TOSCA-I 2÷3.5%) has been achieved by increasing the primary flight path from 12 m to 17 m. A chopper has been added in order to avoid neutron frame overlap and to reduce the fast neutron background. Additional diffraction capability will be installed in the near future.

We report good agreement between neutron and X-ray measurements of the static structure factor S(Q) of liquid mercury, as well as the first determination of its isothermal density derivative from neutron measurements

The back-scattering part (phase I) of the new incoherent inelastic neutron spectrometer, TOSCA, replacing the old TFXA, has been successfully installed at the ISIS pulsed neutron spallation source. The results, owing to the much larger detector area, show a significant enhancement of the counting rate and an improvement of the resolution. In spring 2000, the forward scattering bank (phase II) will be completed and, during the final installation, the whole instrument will be moved from the present 12 m primary flight path to 17 m. This will result in a large improvement of the overall resolution (from 2-3% to 1-1.5% of the energy transfer). A chopper will be added in order to avoid neutron frame overlap and to reduce the fast neutron background. In addition, TOSCA will have a diffraction capability.

The study of the density dependence of neutron diffraction spectra in dilute gases, already applied in recent papers to the determination of two- and three-body interaction properties in monatomic insulating fluids, is extended here to the case of a molecular fluid. Deuterated methane has been chosen for its good neutron scattering properties and its small molecular anisotropy. The centre-of-mass static structure factor S(Q) has been measured at four densities between 0.55nm-3 and 1.84nm-3 and at a temperature just below critical. Density analysis of the Fourier transform c(Q) of the direct correlation function makes it possible to show that valuable information on the intermolecular interactions is contained in the neutron data.

Neutron diffraction on low-density systems offers the possibility of a direct probing of the intermolecular forces. This method, used in the last few years for monatomic systems like rare gases, is applied here to the case of gaseous CD4 at low densities on the 180 K isotherm. The intermolecular scattering has been analysed in order to extract C(q), i.e. the Fourier transform of the direct correlation function. The zero-density limit of C(q), which depends on the (spherical average of the) pair potential only, is compared to the same quantity obtained from effective intermolecular pair potentials found in the literature.