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. Set of hyperspectral images of melanoma-keratinocytes co-culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label-free spectral identification of cell populations (right). J. Biophotonics 9, No. 6, 619–625 (2016) / DOI 10.1002/jbio.201500122 © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim