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We present the development of a cost-effective, sensitive and specific diagnostic methodology to improve the reliability of the cytopathology diagnosis. The methodology will be based on a new cytology protocol where immunolabeling is performed on fresh cells before fixation using spectrally distinctive plasmonic NPs conjugated with antibodies as optical biomarkers. Metallic NPs, typically gold, silver and Au/Ag alloys, are widely used due to their unique plasmonic properties, photo-stability, water solubility and biocompatibility for in vitro and in vivo biomedical applications. The very distinctive NPs chromatic signature depends on their composition, size, and geometry and provides excellent opportunities for a reliable multicolor imaging and multiplexed immunolabeling. The presented methodology includes a new multispectral and hyperspectral dark-field microscopy for reliable multiplexed and quantitative immunoplasmonic markers optical detection. We applied two optical encoding strategies of immunoplasmonic microscopy (IPM) for immunoplasmonic NPs detection in the NPs-cells complex. The first method is based on reflected light microscopy mode (Patskovsky, S. et al J Biophotonics 8 (5), 401-407 (2015))combined with compact hyperspectral scanning source. It provides spectral differentiation, precise spatial localization and multiplexed quantification of NPs labels. The second approach uses a multispectral side-illumination dark-field microscopy that allows to design a compact module for optical imaging and spectroscopic identification of individual plasmonic NPs in fixed or live cell preparations. The presented approach can provide a convenient and routine method for immunoplasmonic markers visualization by the pathologist. It can be easily adaptable to the microscopes currently used in the clinical setting thus facilitating and accelerating its adoption.
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