Multiplexed Plasmonic Nano-Labeling for Bioimaging of Cytological Stained Samples

SIMPLE SUMMARY: The improvement in the reliability and precision of traditional cytopathological examination protocols (semi-quantitative cancer diagnostics) is a persisting challenge. Many developed high-tech diagnostic approaches have also been declined due to their complexity, non-complementary, and problematic integration with standard pathology laboratory equipment and protocols. In this study, a complementary bioimaging approach based on plasmonic nanoparticles (NPs), due to their stable, strong scattering feature, is therefore developed. This type of approach resists against a strong background of the cytological counterstaining while simultaneously delivering ancillary diagnostic information by using the same cytological stained samples. The direct observation and analyses of four types of plasmonic NPs with different scattering colors on hematoxylin and eosin (H&E) paraffin-embedded specimens are demonstrated. This is performed while using a well-designed adapter for side-illuminated (SI) dark-field conventional microscopy without interfering with traditional cytopathology strategies. This state-of-the-art integrated bioimaging approach (observation of plasmonic NPs on H&E-stained cytology samples) constitutes an indispensable tool that improves not only cancer diagnosis but also daily care. ABSTRACT: Reliable cytopathological diagnosis requires new methods and approaches for the rapid and accurate determination of all cell types. This is especially important when the number of cells is limited, such as in the cytological samples of fine-needle biopsy. Immunoplasmonic-multiplexed- labeling may be one of the emerging solutions to such problems. However, to be accepted and used by the practicing pathologists, new methods must be compatible and complementary with existing cytopathology approaches where counterstaining is central to the correct interpretation of immunolabeling. In addition, the optical detection and imaging setup for immunoplasmonic-multiplexed-labeling must be implemented on the same cytopathological microscope, not interfere with standard H&E imaging, and operate as a second easy-to-use imaging method. In this article, we present multiplex imaging of four types of nanoplasmonic markers on two types of H&E-stained cytological specimens (formalin-fixed paraffin embedded and non-embedded adherent cancer cells) using a specially designed adapter for SI dark-field microscopy. The obtained results confirm the effectiveness of the proposed optical method for quantitative and multiplex identification of various plasmonic NPs, and the possibility of using immunoplasmonic-multiplexed-labeling for cytopathological diagnostics.