Fluorescence excitation analysis by two-photon confocal laser scanning microscopy: a new method to identify fluorescent nanoparticles on histological tissue sections

Edmond Kahn,¹ Nicolas Tissot,³ Perrine Frere,³ Aurélien Dauphin,³ Mohamed Boumhras,^2,4 Claude-Marie Bachelet,³ Frédérique Frouin,¹ Gérard Lizard<sup>2

1</sup>Institut National de la Santé et de la Recherche Médicale (INSERM) U678/UMR-S UPMC, CHU Pitié-Salpêtrière, Paris, France; ²Equipe Biochimie du Peroxysome, Inflammation et Métabolisme Lipidique EA7270, Faculté des Sciences Gabriel, Université de Bourgogne-INSERM Dijon, France; ³Plateforme d'Imagerie cellulaire, UPMC, Paris, France; ⁴Laboratory of Biochemistry and Neuroscience, Applied Toxicology Group, Faculty of Science and Technology, Settat, Morocco

Abstract: In the present study, we make use of the ability of two-photon confocal laser scanning microscopes (CLSMs) equipped with tunable lasers to produce spectral excitation image sequences. Furthermore, unmixing, which is usually performed on emission image sequences, is performed on these excitation image sequences. We use factor analysis of medical image sequences (FAMIS), which produces factor images, to unmix spectral image sequences of stained structures in tissue sections to provide images of characterized stained cellular structures. This new approach is applied to histological tissue sections of mouse aorta containing labeled iron nanoparticles stained with Texas Red and counterstained with SYTO13, to obtain visual information about the accumulation of these nanoparticles in the arterial wall. The possible presence of Texas Red is determined using a two-photon CLSM associated with FAMIS via the excitation spectra. Texas Red and SYTO13 are thus differentiated, and corresponding factor images specify their possible presence and cellular localization. In conclusion, the designed protocol shows that sequences of images obtained by excitation in a two-photon CLSM enables characterization of Texas Red-stained nanoparticles and other markers. This methodology offers an alternative and complementary solution to the conventional use of emission spectra unmixing to localize fluorescent nanoparticles in tissue samples.

Keywords: FAMIS, spectral excitation sequences, Texas Red, tunable excitation, unmixing