Cyanine5.5 amine (Cy 5.5 amine), a Cy5.5 analogue, functions as a near-infrared (NIR) fluorescent dye with excitation and emission wavelengths of 648 nm and 710 nm, respectively. This compound is utilized in synthesizing Cy5.5-labeled nanoparticles, facilitating tracking and imaging through confocal microscopy with minimal fluorescence background, as evidenced in studies [1] [2].

Real-time monitoring of Cy5.5-labeled nanoparticles (Cy5.5-PLGA) in retinal blood vessels was performed following these steps: Freshly prepared Cy5.5-PLGA suspensions (resuspended in 0.5 ml 1% poloxamer 188, vortexed gently, and left for 30 min at ambient temperature before use) were administered intravenously (0.5 mL). Rats were anesthetized, eyes treated with Neosynephrine-POS 5%, and Vidisic eye gel was applied. Rats were positioned under a confocal scanning microscope with a cannula in the tail vein. Fluorescence in the retina was observed and imaged at different time points (0, 1, 3, 5, 15, 30, 60, 90 min) while rats were kept on a heating plate. Post-imaging, rats were euthanized with an overdose of anesthetic, eyeballs enucleated, and placed in cooled HEPES buffered solution (135 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 7 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH7.4). The anterior segment and vitreous body were removed, and the retina was carefully separated and flattened on a culture plate. Retinal whole mounts were incubated with 0.1 mg/mL Hoechst 33342 in HEPES solution for 20 min for nuclei staining, fixed with 4% paraformaldehyde solution for 20 min, washed with HEPES solution, and imaged immediately with a microscope.