Cyanine5.5 amine is an amino-modified Cy5.5 near-infrared (NIR) fluorescent dye with excitation/emission wavelengths of 648/710 nm. It offers the advantage of low fluorescence background and is suitable for group conjugation and in vivo imaging.

Instructions:
Real-time monitoring of Cyanine5.5 amine-labeled nanoparticles (Cy5.5-PLGA) in retinal blood vessels was conducted as follows: Freshly prepared Cy5.5-PLGA suspensions (resuspended in 0.5 ml 1% poloxamer 188, gently vortexed, and left for 30 min at ambient temperature before use) were administered intravenously (0.5 mL). Rats were anesthetized, their eyes treated with Neosynephrine-POS 5%, and Vidisic eye gel was applied. The 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 the rats were kept on a heating plate. After imaging, the rats were euthanized with an overdose of anesthetic, their 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; pH 7.4). 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 for 20 min, washed with HEPES solution, and imaged immediately with a microscope.