History and Science of Immunofluorescence

During the last years, many new technologies, like laparoscopy, endoscopy, and robotics, have been introduced in surgery to improve daily practice. Among these techniques, “fluorescence-guided surgery” (FGS) is an intraoperative imaging system allowing the identification of structures through fluorescent probes and dedicated technology. The main aim is to improve surgical guidance. This chapter will discuss the history of immunofluorescence, basic principles of fluorescence, fluorescent probes in surgery, and clinical imaging systems. Since its first clinical application in the surgical field in 1947, fluorescence has undergone a huge development and diffusion. To date, fluorescence has several applications in every surgical specialty like perfusion assessment, cholangiography, lymphography, tumor identification, and ureter identification. The technique implies the administration of a fluorescent probe that is excited by light excitation source and emitted by dedicated instruments, with consequent emission of a fluorescent signal that is detected and visualized on a screen. Among the fluorescent probes already approved for clinical use, like methylene blue, fluorescein sodium, and 5-aminolevulinic acid, the most diffused is indocyanine green (ICG). ICG has some peculiar features that promote its widespread use: it is virtually harmless, due to the lowest rate of adverse effects reported and to the very high toxic dose for the human body, and it is not expensive. Many probes are currently under clinical development and will be on the market soon. A growing number of companies developed new systems for FGS with excitation and emission spectra in the nearinfrared wavelength range, which are suitable for both fluorescence and white light imaging.