Fluorescence and Nuclear Magnetic Resonance (NMR) Spectroscopy Murphy, B. (2017). Fluorescence and Nuclear Magnetic Resonance Spectroscopy: Lecture 3. Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago. FLUORESCENCE SPECTROSCOPY • Electron is excited by absorption and then emits fluorescence upon relaxation • Stokes shift = difference between excited and emitted wavelengths • Fluorophore = molecules or functional groups that have the capacity to exhibit fluorescence o Require extended conjugation of pi bonds o More conjugated → less energy required for excitement → longer wavelength can be used for excitation • Fluorescent probes used to identify biological processes o Green fluorescent protein (GFP) – fluoresces green light when exposed to light in the blue to UV range ▪ Can make its own color using oxygen only ▪ Slight modifications can allow for different colors to be emitted. Gives researcher a toolbox of probes for in vivo imaging studies o Can study specific proteins or cellular movements → disease states ▪ Must be careful → too much modification of the protein can impact its natural functioning • Protein tagging o Can add the fluorescent probe to the C- or N-terminus. Glycine allows for more flexibility • Cellular tagging o Can visualize the G1 phase and the S/G2/M phase • Weakness- hemoglobin and melanin can also absorb fluorescent light o Optimal viewing window is near IR region, not visible light region o Near IR probes – increase tissue penetration and resolution of image ▪ Can use small organic molecules or inorganic nanoparticles ▪ Just need a certain degree of conjugation • Forster resonance energy transfer (FRET) – studying energy transfer between fluorophore molecules → allows study of protein interactions in the cell o The excited energy fluorophore passes its energy to the lower energy fluorophore via a dipole-dipole interaction • Photosensitizers – dyes that can generate reactive oxygen species (ROS) light