Quarterly Reviews of Biophysics 8, 2 (1975), pp. 237-316 Printed in Great Britain The application of fluorescent probes in membrane studies ANGELO AZZI Istituto di Patologia generale e Centro per lo Studio della Fisiologia dei Mitochondri, Universitd di Padova, 35100 Padova, Italy I. INTRODUCTION 238 II. FLUORESCENT PROBES 240 III. THE METHOD 240 1. Perturbation by probes 245 2. Location of probes 249 3. Chemical parameters 254 4. Physical parameters 257 (a) Wavelength of emission maximum 257 (b) Quantum yield 259 (c) Lifetime of the excited state 263 (d) Polarization 264 IV. THE APPLICATION OF FLUORESCENT PROBES 268 1. Membrane polarity 268 2. Membrane fluidity 270 (a) Microviscosity 271 (b) Lateral diffusion 274 (c) Effect of cholesterol 276 (d) Effects of ions 278 (e) Effect of temperature 280 3. Accessibility 282 4. Distances 283 5. Electrical potentials 285 (a) Surface potential 286 (b) Transmembrane potential 296 [237]