[77] Spinning Sample Raman Spectroscopy at 77°°K: Bacteriorhodopsin's Primary Photoproduct

This chapter presents the liquid N₂-temperature spinning sample Raman cell that has enabled to obtain excellent spectra of bacteriorhodopsin's (BR) primary photoproduct (K). This apparatus is useful for observing Raman spectra of other photosensitive molecules at low temperatures. The technique begins with the pump-and-probe method, the primary photoproduct in visual excitation. For bacteriorhodopsin as well as rhodopsin, cooling to 77°K blocks the thermal decay of the primary photoproduct. The sample is spinned and illuminated successively with spatially separated pump and probe beams. The BR sample is converted to K each time it passes through the 514-nm pump beam. The K that is produced then spins to the red probe beam, where its Raman spectrum can be observed, unobscured by the pump beam fluorescence. Elimination of the pump beam fluorescence is the major reason for spinning the sample in this experiment. Besides its usefulness for this dual-beam pump-probe Raman technique, the spinning cell has the additional advantage that it allows to overcome thermal or photochemical degradation of the sample caused by the high laser intensities needed to obtain satisfactory signal levels.