Development of a Raman Microspectrometer for Ultraviolet Excitation

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Raman microspectroscopy is a useful method to investigate the structures of micrometer-sized particles in a non-destructive manner. On the other hand, ultraviolet resonance Raman spectroscopy provides key information on the structures of proteins and nucleic acids. In this study, we have developed an ultraviolet Raman microspectrometer to exploit the advantages of both spectroscopic methods. The microspectrometer consists of a continuous-wave ultraviolet laser, an ultraviolet microscope, and a Raman spectrometer. The ultraviolet laser beam is obtained by frequency-doubling visible laser light from an Ar^+ laser. The ultraviolet microscope is specially designed for high throughput in the ultraviolet region and high spatial resolution of microscopic images by employing the confocal arrangement. Samples under a microscope objective are irradiated with ultraviolet laser light and the Raman scattered light was collected with the same objective and then guided to the Raman spectrometer. We have succeeded to record Raman spectra of proteins and nucleic acids by using the newly developed ultraviolet Raman microspectrometer. Detailed examination of the spectrometer performance has shown that the throughput of microscope objective is the most important factor that determined the quality of spectra to be obtained. To improve the performance of the microspectrometer, we are planning to design a new objective. The present research has established the basis for future development of an ultra-sensitive ultraviolet resonance Raman microspectrometer, which will be applicable to structural analysis of molecules and molecular assemblies even in living cells.