2. Instrumentation

A Raman spectrometer useful for in vivo measurements should be an integrated system that can provide real-time spectral acquisition and analysis [1]. A Raman system for in vivo measurements includes a light source, sample light delivery and collection, spectrograph with detector, and the computer interface. Lasers are the excitation source for Raman spectroscopy due to the fact they can provide sufficient power to the sample in order to detect Raman spectra in a reasonable integration time. However, it is necessary to consider important issues such as power, integration time, and wavelength of the laser to optimize the Raman system for in vivo biomedical applications. For example, to avoid tissue damage, the maximum permissible exposure (defined by ANSI) and temperature increase must be considered. Therefore, a correct laser power selection depends on achieving a good signal to noise and to minimize tissue damage. In biological tissue, the fluorophores can generate signals that mask or overwhelm the weak Raman signal, and to avoid fluorescence background, multiple approaches have been proposed including the excitation in the near infrared (NIR) [2]. It is known that most biological fluorophores have no peak emission in this region of the spectrum, which results in lower fluorescence background compared to visible or UV excitation. Due to these advantages, most of the Raman spectroscopy systems for skin diagnosis use a 785-nm diode laser as the excitation source, since it provides low-cost light source that generates low fluorescence and can penetrate deep into human tissue. In sample light delivery and collection, the most used method for clinical applications is optical fibers. The Raman fiber probe design varies depending on the clinical application. In the case of Raman spectroscopy of the skin, the probe consists of a single central delivery fiber surrounded by several collection fibers. The selection of a suitable detection system is an important issue for Raman spectroscopy. The

Figure 1. Schematic of a typical Raman system for in vivo biomedical applications.

typical Raman detection system used for biomedical applications consists of a spectrograph attached to a cooled charge coupled device (CCD). Most CCDs use a thermoelectric (TE) system to cool the detector down to 70C in order to reduce thermal noise. The detection system also requires a spectrograph coupled to the Raman probe and to the CCD. It is recommended the spectrograph have a spectral resolution of 8–10 cm<sup>1</sup> in order to provide detailed information of biological Raman bands. The spectral resolution depends on spectrograph optical parameters, the diffraction grating, and the CCD pixel size. A schematic of the typical arrangement of these components is shown in Figure 1.
