With the issue of background fluorescence solved, Raman spectroscopic analysis has become an analytical method of choice in an extremely wide range of biological applications. Some of the more obscure applications of this technique include everything from determining the molecular structure of the skin of a 5200 year old frozen man to the analysis and authentication of foods such as olive oil and Japanese sake [20-22]. One of the more significant applications has been in pharmaceutical research and development, where Raman spectroscopy has been applied in duties ranging from shelf-life assessment and drug formula characterization to non-invasive pharmacokinetic analysis [9,23,24].
Of even greater consequence, perhaps, has been Raman spectroscopy's contribution to detailed cellular analysis. Modern techniques have allowed for the Raman spectroscopic analysis of cells in vivo without the need of fixatives, thereby providing extremely detailed analysis of cells in their natural state . Such analytical potential has been put to good use in not only completing spectral maps but also monitoring the changes over time of numerous varieties of cells, including bacteria and many eukaryotes [25,26]. For example, Raman spectroscopy has been applied to everything from studying lipid droplet and other particulate levels in human cells to finding lignin radicals in plant cell walls and monitoring bacterial levels in drinking water [27-29]. Additionally, as is shown in table 1, the necessary steps and time required in sample preparation is much less in Raman spectroscopy than with other analytical methods.
Of particular interest has been the application of Raman spectroscopy in medicine. The technique's ability to provide detailed images of cells has allowed for the comparative analysis between numerous healthy tissues and their diseased states. Such analytical potential has been especially suited in the diagnosis of numerous cancers, including: intestinal, stomach, laryngeal, brain, breast, mouth, skin, and others [30-37]. Other applications of Raman spectroscopy outside of cancer have included bone quality assessment for improved estimates of the risk of fracture, corneal hydration gradient analysis, rapid identification of bacterial and fungal infection, and even antibiotic susceptibility testing [23,38-43].
Recently, Raman spectroscopy has been coupled with modern fiber optic technology to accurately measure tissue spectra in vivo without the need of biopsy. This method employs a small fiber optic probe that both has the capability to reach less assessable organs and only requires less than two seconds to collect spectra . As such, it is very useful for determining the spectra of cells in their most natural state, and therefore ensures more accurate results. This method has been successfully used in the detection of atherosclerosis and cervical cancers, among other diseases [45,46]. Use of higher, near UV wavelengths has solved the initial problems this technology experienced with fluorescent interference .
The use of Raman spectroscopy in differential medicine is not limited to tissues and cells; it also has applications in virology. The technique has been put to good use in determining the structures and stereochemistry of both the protein and nucleic acid components of viruses, even going so far as to being able to distinguish between different types of right-handed DNA alpha-helixes [48-53]. Raman spectroscopy has also been used to help better characterize the conformational changes that occur leading to viral procapsid and capsid assembly [54-56]. As such, Raman spectroscopy holds the potential to distinguish between even the most similar viruses, thereby increasing its possible role even further in diagnostic medicine.
Limits of Raman spectroscopy
The analytical capabilities of Raman spectroscopy are limited by its inability to manipulate, and therefore thoroughly analyze the biological molecules under study without making physical contact. This limitation has been resolved by coupling Raman spectroscopy with a technology called optical tweezers. The new method, termed Raman tweezers, uses optical tweezers to manipulate a sample without contact with it so that it remains unchanged for Raman spectroscopic analysis.