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This review provides an introduction for a virologist into the world of …


Home » Biology Articles » Methods & Techniques » Raman spectroscopy: the gateway into tomorrow's virology » Limits of Raman spectroscopy

Limits of Raman spectroscopy
- Raman spectroscopy: the gateway into tomorrow's virology

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.

Raman tweezers

Raman tweezers is a relatively new technology that couples Raman spectroscopy with optical tweezers to achieve previously unheard of sample control and resolution. Optical tweezers is a system that focuses a near-infrared laser on a sample to fix it in an optical trap from which it may then be maneuvered and controlled. The technique, which was first developed by Arthur Ashkin et al. in 1986, has the ability to control objects ranging in size from 5 nm to over 100 mm, whether they be atoms, viruses, bacteria, proteins, cells, or other biological molecules [57,58]. Perhaps most importantly, optical tweezers allows for the analysis of the sample without physically touching it or needing to absorb it to a surface, thereby leaving it in a less disturbed and more natural state [59]. As such, Raman tweezers has the capability to analyze a molecule from every angle and therefore provide more accurate information about identity, structure, and conformation than can Raman spectroscopy alone. Optical tweezers provides the further advantages of eliminating stray light and fluorescence as well as, in holding a molecule in place in an optical trap, allowing for the best possible excitation and collection of Raman spectra [60]. This optical trap also allows Raman tweezers to easily separate molecules for isolated study, such as their response to different conditions and/or treatments [61]. A schematic describing the set-up of a Raman tweezers is shown in Figure 1. The results of Raman tweezers are depicted in the form of a spectrograph (Raman spectrum profiles). Each peak on the spectrograph represents a particular molecule (example: DNA, amino acid, and amide) in the sample. The set of peaks on a spectrograph is different for every unique molecule, thereby allowing Raman tweezers to create spectroscopic "fingerprints" of molecules that can be used as reference in analytical studies.

The one major drawback of using Raman tweezers instead of Raman spectroscopy, however, is its inability to be used with fiber optic probes and therefore be applied to in vivo tissue analyses. Despite this drawback, Raman tweezers is a highly useful marriage of Raman spectroscopy and optical tweezers that further enhances Raman spectroscopy's analytical capabilities.

Current applications of Raman tweezers

The potential of Raman tweezers is staggering. The technique holds all the promise of Raman spectroscopy, including the potential to identify almost any biological molecule and disease, and adds to it both a greater level of control and analytical capability as well as the capability of observing a sample in its natural state. As such, Raman tweezers is likely to surpass Raman spectroscopy in use for biological analysis.

To date, only a handful of biological molecules and processes, including red blood cells, lipoproteins, cell membrane components and T cell activation, have been studied with Raman tweezers [62-65]. Notably, Ramser and Enger et al. have taken advantage of Raman tweezer's ability to suspend red blood cells to study their reaction in vivo under different conditions [66]. Raman tweezers has also been employed in the study of disease in not only identifying pathogenic bacteria and spores but also discerning healthy from virally infected cells [62,67-69]. Thus, even though Raman tweezers cannot yet be coupled with fiber optics for human in vivo tissue analysis, its ability to manipulate a sample without physically coming into contact with it has allowed a degree of detailed analysis not possible with Raman spectroscopy alone.

Future applications of Raman tweezers in virology

Raman Tweezers, while yet far having proven itself an enlightening diagnostic tool in virology, is still in its infancy. With proper nurturing, this technique has the potential to blossom into a truly brilliant and highly useful tool in the virologist's arsenal. As the resolution of Raman spectrographs increases, so will their analytical capabilities. It is likely in the not too distant future, that this technology will allow scientists to go beyond their current capability of distinguishing infected from healthy cells to being able to distinguish between differentially infected cells. Given a detailed library of spectra, a researcher could potentially even be able to characterize an unknown virus' structure, components, and lytic or latent state of infection. Furthermore, the technique's optical tweezers would allow for the study of the more temperamental cell lines, such as 293, that die more easily upon physical contact. All of these analytical capabilities would give the virologist a much clearer window to study viruses.

One could also use this technique's capabilities to not only characterize a virus, but also monitor the efficacy of antiviral treatments and determine viral load, among other applications. While all of these potential applications can be done today through alternative means, these processes must be completed separately and are time consuming. Raman tweezers greatly simplifies this process by providing a comprehensive analytical system that is both able to collect all the necessary data at once and able to do so in a very short time, thereby making it extremely cost effective. The process is so fast in fact (Table 1) that the progression of an infection or treatment could be studied in relative real time. This would serve investigators as an enormous tool with which to study viral processes as they progress, instead of just being able to study them from specific and distant time points. Such immediate and detailed analysis has potentially great applications in medicine in allowing for quick diagnosis and monitoring of virally infected patients. Through running a few drops of a patient's blood through a Raman spectrograph and reading their spectra, their care could be tailored to the state of their infection and the efficacy of the drugs to treat that infection. In addition, asymptomatic virally infected patients could be easily identified and treated before potentially harmful symptoms manifest themselves [70]. As such, Raman tweezers could prove to be one of the most effective analytical tools not only in the researchers', but also clinicians' repertoire.


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