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A brief history on spectroscopy
- Raman spectroscopy: the gateway into tomorrow's virology

Spectroscopy was born in 1801, when the British scientist William Wollaston discovered the existence of dark lines in the solar spectrum. Thirteen years later, Jospeh von Fraunhofer repeated Wollaston's work and hypothesized that the dark lines were caused by an absence of certain wavelengths of light [2]. It was not until 1859, however, when German physicist Gustav Kirchhoff was able to successfully purify substances and conclusively show that each pure substance produces a unique light spectrum, that analytical spectroscopy was born. Kirchhoff went on to develop a technique for determining the chemical composition of matter using spectroscopic analysis that he, along with Robert Bunsen, used to determine the chemical make up of the sun [3].

The end of the nineteenth and beginning of the twentieth centuries was marked by significant efforts to quantify and explain the origin of spectral phenomena. Beginning with the simplest atom, hydrogen, scientists including Johann Balmer and Johannes Rydberg developed equations to explain the atom's frequency spectrum. It was not until Niels Bohr developed his famous model in 1913 that the energy levels of the hydrogen spectrum could accurately be calculated. However, Bohr's model failed miserably when applied to other elements that had more than one electron. It took the development of quantum mechanics by Werner Heisenberg and Erwin Schrodinger in 1925 to universally explain the spectra of most elements [4].

From the discovery of unique atomic spectra developed modern spectroscopy. The three main varieties of spectroscopy in use today are absorption, emission, and scattering spectroscopy. Absorption spectroscopy, including Infrared and Ultraviolet spectroscopy, measures the wavelengths of light that a substance absorbs to give information about its structure. Emission spectroscopy, such as fluorescence and laser spectroscopy, measures the amount of light of a certain wavelength that a substance reflects. Lastly, scattering spectroscopy, to which Raman spectroscopy belongs, is similar to emission spectroscopy but detects and analyzes all of the wavelengths that a substance reflects upon excitation [5].

Raman spectroscopy

Raman spectroscopy is named after the famous Indian physicist Sir Chandrasekhara Venkata Raman who in 1928, along with K.S. Krishnan, found that when a beam of light transverses a transparent chemical compound, a small fraction of that beam will emerge from the compound at right angles to and of a different wavelength from the original beam [6]. Raman received the Nobel Prize in 1930 for his work on this phenomenon, which has since been known as the Raman effect [6].

Normally, when a beam of light is shined through a transparent substance, the molecules of the substance that absorb those light wavelengths are excited into a partial quantum state (or higher vibrational state) and emit wavelengths of equal frequency as the incoming wavelengths such that there is no net change in energy between the light and the substance. Such light wavelengths are said to be elastically scattered in a process known as Rayleigh scattering [7]. On rare occasion (approximately 1/100,000 cases), the Raman Effect occurs and the molecule absorbing the incoming wavelength's energy emits a wavelength of a different frequency/energy. Of these rare occurrences, the most common are those in which a molecule releases a wavelength of lesser energy than the incoming wavelength, thereby absorbing some of the incoming wavelength's energy. These events are referred to as Stokes shifts [8]. The opposite effect may also occur, referred to as anti-Stokes shifts, in which a molecule releases a wavelength of higher energy than the wavelength it absorbs [6]. Anti-Stokes shifts are very rare; however, this is possible under certain circumstances wherein the absorbing molecule is in a partially elevated energy state prior to absorbing the incoming wavelength in order to emit a wavelength of even greater energy [4]. The ratio of these aberrant high to low wavelengths can be measured to give what is known as a Raman signal. The Raman signal given off by every type of molecule, by the interaction between different molecules, and by different thicknesses of molecules is unique, and as such, may be used to analyze a molecular species both qualitatively and quantitatively.

Raman spectroscopy is performed by illuminating a sample with a laser. The reflected light is collected with a lens and sent through a monochromator that typically employs holographic diffraction gratings and multiple dispersion stages to achieve a high degree of resolution of the desired wavelengths [6]. A charge-coupled device (CCD) camera or less commonly, photon-counting photomultiplier tube (PMT) then detects and measures those wavelengths, which are then compared to a library of known wavelengths of molecules in order to determine the composition of the tested substance [1]. Alternatively, a Fourier transform technique may be employed in which a Fourier transform is used to convert an interferogram produced from a sample into a highly accurate spectrum [9]. Unlike conventional methods, the Fourier transform technique may only be used in the near-infrared spectrum [9].

While initial Raman spectroscopy was unable to analyze most biological samples due to the interference from the background fluorescence of water, buffers, and/or mediums present in the sample, two new types of Raman spectroscopy have been developed that solve this problem. Both types, near-infrared (NIR) and ultraviolet (UV) Raman spectroscopy, rely on using wavelengths well away from those of fluorescence. Near-infrared Raman spectroscopy relies on long near-infrared wavelengths while ultraviolet Raman spectroscopy relies on short wavelengths to avoid interference from mid-wavelength fluorescence, as shown in figure 1. UV Raman spectroscopy has a slight advantage over NIR Raman spectroscopy in better avoiding interference due to fluorescence [10].

There are four major types of Raman spectroscopy in use today: surface enhanced Raman spectroscopy (SERS), resonance Raman spectroscopy (RRS), confocal Raman microspectroscopy, and coherent anti-Stokes Raman scattering (CARS) [1]. SERS, which absorbs molecules onto a rough gold or silver surface, has the advantage of providing anywhere from a thousand to ten-million fold enhancement of the Raman signal [11,12]. In addition, the use of gold or silver in this technique removes any interference from fluorescence [13]. Unfortunately, SERS can only be used to analyze charged analytes, and therefore has only limited use in biological applications [11]. RRS also provides a marked increase in the Raman signal, but does so by taking advantage of the one hundred to one million fold signal enhancement that a molecule emits when exited at a wavelength near its transition state [14]. Unfortunately, RRS is sometimes hindered by fluorescent interference [1]. Recently, SERS and RRS have been combined to produce surface enhanced resonance Raman scattering (SERRS), a system that combines the signal enhancement of both RRS and SERS and the SERS's avoidance of fluorescence to produce ultra-sharp spectrographs. To date, SERRS has proven to be extremely useful in DNA detection [15].

The second two types of Raman spectroscopy, confocal Raman microspectroscopy and coherent anti-Stokes Raman scattering (CARS) are not only able to analyze nearly all biological samples, but also avoid any fluorescent interference. Both confocal Raman microspectroscopy and CARS spectroscopy get around this problem of fluorescence in unique ways. Confocal Raman microspectroscopy eliminates any lingering fluorescence by measuring the Raman spectra of micro regions of a sample one at a time such that the effects of fluorescence are eliminated while high resolution is maintained [16]. Because this method measures micro regions individually, it also has the advantage of being able to detect and isolate small individual biological molecules that other techniques cannot. The major disadvantage of using confocal Raman microspectroscopy, is the long time (several hours) the technique requires to produce a Raman image [17]. CARS spectroscopy eliminates the effects of fluorescence by combining the beams from two lasers to create a single high energy beam that is so strong that the Raman spectra it produces can be detected over background fluorescence [16,18]. This system also has the advantage, since it computes nonlinear (quadratic, cubic, and quartic) functions of the electromagnetic field strength, of being able to determine a molecule's chirality [19]. The largest drawback of CARS despite current work to resolve it, is its relative inability to distinguish between small equally sized molecules [16].

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