- Vibrational spectroscopy - When a molecule absorbs energy, vibration can be induced in its molecular bonds. This can occur if infra-red (IR) light is absorbed by a molecule or if radiation (typically in the visible-IR wavelength range) is inelastically scattered (as shown in the diagram below). Molecular bonds usually have very distinct vibrational signatures. This means that they absorb (or scatter) light of different, and very specific wavelengths. For this reason, IR absorption spectroscopy has been widely used to accurately identify the chemical structures of unknown compounds by determining which molecular bonds are present through measurements of their vibrational signatures.
The figure on the right provides a simple depiction of inelastic scattering - incident light induces molecular vibrations leading to changes in the wavelength (λ) of the scattered photons.
- Raman scattering - Named after one of its discoverers, Raman scattering is an optical transition that occurs when light inelastically scatters from a target molecule. Elastic scattering – otherwise known as Rayleigh scattering – is defined as a photon scattering from a sample without a change in energy occurring. As such, the wavelength of the impinging photon remains unchanged. Raman (inelastic) scattering is different and involves a transfer of energy between the photon and the scattering medium, with this transfer of energy manifesting itself as a change in the vibrational energy state of the molecule. As described in the figure, scattering of light involves an interaction between a photon and a molecule. During this interaction, the scattering molecule is instantaneously promoted to an excited energy state – known as a ‘virtual state’ due to its infinitely short existence. After this excitation event, the molecule decays back to its ground electronic state, which contains multiple vibrational energy levels (vibronic modes). The figure on the right shows a Jablonski diagram illustrating some important optical transitions that can occur in a scattering/absorbing/fluorescent molecule.
In the case where the molecule falls back into the vibronic mode that it started in, there is no transfer of energy between the photon and the scattering molecule, and we observe an elastic scattering event. In the alternative event that the molecule falls into a different vibrational energy level, its energy changes and there is a concomitant transfer of energy between the photon and the scatterer (Raman scattering). Most often, energy is transferred from the photon to the scattering molecule; the molecule gains vibrational energy and the wavelength of the photon increases (Stokes shift) as it loses energy. The inverse can occur when a photon scatters from a molecule that is already in an excited vibrational state. Scattering can then perturb the molecule such that it falls into a lower energy vibronic mode, with energy being transferred from the molecule to the photon. The energy of the photon increases and we observe a decrease in its wavelength, known as an anti-Stokes shift. Raman spectroscopy involves measuring the spectrum of light scattered from a sample of interest in order to ascertain information about its vibrational energy level structure. This has a variety of applications including identification of unknown compounds based on the vibrational signatures of their constituent bonds, or discrimination of different tissue disease states based on changes in their molecular make-up or metabolic activity.
- Direct detection of bacteria using Raman spectroscopy - Raman spectroscopy has been widely used for the detection of bacteria for a variety of applications (e.g. [1-4]). Importantly, many bacteria have been observed to show distinct Raman signatures and spectra can be acquired without the use of exogenous contrast agents. Additionally, Raman spectra can reveal the presence of bacteria directly, rather than relying on secondary markers of bacterial infection (such as inflammation or tissue oxygen saturation) that can be affected by outside factors. However, to date, very little research has been published using Raman spectroscopy to detect bacteria in vivo due to inherently low signal levels and background signals often observed in optical fibres.
At the Hamlyn Centre we are designing and building miniature sensors that can detect infection in hospital patients. These sensors use light to detect the presence of bacteria, which is the cause of many dangerous infections. When light of a specific colour (wavelength) illuminates a region of human tissue (such as the skin), the molecules that make up that tissue can start to vibrate. This means that energy has been transferred from the light to the vibrating molecules. By detecting the wavelength of the light that is reflected back, we can learn about the types of molecules that the light has interacted with. Importantly, we will detect specific signals from different bacteria and this will allow us to find out whether there is any bacteria present and whether or not it is likely to cause an infection. This method of using light to study molecular vibrations is known as Raman spectroscopy. We aim to develop miniature Raman spectrometers that can be worn on a patient’s body or even implanted beneath their skin. We hope that these systems will help to speed up and improve the diagnosis of infections that occur after patients have surgery.
Raman spectroscopy reveals the vibrational energy level structure of a sample and has been shown to detect and effectively discriminate between different bacteria. Despite these promising results, it has not been regularly used in vivo or in clinical settings, nor has it been used extensively with optical fibres employed to allow remote measurements. We are developing a compact optical fibre based Raman spectrometer designed for the clinical detection of bacteria. This device consists of an IR (785 nm) laser diode for illumination and a small spectrometer for detection. All optical and electrical components are mounted on a small optical breadboard and enclosed within an anodized aluminium box to allow laser-safe use in a clinical environment. Light is delivered to and collected from a measurement site using a custom made bifurcated optical fibre. This fibre has two proximal arms, each containing a single optical fibre – one for light delivery and one for light collection. The two fibres have a common distal end that can be easily placed by hand on a desired sample. The fibre’s outer sheath is made from Teflon and the distal ferrule is constructed using stainless steel, both of which can be easily cleaned with ethanol after use on a clinical sample. Due to its compact nature, our Raman spectrometer can be easily transported to and used within a clinical environment.
What Are We Using It For?
Using the compact, clinically viable Raman spectrometer described above, we plan to interrogate multiple clinical samples ranging from bacterial cell cultures, to blood and urine samples, and finally to in vivo surgical sites in animals and humans. In doing so, we aim to demonstrate the feasibility of Raman spectroscopy as a clinical tool that permits rapid detection of bacteria. Alongside this clinical research path, we are also working towards developing miniaturised Raman infection sensors that can be worn by or even implanted within a patient. This will entail a number of engineering challenges and the end products will be of significant novelty in the field. Miniaturised Raman spectrometers are desirable not only in medicine but also within other sensing fields. For example, handheld Raman spectrometers are being developed for the Police and security services to use in the detection of substances such as explosives and illegal drugs. Clearly, in these scenarios, the smaller and lighter the spectrometer is the better. As such, miniaturisation of a Raman spectrometer will entail an important and significant technological advance.
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E. Kastanos et al, J. Raman Spectrosc. 41(9): 958-963 (2010).
X. Yang et al, Anal. Chem. 83: 5888-5894 (2011).
E. Kastanos et al, Int. J. Spectrosc. 12: 195317 (2012).