ESTABILIDAD AMBIENTAL (EA)
3.13 ESTRUCTURA ORGANIZACIONAL
Conventionally, in order to reduce the contribution of fluorescence in the Raman spectra for bio-medical applications, excitation in the NIR region is opted. This wavelength region is a good choice since the absorption coefficient is very low in general for biological samples in the NIR region. Also to further suppress the background contribution a set of filters are used along the optical path. Even with the use of NIR excitation and filters there would be residual contribution due to the auto- fluorescence from the sample, which poses a significant challenge since the Raman signal and the fluorescence signal occur in the same fingerprint region. The next section details the different techniques that are used to suppress this fluorescence background.
Traditionally, the routine to remove the background in the acquired Raman spectra consist of applying mathematical methods such as background-subtraction procedures and polynomial fit of the background 58. This method is computationally straight forward however, multiple fluorescence peaks, negative errors in fit can lead to artefacts in the final processed spectra. One such example illustrating this point is shown in Figure 25 of section 2.6.
The solution to avoid such artefact is to suppress fluorescence background using physical principles associated with the Raman scattering process. A simple approach to remove fluorescence background is to exploit the life time difference between the Raman and fluorescence processes and this approach is called Time Resolved Raman Spectroscopy (TRRS)59, 60. Raman scattering is instantaneous and has a short life time (~ 10-11-10-13) in comparison to the fluorescence background (~10-9-10-7), therefore by using a pulsed laser source and gating the signal collection time a dominant part of the fluorescence can be rejected. However, this technique doesn’t guarantee complete removal of the background. Also, this method requires a complete understanding of the fluorescence characteristics of the sample in order to pick the detector gating time to effectively collect the entire Raman signal and reject the fluorescence background.
The Polarization modulation technique is another technique for background rejection which exploits the fact that the polarization properties of the Raman signal and the fluorescence signal are different61. When the Raman signal is highly polarised the
fluorescence signal is totally depolarised. Hence in this case the Raman signal is acquired with parallel and perpendicular polarised light and a difference between these two signals should ideally give a background free Raman spectra. However, it was reported that it is not possible to retrieve the complete Raman features while using this technique. Phase sensitive detection scheme is another fluorescence rejection method where the sample that is analysed is periodically modulated in position along with single channel lock-in detection using a photomultiplier tube62, 63. This method is slow and demands longer acquisition time to collect Raman signal, especially for biological samples.
WMRS, also known as Frequency Modulated Raman Spectroscopy, is a fluorescence rejection technique introduced in the 1970s64, 65. This technique is based on the fact that when the wavelength of the exciting light is slightly modulated (5A0), the Raman bands also get modulated while the fluorescence background, which is insensitive to small changes in the excitation wavelength, remains a constant. This varying Raman signal can then be extracted from the constant fluorescence background. Lock-in-detection scheme was used in the past for extracting the Raman signal. Later, it was demonstrated that by incorporating statistical techniques such as EM algorithm and PCA that picks up the largest variation in the data (which corresponds to the Raman signal) from the non-varying one (that corresponds to the fluorescence background) the lock-in-detection scheme can be avoided66, 67. Shifted Excitation Raman Difference Spectroscopy68 and Multi-excitation Raman Spectroscopy66 are basically variations of this technique.
Even though the proof of principle of WMRS has been demonstrated in the late 70’s where the excitation source was modulated incorporating lock-in-detection scheme65, 69- 71
, due to the limitations in the instrumentation at that time, the technique remained unexplored 69. Later with advancements in the excitation source, detectors, and robust statistical algorithms WMRS could be extended for biological samples. It was demonstrated that by incorporating statistical techniques such as Principal Component Analysis and Expectation Maximisation algorithm that picks up the largest variation in the data (which corresponds to the Raman signal) from the non-varying one (that corresponds to the fluorescence background), WMRS can be taken to the next level
where the lock-in-detection scheme can be avoided 66, 72. This makes it easy to integrate WMRS into existing Raman systems, which only require replacing the excitation laser with one that can be modulated. Unlike TRRS and PMRS, this technique does not depend on the temporal and polarization properties of the Raman signal itself therefore avoiding any loss in the Raman information during fluorescence suppression. However, this technique required further optimization particularly for biological samples whose Raman bands are usually broader than pure chemicals as shown in Figure 27 of section 3.3. Hence a specific protocol is required to be developed while probing biological samples.