Charge-shifting CCD operation enables fast Shifted Excitation Raman Difference Spectroscopy
Shifted excitation Raman difference spectroscopy (SERDS) is a powerful technique for the analysis of various fluorescent specimens. SERDS is based on the subsequent excitation of the sample at two slightly different laser wavelengths and the recording of two corresponding Raman spectra one after the other. While Raman signals follow the shift in excitation wavelength, static interference (e.g. from fluorescence or ambient light) will remain unaffected and can be removed by subtraction of the two Raman spectra. In certain scenarios short measurement times can be required. Examples include fast-changing backgrounds, e.g. due to fluorescence quenching or movement of handheld Raman probes when analyzing heterogeneous samples.
Regarding the laser source, fast switching between the two wavelengths is readily achievable, as e.g. demonstrated up to a frequency of 1,000 Hz applying both external cavity diode lasers  and distributed Bragg reflector Y-branch diode lasers  developed at FBH. On the detection side, however, the read-out and digitization steps after each exposure present a fundamental limit to fast alternating detection of the two Raman spectra required for SERDS. Through a prolific collaboration with UKRI Science and Technologies Facilities Council’s Rutherford Appleton Laboratory (United Kingdom) an approach to overcome this issue has been investigated. Here, SERDS is combined with fast charge-shifting CCD operation in the kilohertz range that has been already successfully demonstrated for the rejection of ambient light interference in conventional Raman spectroscopy . Key components of the approach, permitting for short exposure times and no delay (e.g. read-out) between exposures, are a custom 830 nm dual-wavelength diode laser and a specialized CCD.
The charge-shifting process is depicted in Fig. 1 and starts with the illumination of a defined area on the CCD by scattered light excited at the first laser wavelength generating charges in this area (blue color). All charges on the CCD chip are then shifted upwards so that the previously accumulated charge is located in an unilluminated area at the top. Charges generated by scattered light excited at the second laser wavelength are then stored in a different area (golden color) that is now illuminated. Subsequently, all charges are shifted down and further charges are generated due to scattered light excited at the first laser wavelength. This cycle is repeated until sufficient signal height is achieved, and the process ends with one single read-out step after many thousands of cycles.
To assess the performance of our approach, six natural rock samples were exemplarily selected as heterogeneous and fluorescent specimens and moved irregularly during spectral acquisition. Fig. 2a displays Raman spectra of basalt rock excited at 828.85 nm and 829.40 nm recorded in charge-shifting mode at 1,000 Hz as well as the corresponding SERDS spectrum calculated as difference of the two Raman spectra. Background interferences are effectively removed in the SERDS spectrum allowing for identification of characteristic Raman signals of calcite (CaCO3). A comparison of charge-shifting and conventional CCD read-out is given in Fig. 2b showing the average and standard deviation of 20 spectra each. In case of slow spectral acquisition at 5.4 Hz in conventional mode pronounced baseline distortions due to sample heterogeneity remain in the spectra. Fast charge-shifting acquisition permits for effective removal of background interference and improved reproducibility of repeat spectra (see reduced standard deviation (gray area)).
For a quantitative evaluation, the cumulative standard deviation in the spectral range from 200 cm-1 to 1,600 cm-1 was determined for spectra from all six investigated rocks recorded in both read-out modes. The results presented in Fig. 3 highlight the advantages of the charge-shifting concept. Here, better reproducibility between repeat measurements of the heterogeneous rocks and reduced susceptibility to variations during the measurement become evident. The decreased variability of spectra obtained from one rock species in charge-shifting mode has proven beneficial for the discrimination between different rock species. Improved classification using the charge-shifting mode (sensitivity: 99 %, specificity: 94 %) compared to conventional read-out (sensitivity: 90 %, specificity: 92 %) could be demonstrated [4, 5]. The charge-shifting technique will be particularly beneficial for a wide range of (spectroscopic) applications where rapidly changing background interference due to sample heterogeneity, dynamically evolving systems and ambient light variations currently impose major challenges.
This study was funded by the UKRI Science and Technology Facilities Council through the Proof-of-Concept project PoCF1516-13. Our recent publication about the charge-shifting SERDS approach  has been awarded the 2020 William F. Meggers Award for outstanding contributions to Applied Spectroscopy.
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