Wide Field Spectral Imaging with Shifted Excitation Raman Difference Spectroscopy Using the Nod and Shuffle Technique

Abstract

Wide field Raman imaging using the integral field spectroscopy approach was used as a fast, one shot imaging method for the simultaneous collection of all spectra composing a Raman image. For the suppression of autofluorescence and background signals such as room light, shifted excitation Raman difference spectroscopy (SERDS) was applied to remove background artifacts in Raman spectra. To reduce acquisition times in wide field SERDS imaging, we adapted the nod and shuffle technique from astrophysics and implemented it into a wide field SERDS imaging setup. In our adapted version, the nod corresponds to the change in excitation wavelength, whereas the shuffle corresponds to the shifting of charges up and down on a Charge-Coupled Device (CCD) chip synchronous to the change in excitation wavelength. We coupled this improved wide field SERDS imaging setup to diode lasers with 784.4/785.5 and 457.7/458.9 nm excitation and applied it to samples such as paracetamol and aspirin tablets, polystyrene and polymethyl methacrylate beads, as well as pork meat using multiple accumulations with acquisition times in the range of 50 to 200 ms. The results tackle two main challenges of SERDS imaging: gradual photobleaching changes the autofluorescence background, and multiple readouts of CCD detector prolong the acquisition time.

Assessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples

“…Upon multiple iterations, this acquisition technique distributes the photobleaching more evenly among the spectra measured with different excitation wavelengths and hence efficiently compensates photobleaching. 29,53 Since this was not feasible with the used laser source, an advanced difference optimization approach was incorporated in the preprocessing step to correct a baseline before the difference optimization and calculation of the difference spectra. Then, SERDS outperforms common baseline correction methods offering a better correction and spectral interpretation, in particular for very high back-grounds.…”

“…The instrumental method of shifted excitation Raman difference spectroscopy (SERDS) 27 can be regarded as a wavelength modulated method, which has already been employed for biological samples. [28][29][30][31][32][33][34][35][36][37][38][39] In SERDS, two Raman spectra are acquired at the same spatial position with two slightly shifted excitation wavelengths. The shift in excitation wavelength should be chosen according to the full width at half maximum (FWHM) of the expected Raman bands and is limited by the transmission of the bandpass filter for laser clean-up in the setup.…”

The role of Raman spectroscopy in biopharmaceuticals from development to manufacturing

“…Yet, using a longer wavelength does require longer signal integration times because the Raman effect is inversely proportional to the excitation wavelength to the 4 th power. Time-resolved or SERDS may provide a faster means of reducing fluorescence, and these approaches show promise in reducing fluorescence in Raman spectra of biological materials [ 19 , 20 ].…”

Looking for a perfect match: multimodal combinations of Raman spectroscopy for biomedical applications

“…To correct for AF background in Raman spectra, there is a multitude of computational and instrumental techniques. 55 – 60 Nevertheless, the AF signal also carries valuable information about a sample, namely, its endogenous fluorophores, which could add beneficial information to improve classification rather than removing it by means of sophisticated chemometric algorithms. 61 – 64 Obviously, a trade-off between a sufficiently strong Raman signal and manageable AF signal is still of utmost importance.…”