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

Christoph Krafft,Martin M. Roth,Christer Sandin,Genoveva Micheva,Günther Tränkle,Florian Korinth,Bernd Sumpf,Martin Maiwald,Jürgen Popp,Clara Stiebing,Tanya Urrutia,André Müller,Elmar Schmälzlin

doi:10.3390/s20236723

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.

Highlights

Raman spectroscopy provides a label-free, non-destructive insight into the biochemical composition of a sample
Higher signal intensity corresponds to higher brightness, and the horizontally arranged traces correspond to complete Raman spectra of two intertwined Raman images taken at two different excitation wavelengths λ1 and λ2
The total acquisition time of a Raman image, in our case consisting of 400 spectra of a 20 by 20 fiber array, is orders of magnitude faster than in point-by-point Raman imaging

Summary

Introduction

Raman spectroscopy provides a label-free, non-destructive insight into the biochemical composition of a sample. It can be used for the pathological assessment of biological samples such as cells and tissues [1,2,3,4,5]. Since the fluorescence absorption cross-section is much larger than the Raman scattering cross-section, even trace amounts of autofluorescent molecules often result in high-intensity spectral backgrounds. This high background can mask the Raman fingerprint information and increases the shot noise in spectra [7]. Depending on the complexity of the algorithm, the computational data processing can be time consuming

Methods

Results

Conclusion