Label‐free Raman spectroscopic imaging to extract morphological and chemical information from a formalin‐fixed, paraffin‐embedded rat colon tissue section

Abstract

SummaryAnimal models and archived human biobank tissues are useful resources for research in disease development, diagnostics and therapeutics. For the preservation of microscopic anatomical features and to facilitate long‐term storage, a majority of tissue samples are denatured by the chemical treatments required for fixation, paraffin embedding and subsequent deparaffinization. These aggressive chemical processes are thought to modify the biochemical composition of the sample and potentially compromise reliable spectroscopic examination useful for the diagnosis or biomarking. As a result, spectroscopy is often conducted on fresh/frozen samples. In this study, we provide an extensive characterization of the biochemical signals remaining in processed samples (formalin fixation and paraffin embedding, FFPE) and especially those originating from the anatomical layers of a healthy rat colon. The application of chemometric analytical methods (unsupervised and supervised) was shown to eliminate the need for tissue staining and easily revealed microscopic features consistent with goblet cells and the dense populations of cells within the mucosa, principally via strong nucleic acid signals. We were also able to identify the collagenous submucosa‐ and serosa‐ as well as the muscle‐associated signals from the muscular regions and blood vessels. Applying linear regression analysis to the data, we were able to corroborate this initial assignment of cell and tissue types by confirming the biological origin of each layer by reference to a subset of authentic biomolecular standards. Our results demonstrate the potential of using label‐free Raman microspectroscopy to obtain superior imaging contrast in FFPE sections when compared directly to conventional haematoxylin and eosin (H&E) staining.

Developing Raman spectroscopy as a diagnostic tool for label‐free antigen detection

“…The pseudo‐color Raman comparison (created from the PC2 loading in the PCA) arises from DNA/RNA signals. Peaks characteristic of DNA have previously been assigned at 782, 1100, 1335, 1573 and 1662 cm ‐1 and corresponding peaks in the loadings of PC2 could be found at 780 and 1105 cm −1 in our spectra. Even though the major peak in the loading identified around 556 cm −1 could not be assigned to DNA, the IHC/RS comparison for the Ki67 marker shows good correlation with DNA since the greatest scattering intensity is also mapped to crypt bases, where cells are actively proliferating.…”

“…It is likely that spectral contamination due to fluorescence and paraffin retention are the cause of these inconsistencies in the loadings. Paraffin retention can contaminate Raman spectra across the whole biological region, masking peaks of interest and detrimentally contributing to PC loadings; particularly those associated with discriminating lipids . Additionally, mucin‐related signals will be highly influenced by paraffin since we have observed that it is commonly retained in mucus and that the vigorous deparaffination required to remove trapped paraffin sometimes also removes the non‐attached mucus layer.…”

“…The major spectral contributions used to generate the PC1 red pseudo‐color image can be attributed to amino acid scatter, which have easily distinguishable spectra, even in complex biological samples . Intense peaks corresponding to proline at 852 cm −1 , phenylalanine at 1004 cm −1 and the CH 2 bending mode of proteins at 1449 cm −1 all indicate that protein scatter is the major contributor to PC1 .…”

Application of Raman spectroscopy in comparative study of antiobesity influence of oxytocin and freeze‐dried extracts of Uvariodendron anisatum Verdeck (Annonaceae) in Sprague Dawley rats

“…The spectral similarity between Raman spectral data from UAV‐treated, OT‐treated, obese, and nonobese rats was computed using cosine similarity. Cosine similarity is defined as follows: where O i and U i denotes Raman intensity at wavenumber i of spectra of the two data sets being compared. Identical (dissimilar) spectral data sets will give a value of 1 (0), whereas directly opposite data sets give a value of −1.…”

Utility of Raman spectroscopy in diabetes detection based on biomarker Raman bands and in antidiabetic efficacy studies of herbal extract Rotheca myricoides Hochst

“…To compute the spectral similarity between spectral data from blood of diabetic rats (D) and those from a sum of individual average spectra from creatine monohydrate, valine, leucine, isoleucine, glucose, and fructose solids (Solid, S; i.e., Raman spectra of creatine + valine + leucine + isoleucine + glucose + fructose; black line, solid), cosine similarity metric was used. Cosine similarity is defined as , where D i and S i denote Raman intensity at wave number i of spectra from blood of diabetic rats and from the combined solid spectra respectively.…”