title: Label-free Raman spectroscopic imaging to extract morphological and chemical information from a formalin-fixed, paraffin-embedded rat colon tissue section.
abstract: Animal 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.
authors: Department of Cell and Developmental Biology, University College London, London, UK.
authors: Riana
authors: R
authors: Gaifulina
authors: Department of Cell and Developmental Biology, University College London, London, UK.|CoMPLEX, University College London, London, UK.
authors: Andrew Thomas
authors: AT
authors: Maher
authors: Biophotonics Research Unit, Gloucestershire Royal Hospital, Gloucester, UK.
authors: Catherine
authors: C
authors: Kendall
authors: Department of Statistical Science, University College London, London, UK.
authors: James
authors: J
authors: Nelson
authors: Department of Gastrointestinal Pathology, University College Hospital, London, UK.
authors: Manuel
authors: M
authors: Rodriguez-Justo
authors: Spectroscopy Products Division, Renishaw Plc, Wotton-under-Edge, UK.
authors: Katherine
authors: K
authors: Lau
authors: Department of Cell and Developmental Biology, University College London, London, UK. g.thomas@ucl.ac.uk.
authors: Geraint Mark
authors: GM
authors: Thomas
mesh_terms: D000818:Animals; D003106:Colon / Q000033:anatomy & histology* / Q000737:chemistry*; D005404:Fixatives; D005557:Formaldehyde; D007413:Intestinal Mucosa / Q000033:anatomy & histology / Q000737:chemistry; D016612:Paraffin Embedding / Q000379:methods; D025341:Principal Component Analysis; D017208:Rats, Wistar; D013059:Spectrum Analysis, Raman / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005404:Fixatives; D005557:Formaldehyde
keywords: Raman microspectroscopy; biochemistry; formalin-fixed paraffin-embedded; haematoxylin and eosin; paraffin; principal component; rat colon