Complementary diagnostic methods to conventional histopathology are currently being investigated for developing rapid and objective molecular-level understanding of various disorders, especially cancers. Spectral histopathology using vibrational spectroscopic imaging has been put in the frontline as potentially promising in this regard as it provides a “spectral fingerprint” of the biochemical composition of cells and tissues. In order to ascertain the feasible conditions of vibrational spectroscopic methods for tissue-imaging analysis, vibrational multimodal imaging (infrared transmission, infrared-attenuated total reflection, and Raman imaging) of the same colon tissue has been implemented. The spectral images acquired were subjected to multivariate clustering analysis in order to identify on a molecular level the constituent histological organization of the colon tissue such as the epithelium, connective tissue, etc., by comparing the cluster images with the histological reference images. Based on this study, a comparative analysis of important factors involved in the vibrational multimodal imaging approaches such as image resolution, time constraints, their advantages and limitations, and their applicability to biological tissues has been carried out. Out of the three different vibrational imaging modalities tested, infrared-attenuated total reflection mode of imaging appears to provide a good compromise between the tissue histology and the time constraints in achieving similar image contrast to that of Raman imaging at an approximately 33-fold faster measurement time. The present study demonstrates the advantages, the limitations of the important parameters involved in vibrational multimodal imaging approaches, and their potential application toward imaging of biological tissues.

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