Infrared Imaging Reveals Changes in Chemical Composition in Lung Tissue with Idiopathic Pulmonary Fibrosis Isabella Molina,1 George Kipshidze,2 Vidyani Suryadevara,3 Lisa Miller 4 Hofstra University, 1000 Hempstead Tpke, Hempstead, NY 11549 2 Integrative Neuroscience Dept., SUNY Binghamton University, Vestal, NY, 13902 3 Department of Radiology, Stanford University, Stanford, CA 94305 4 NSLS-II, Brookhaven National Laboratory, Upton, NY, 11973 1
Abstract Idiopathic Pulmonary Fibrosis (IPF) is a chronic and fatal lung disease associated with damage to epithelial tissue and decreased repairing capability of the alveolar compartment as abnormal extracellular matrix (ECM) deposits lead to tissue scarring. Fourier transform infrared imaging (FTIRI) is a promising technique for imaging the biochemical changes related to fibrotic changes, which can be analyzed to mark the progression of IPF. In this study the FTIRI was used to obtain infrared images from 5 human lung tissue samples containing IPF, and 5 normal human lung tissue samples. Samples were embedded in paraffin wax, cross sectioned, placed on low-e microscope slides, and then deparaffinized. Spectral and image analysis revealed differences in lipid composition between IPF samples compared to CNT samples. Specifically, we found IPF samples contain more short chain lipids than CNT samples. There were also minor differences in collagen composition between IPF and CNT animals that will require further multivariate analysis. These findings indicate that FTIRI has the potential to be a promising diagnostic technique for IPF, as analysis of infrared data can reveal information regarding disease progression and provide insight on the chemical composition of IPF lung tissue. Upon participation in the SULI program this summer, I have added IR spectroscopy and FTIR imaging to my skillset. Moreover, I became familiar with various software including ImageJ, OMNIC, and Spotlight. Equally important to the laboratory skills and academic enrichment gained, I also had the invaluable opportunity of networking within the lab and expanding my professional connections and relationships.
Research Goals 1. View fibrosis in IPF to identify spectral features for disease diagnosis 2. Clinical implications - Determine whether FTIR-I can be used to characterize biopsy for disease 3. Chemical implications - Understand composition of IPF lung tissue
Image Analysis A
D
B
E
C
F
Introduction • Current IPF diagnostic methods: • Radiological and histological analysis • Limitations of radiological assessment • Requires chemical staining and histological review by a pathologist • Biochemical characteristics of IPF progression are unknown • FTIR-I can reveal chemical information about biochemical events in IPF tissue
Methods
https://doi.org/10.1016/j.ajpath.2020.09.001
• 10 cross sections of human lung tissue • 5 contain IPF • 5 normal tissue • Spectrum Spotlight 400 FT-IR Imaging System for collection of spectral images • Imaging of one tissue sample takes approximately 2 hours
Spectral Analysis
Figure 2. (a) Visible image survey of an IPF tissue sample. (b) Ratio of 1660 cm-1 / total protein of (a) showing the collagen distribution in IPF. (c) Ratio of 2958 cm-1 / 2916 cm-1 of (a) showing the short-chain lipid distribution in IPF. (d) Visible image survey of a CNT tissue sample. (e) Ratio of 1660 cm-1 / total protein of (d) showing the collagen distribution in CNT. (c) Ratio of 2958 cm-1 / 2916 cm-1 of (d) showing the short-chain lipid distribution in CNT. Scale bar is 1 mm.
Discussion
Figure 1. Average absorbance of 20 spectra per 5 IPF animals (blue) and 20 spectra per 5 CNT animals (red).
Literature Cited 1. Suryadevara, V., Huang, L., Kim, S.-J., Cheresh, P., Shaaya, M., Bandela, M., Fu, P., FeghaliBostwick, C., Di Paolo, G., Kamp, D. W., & Natarajan, V. (2019). Role of phospholipase D in bleomycin-induced mitochondrial reactive oxygen species generation, mitochondrial DNA damage, and pulmonary fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 317(2). https://doi.org/10.1152/ajplung.00320.2018 2. Suryadevara, V., Nazeer, S. S., Sreedhar, H., Adelaja, O., Kajdacsy-Balla, A., Natarajan, V., & Walsh, M. J. (2020). Infrared Spectral Microscopy as a tool to monitor lung fibrosis development in a model system. Biomedical Optics Express, 11(7), 3996. https://doi.org/10.1364/boe.394730
• FTIR-I demonstrates compositional differences between IPF and normal lung samples • Analysis revealed that differences in lipid composition exist between IPF samples compared to CNT samples. IPF contains more short chain lipids than CNT • There were minor differences in collagen composition between IPF and CNT • These data can be utilized as a diagnostic tool as it provides information characteristic to that of fibrosis which may mark disease progression
Acknowledgements I would like to thank Brookhaven National Laboratory and the Office of Educational Programs for the opportunity to participate in the 2022 SULI Program. Special thank you to Dr. Vidyani Suryadevara for preparing the samples used for this study and for her insight and guidance. This project was supported in part by the U.S. Department of Energy, Office of Science, Office of Workforce Development for Teachers and Scientists (WDTS) under the Science Undergraduate Laboratory Internships Program (SULI).