Abstract: SA-PO449
Label-Free Multimodal Imaging of Diabetic Kidney Disease Tissue
Session Information
- Diabetic Kidney Disease: Basic - II
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 701 Diabetic Kidney Disease: Basic
Authors
- Fung, Anthony A., University of California San Diego, La Jolla, California, United States
- Jain, Sanjay, Washington University in St Louis, St Louis, Missouri, United States
- Shi, Lingyan, University of California San Diego, La Jolla, California, United States
Group or Team Name
- HuBMAP.
Background
Accurate biomarker detection without sample destruction is critical for better utilization and preservation of biopsies. We developed a label-free multimodal imaging platform, combining hyperspectral SRS, MPF, and SHG. This approach reduces sample prep, enables the use of smaller biopsies, and details structural and biochemical changes in both 2D and 3D. Several histological stains are produced from the same sample without the need for serial reconstruction, and avoids unintended sample preparation effects such as lipid droplet fusion. Key insights include lipid subtype ratios, mesangial expansion, collagen fibrotic changes, and oxidative stress. Compatible with conventional methods, this platform affirms and deepens our understanding of DN to improve patient outcomes.
Methods
FFPE and fresh frozen renal tissue, subsequently fixed in 4% PFA, were sliced to 200μm and cleared with 8M urea and 0.2% TritonX. These samples were placed on glass slides and sealed under coverglass in water. SRS images used a tunable 500mW pump and 330mW 1031nm Stokes beams with a pixel dwell time of 40μs and a spectral resolution of 15cm-1. SHG was captured using the Stokes beam.
Results
3D volumetric imaging provided more accurate mesangial volume fractions than single plane measurements, enabling the visualization of this expansion in DN. Relative distribution of ceramides and TAGs, as well as a greater degree of cholesterol esterification were observed in DN tubules. We also observed collagen fiber thickening and disarray, and oxidative stress. Findings were corroborated by routine IHC and IF.
Conclusion
This is the first all-in-one optical platform to visualize lipid subtype dysregulation, collagen thickening and anisotropy, oxidative stress, and mesangial expansion in DN in 2D and 3D.The spatial and chemical resolution afford multiplexed views of DN and maximizes sample utilization while maintaining flexibility.
Fig 1.(A) Multiphoton laser microscopy system capable of (B) SHG imaging and (C) anisotropy analysis of collagen, (D-F) autofluorescence measurement of NAD[P]H and flavins, (G) SRS HSI, (H) 3D digital histology. (I) HSI segmentation. (J) Structure spectra plots.
Funding
- NIDDK Support