Abstract: TH-PO384

Multispectral Fluorescence Unmixing for Large-Scale Three-Dimensional Imaging and Quantitative Tissue Cytometry of Human Kidney Tissue

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Winfree, Seth, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Kelly, Katherine J., Indiana University , Indianapolis, Indiana, United States
  • Phillips, Carrie L., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Eadon, Michael T., Indiana University Division of Nephrology, Indianapolis, Indiana, United States
  • Sutton, Timothy A., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Dunn, Ken, Indiana University, Indianapolis, Indiana, United States
  • Dagher, Pierre C., Indiana University, Indianapolis, Indiana, United States
  • El-Achkar, Tarek M., Indiana University, Indianapolis, Indiana, United States
Background

Large-scale confocal fluorescence microscopy combined with three-dimensional tissue cytometry (3DTC) has the potential to provide quantitative data like flow cytometry, while preserving the localization and distribution of cells in intact kidney tissue. Confocal imaging is typically limited to 4 markers, dictated by available lasers and spectral bleed-through, limiting cell identification to 2 or 3 types.

Methods

To extend the palette of simultaneously distinguishable fluorophores (up to 8 colors), we implemented fluorescence spectral unmixing. Using this approach, we labeled proximal and distal tubular cells along with 4 basic types of immune cells in single human kidney tissue sections.

Results

Large-scale 3D imaging of these sections generated data suitable for 3DTC with our recently developed Volumetric Tissue Exploration and Analysis (VTEA) software tool. Using VTEA, we determined the abundance and localization of each labeled cell type. Furthermore, we explored the distribution of leukocytes in relation to nephron sub-segments and showed clustering of neutrophils around proximal tubules.

Conclusion

Multi-fluorescence labeling with spectral unmixing enhances our ability to simultaneously visualize and detect various cell types withn the kidney in 3D, and quantify the association of immune cells with nephron sub-segments in situ.

Large-scale 3D multi-fluorescence imaging of a human nephrectomy section (≈ 7 x 3 x 0.05 mm3, left), and tissue cytometry analysis (middle and right) using VTEA. Gates on the scatter plot identify CD68+ (macrophages) and MPO+ cells (neutrophils). The gated cells were mapped to the original volume to identify their spatial distribution (middle). A 3D rendered sub-volume demonstrates the resolution and precision of gating (lower right, gated cells identified by nuclear overlays). Scale bar = 500µm.

Funding

  • NIDDK Support