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Fix or Freeze? Spectral Differences Arising from Tissue Preparation in Chemical Imaging.

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Tissue preparation significantly impacts mid-infrared spectrochemical imaging (MIRSI) data. Fresh frozen tissues retain more biochemical detail than formalin-fixed, paraffin-embedded tissues, affecting spectral analysis and AI applications.

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Area of Science:

  • Biomedical Imaging
  • Spectroscopy
  • Biochemistry

Background:

  • Mid-infrared spectrochemical imaging (MIRSI) is a label-free technique for visualizing tissue biochemistry.
  • Understanding MIRSI's sensitivity to sample preparation is crucial for accurate data interpretation in research and drug development.

Purpose of the Study:

  • To systematically compare spectral data from fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) rat kidney and liver tissues.
  • To characterize preparation-induced differences in MIRSI data using various spectral analysis techniques.

Main Methods:

  • Quantum cascade laser (QCL)-based MIRSI was used to analyze FF and FFPE rat kidney and liver tissues.
  • Spectral data processing included uniform manifold approximation and projection (UMAP), correlation matrices, and second-derivative analysis.

Main Results:

  • FF tissues preserved a wider range of biochemical signals and innate chemical composition compared to FFPE tissues.
  • FFPE tissues exhibited reduced spectral diversity and absorption signal intensity.
  • A unique spectral band at 1026 cm-1, indicative of formalin-induced collagen crosslinking, was observed in FFPE samples.

Conclusions:

  • Tissue preparation methods substantially alter chemical and morphological information obtained via MIRSI.
  • Careful consideration of tissue processing protocols is essential for MIRSI workflows, especially those involving AI-based spectral analysis.