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Updated: May 19, 2026
07:54
Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation
Published on: March 12, 2015
Fix or freeze? Spectral differences arising from tissue preparation in chemical imaging
Tianyi Zheng1, Wihan Adi1, Paul J Campagnola1
1Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA. filiz.yesilkoy@wisc.edu.
The Analyst
|May 18, 2026
Summary
Tissue preparation significantly impacts mid-infrared spectrochemical imaging (MIRSI) data. Fresh frozen tissues retain more biochemical information than formalin-fixed, paraffin-embedded tissues, affecting AI-based analysis.
Area of Science:
- Biomedical Imaging
- Spectroscopy
- Biochemistry
Background:
- Spectrochemical imaging offers label-free visualization of tissue biochemistry via vibrational signatures.
- Mid-infrared spectrochemical imaging (MIRSI) is crucial for disease research, biomarker discovery, and drug development.
- The influence of tissue preparation on MIRSI data interpretation is not well understood.
Purpose of the Study:
- To systematically compare spectral data from fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) rat kidney and liver tissues using quantum cascade laser (QCL)-based MIRSI.
- To characterize preparation-induced spectral differences using data processing techniques like UMAP and second-derivative analysis.
Main Methods:
- Quantum cascade laser-based mid-infrared spectrochemical imaging (QCL-MIRSI) was used.
- Rat kidney and liver tissues were processed using fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) methods.
- 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 lower absorption signal intensity.
- A distinct spectral band at 1026 cm-1 was observed in FFPE samples, likely due to fixation-induced chemical changes.
Conclusions:
- Tissue preparation methods significantly alter chemical and morphological information obtained through MIRSI.
- Careful consideration of tissue processing protocols is essential for reliable chemical imaging and AI-driven spectral analysis.
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