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Bioinformatik Uni Leipzig

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Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging


Wolfram Steller, Reiner Salzer, Christoph Krafft
Institute for Analytical Chemistry
Dresden University of Technology

Ulf-Dietrich Braumann, Hans Binder
Interdisciplinary Centre for Bioinformatics
University of Leipzig

Jens Einenkel
Department of Gynaecology and Obstetrics
University of Leipzig

Lars-Christian Horn
Institute for Pathology
University of Leipzig



Figure 1: IR image of the transition from cervical stroma to squamous epithelium. The upper part shows a H&E-stained tissue for direct comparison. The part below shows the result of hierarchical cluster analysis. The spectra were classified using a ten-color-coding (see horizontal dashed line in the dendrogram). They refer to cervical stroma (pink), inflammation (cyan, blue), basal cell layer (red), transition from basal to parabasal cell layer (light green, dark green), parabasal cell layer (yellow), intermediate cell layer (light orange, dark orange), superficial cell layer (brown).
Background: Infrared (IR) spectroscopy relies on the absorption of radiation which excites molecular vibrations. Biological macromolecules such as proteins, nucleic acids, and lipids have specific, fingerprint-like IR spectra. Because these spectral fingerprints are directly determined by molecular structures and cellular chemistry, IR spectroscopy has the promising potential to detect abnormal changes in cells and tissues on the basis of biochemical alterations related to the pathogenic state of the cell. Further advantages of IR spectroscopic-based techniques are minimal requirements for sample preparation and fast detection. Note that the term “imaging” refers to the parallel detection of all pixels across the observed area by an array detector, which comprises several thousands elements and thus enable correspondingly fast measurement.


Figure 1: Seven different H&E-stained tissue areas (1 to 7 from top to bottom) were compared with the respective IR images referring to two different spectral windows (middle: 950–1480 cm-1 ; right: 1420-1480 cm-1 ). The right coloring scheme highlights basal cell layer (yellow), dysplasia (orange), squamous cell carcinoma (red), non-basal cell layer and non-malignant tissue (blue). Bar=200 µm.
Results: We applied IR spectroscopic imaging coupled with microscopy to investigate thin sections of cervix uteri encompassing normal tissue, precancerous structures, and squamous cell carcinoma. 122 images were recorded with a 64×64 focal plane array detector which provides 499,712 IR spectra. They were grouped using a fuzzy C-means clustering approach followed by hierarchical cluster analysis. The resulting false color maps were correlated with the morphological characteristics of adjacent sections of hematoxylin and eosin- (H&E-) stained tissue. Whereas cervical stroma, epithelium, inflammation, blood vessels, and mucus could be distinguished in IR images by analysis of the spectral fingerprint from 950-1480 cm-1, carcinoma overlap with normal basal cell layer where cancerogenesis preferentially occurs. However, the detailed analysis in the spectral window 1420–1480 cm-1 enables, for the first time, IR spectroscopic distinction between the basal cell layer, dysplastic lesions and squamous cell carcinoma within a particular sample (Figure 2, right part).

Conclusions and outlook: The joint application of IR microspectroscopic imaging and multivariate spectral processing combines diffraction-limited lateral optical resolution on the single cell level with highly specific and sensitive spectral classification on the molecular level. Compared with previous reports our approach constitutes a significant progress in the development of optical molecular spectroscopic techniques toward an additional diagnostic tool for the early histopathological characterization of cervical cancer. Publication: Steller, W., Einenkel, J., Horn, L.C., Braumann, U.D., Binder, H., Salzer, R., Krafft, Ch. (2006). Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging. Analytical and Bioanalytical Chemistry.

Publication:
Steller, W., Einenkel, J., Horn, L.C., Braumann, U.D., Binder, H., Salzer, R., Krafft, Ch. (2006)
Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging
Analytical and Bioanalytical Chemistry.

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