Spectral imaging of multi-color chromogenic dyes in pathological specimens

Merryn V.E. Macville, Jeroen A.W.M. Van Der Laak, Ernst J.M. Speel, Nir Katzir, Yuval Garini, Dirk Soenksen, George McNamara, Peter C.M. De Wilde, Antonius G.J.M. Hanselaar, Anton H.N. Hopman, Thomas Ried

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


We have investigated the use of spectral imaging for multicolor analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.

Original languageEnglish
Pages (from-to)133-142
Number of pages10
JournalAnalytical Cellular Pathology
Issue number3
StatePublished - 2001
Externally publishedYes


  • Absorption
  • Bright-field microscopy
  • Fourier-transform spectroscopy
  • In situ hybridization, immunocytochemistry
  • Multicolor
  • Optical density
  • Pathology
  • Spectral imaging


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