Abstract
We have investigated the use of spectral imaging for multi‐color 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. Figures on http://www.esacp.org/acp/2001/22‐3/macville.htm.
Highlights
Spectral imaging of fluorescent dyes has been successfully applied to spectral karyotyping (SKY) for the genome-wide screening of chromosomal aberrations in metaphase cells [18], and is being used in clinical and tumor cytogenetics [7,19,20,25]
The absorption spectra of PO-substrates DAB, AEC, TMB, and alkaline phosphatase (AP)-substrates Fast Red (FR) and New Fuchsin (NF) were measured in single-color in situ hybridization (ISH) experiments on T24 cells without nuclear counterstaining (Fig. 1A)
Complementing the morphologic data with molecular data in situ can refine the diagnosis and staging of certain types of cancer. These include (a) phenotypic protein expression data obtained by means of immunocytochemistry (ICC) with antibodies against products of commonly deregulated oncogenes or tumor suppressor genes, (b) protein function assays by enzyme histochemical procedures, and (c) molecular cytogenetic detection by in situ hybridization (ISH) of genomic alterations of chromosomes or chromosome loci
Summary
Spectral imaging of fluorescent dyes has been successfully applied to spectral karyotyping (SKY) for the genome-wide screening of chromosomal aberrations in metaphase cells [18], and is being used in clinical and tumor cytogenetics [7,19,20,25]. Dedicated software for SKY and spectral FISH enables analysis of combinatorial labeled nucleic acid probes in metaphase and interphase cells, respectively. Spectral imaging may be used for conventional transmission light microscopy using chromogenic dyes [10,16]. Such an application might be of great clinical importance, because diagnosis of neoplasms frequently requires application of immunocytochemistry (ICC) and in situ hybridization (ISH) for the assessment of phenotypic and genetic biomarkers to complement the cy-. Bright-field microscopy optics can be used to detect up to three ISH targets simultaneously, together with a nuclear or cytoplasmic dye for morphology assessment. Color recognition of weak or small ISH signals, such as for chromosome locusspecific probes, has been impossible
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
