Abstract
Background: The current appreciation of the biological complexity of disease has led to increasing demands on pathologists to provide clinically relevant, quantitative morphological and molecular information while preserving cellular and tissue context. This can be technically challenging, especially when signals of interest are colocalized. With fluorescence-based methods, sensitivity and quantitative reliability may be compromised by spectral cross-talk between labels and by autofluorescence. In brightfield microscopy, overlapping chromogenic signals pose similar imaging difficulties. Approach: These challenges can be addressed using commercially available multispectral imaging technologies attached to standard microscope platforms, or alternatively, integrated into whole-slide scanning instruments. Assessment: Multispectral techniques, along with other developments in digital analysis, will allow pathologists to deliver appropriate quantitative and multiplexed analyses in a reproducible and timely manner. Caveats apply – as the complexity of the sample preparation and analysis components increases, commensurate attention must be paid to the use of appropriate controls for all stages of the process.
Acknowledgements
Support from the NIH via grants BRP 5R01CA108468 and SBIR 2R43CA088684 is gratefully acknowledged. Thanks are also due to James Mansfield for assistance in the preparation of this manuscript.