Classification accuracy in multiple color fluorescence imaging microscopy

Background. The discriminatory power and imaging efficiency of different mu lticolor FISH (M-FISH) analysis systems are key factors in obtaining accura te and reproducible classification results. In a recent paper, Garini et al . put forth an analytical technique to quantify the discriminatory power (" S/N ratio") and imaging efficiency ('excitation efficiency') of multicolor fluorescent karyotyping systems. Materials and Methods. A parametric model of multicolor fluorescence micros copy, based on the Beer-Lambert law, is analyzed and reduced to a simple ex pression for S/N ratio. Parameters for individual system configurations are then plugged into the model for comparison purposes. Results. We found that several invalid assumptions, which are used to reduc e the complex mathematics of the Beer-Lambert law to a simple S/N ratio, re sult in some completely misleading conclusions about classification accurac y. The authors omit the most significant noise source, and consider only on e highly abstract and unrepresentative situation. Unwisely chosen parameter s used in the examples lead to predictions that are not consistent with act ual results. Conclusions. The earlier paper presents an inaccurate view of the M-FISH si tuation. In this short communication, we point out several inaccurate assum ptions in the mathematical development of Garini et al. and the poor choice s of parameters in their examples. We show results obtained with different imaging systems that indicate that reliable and comparable results are obta ined if the metaphase samples are well-hybridized. We also conclude that so -called biochemical noise, not photon noise, is the primary factor that lim its pixel classification accuracy, given reasonable exposure times. Cytomet ry 41:139-147, 2000. (C) 2000 Wiley-Liss, Inc.