Although the development of HCA has focused on the application of fluorescent probes, chromogenic probes continue to be used extensively for labeling tissue sections. Table 25-3 ^∗∗ compares the advantages and disadvantages of fluorescent and chromogenic probes. Most HCA systems are optimized for the use of fluorescent probes, principally for their high sensitivity, high specificity, broad range of cellular functional readouts, broad range of wavelengths for multiplexing, and ability to engineer cells to express fluorescent proteins and biosensors. Because HCA makes use of automated imaging and quantitative image analysis, there is no need for direct viewing of the labeled specimen, and once the images are acquired, there is no further need for the specimen other than for institutional or clinical requirements. In traditional pathology, on the other hand, chromogenic probes have some advantages. The human brain is still the most sophisticated and reliable image processor for the interpretation of small numbers of images. ⁷⁶ Readily available, low-cost chromogenic probes provide stable and dense labeling for visualization in a transmitted light microscope or by digital image pathology, while simultaneously viewing the contextual morphology of the cells. Although providing somewhat lower resolution and more limited multiplexing than fluorescent probes, chromogenic probes still provide a good labeling strategy where one to three biomarkers per slide can be useful.

Success in the Human Genome Project demanded tools to define the functions of the coding and noncoding portions of the genome, to define the dynamic interplay of cellular constituents within and between cells, and to characterize subpopulations, as well as to define the relationships between populations of cells in higher order biological systems. The field was named cellomics, and the platform technology was named HCA. HCA harnesses the ability to implement combinatorial treatments on large sample sizes by using microplates, patterned microarrays ⁷⁷ and microfluidic devices ⁷⁸ for cells, microplates for small organisms, and mounted sections/microarrays for tissues. These large sample sizes are required for statistical analyses and exploration by computational and systems biology. ^{16,21,63,79,80}