Significant false signals can occur in multiwavelength and multiprobe flow cytometry experiments, according to researchers at Simphotek (Newark, NJ). What's more, these errors can result from unanticipated phosphorescence overlap—which is surprising since phosphorescence has not traditionally been considered a source of error in flow cytometry.
A software tool designed to expose the hidden mechanisms of potential false readings in flow cytometry, SimphoSOFT demonstrates that problematic or false signals from phosphorescence can range from 40% to greater than 500% of the correct (fluorescence) signal—and lead to possible misinterpretations of biological results. For example, it can indicate that cancer cells are not present when, in fact, they are.
In some cases, measuring the signals in wavelength or detector channels before and after each probe is used may mitigate or reduce spectral overlap; however, false results may be unaccounted for due to limited detector sensitivity. SimphoSOFT can select a particular combination of probe molecules ahead of measurements by determining if the probe molecules may be problematic, and can correct and/or check post-experiment measurements by calculating and removing any undetected false signals. In addition to emission intensity, the group's software also calculates photobleaching, singlet oxygen formation, energy transfer, and upconversion in multiple fluorescent probes used in biology and medicine.
The tool may be particularly useful because flow cytometry increasingly involves multiple lasers and multiple probes fluorescing at different wavelengths.