Out-of-Phase Imaging after Optical Modulation (OPIOM) for Multiplexed Fluorescence Imaging Under Adverse Optical Conditions

Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.