Separation of spectrally overlapping fluorophores using intra-exposure excitation modulation.

Multicolor fluorescence imaging is an excellent method for the simultaneous visualization of multiple structures, although it is limited by the available spectral window. More labels can be measured by distinguishing these on properties, such as their fluorescence dynamics, but usually these dynamics must be directly resolvable by the instrument. We propose an approach to distinguish emitters over a much broader range of light-induced dynamics by combining fast modulation of the light source with the detection of the time-integrated fluorescence. We demonstrate our method by distinguishing four spectrally overlapping photochromic fluorophores within bacteria, showing that we can accurately classify all four probes by acquiring just two to four fluorescence images. Our strategy expands the range of probes and processes that can be used for fluorescence multiplexing.