Fig. 2. Image and phasor analysis with spectrum and lifetime analysis in MOSAICA.

a As an example, four different probes are used to target the transcripts of four different genes. The fluorescence is collected using the spectral and Fluorescence Lifetime Imaging and Microscopy (FLIM) instrument to form images where each pixel carries information of the spectra and lifetime. b At each pixel we compute the photon distribution in the spectral and temporal dimension. The phasor transform maps these distributions in each pixel to a position on the phasor space. c The phasor plots reveal the presence of different populations. These populations are identified and then mapped back to the original image. d We color code the pixels based on the combination of the two properties. This allows us to separate by lifetime probes that were emitting with similar spectra and vice-versa, separate by spectra probes that fluoresce with similar lifetimes.