Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission
Fig 5
Images of multilabeled samples where method II is used to resolve the fluorescent signal from sharp emission bands and the background.
Smoothed raw data S (integrated for all the pixels in the image) and RGB images where green color indicates the background signal B from F18, MitoTracker Red, and ATTO647N and red or blue color indicate the fluorescence L(II) from Eu(III) or Tb(III), respectively. The signal of the lanthanides L(II) is integrated inside the spectrally filtered band indicated for each image, whereas the signal B is integrated over the whole spectral region. In the case of Eu(III) the spectral region in gray color was not integrated, since the Eu(III) band in that region was not sharp enough to be resolved by method II. The R, G, and B images that form the merged RGB image are individually normalized in order to resolve the features detected in the background signal and from the lanthanide centered emission. In the bottom row, the resolved spectra L show that several lanthanide(III) ions can be imaged simultaneously if the fluorescent signals are integrated within a specific lanthanide band as illustrated by the dashed lines.