Multicolor PALM (Photo-Activated Localization Microscopy) is gestalt microscopy technique as it requires combined knowledge of genetics, cell biology and biophysics. This experimentally very complex technique is an option for those wanting to directly visualize the interactions on the nm scale, sometimes even on the level of the individual molecules.
Multicolor PALM is quite complex and demanding. When two or more photoactivatable fluorescent proteins are observed in PALM, the crucial step is the manipulation of the expression levels of these proteins in the cell, making them just right; i.e. as close as possible to physiological levels and in balance. Too much of the proteins can create artifacts with oversaturating signals, too little will not provide enough photons for super resolution imaging and a large difference in the expression levels of different proteins creates the environment where a signal of the more abundant protein can suffocate the signal of the less abundant one. Multicolor PALM is thus achieved by the generation of the adequately positioned label and the promoter (genetics), optimization of the growth conditions (cell biology), sample preparation tinkering (biochemistry) and refined image acquisition (biophysics). This is shown on the example of dual color PALM in Saccharomyces cerevisiae cells with Gap1C labeled with mEos3.2 on the N-terminus and under controllable promoter on the plasmid (magenta) and Can1 labeled with mEos3.2 on the C-terminus and expressed from the chromosome under the endogenous promoter (green).