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Biological macromolecular interactions between proteins, transcription factors, DNA and other types of biomolecules,
are fundamentally important to several cellular and biological processes. 3D Multi-channel confocal microscopy and
colocalization analysis of fluorescent signals have proven to be invaluable tools for detecting such molecular
interactions. The aim of this work was to quantify colocalization of the FOXP3 transcription factor in 3D cellular space
generated from the confocal 3D image sets. 293T cells transfected with a conditionally active form of FOXP3 were
stained for nuclei with Hoechst, for FOXP3 with anti-FOXP3 conjugated to PE, and 4-hydroxytamoxifen used as protein
translocation and activation agent. Since the protein signal was weak and nonspecific intensity contributions were strong,
it was difficult to perform colocalization analysis and estimate colocalization quantities. We performed 3D restoration by
deconvolution method on the confocal images using experimentally measured point spread functions (PSFs) and
subsequently a color shift correction. The deconvolution method eliminated nonspecific intensity contributions
originating from PSF imposed by optical microscopy diffraction resolution limits and noise since these factors
significantly affected colocalization analysis and quantification. Visual inspection of the deconvolved 3D image
suggested that the FOXP3 molecules are predominantly colocalized within the nuclei although the fluorescent signals
from FOXP3 molecules were also present in the cytoplasm. A close inspection of the scatter plot (colocalization map)
and correlation quantities such as the Pearsons and colocalization coefficients showed that the fluorescent signals from
the FOXP3 molecules and DNA are strongly correlated. In conclusion, our colocalization quantification approach
confirms the preferential association of the FOXP3 molecules with the DNA despite the presence of fluorescent signals
from the former one both in the nuclei and cytoplasm.
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