Multicolor FPALM can be used for a variety of different probes, including photoactivatable proteins and organic caged or non-caged dyes. We can image multiple species in fixed or living cells.
Samples are labelled with multiple species of photoactivatable proteins or colors of organic dyes. The fluorescence from the sample is directed to the camera. By splitting this fluorescence into channels of different colors (see Figure), we can then use a ratiometric analysis of the data to localize molecules of different colors.
Figure 2 – A fibroblast cell showing the distributions of Dendra2-hemagglutinin (green, A) and PAmCherry-cofilin (red, B). Clusters of the Dendra2-hemagglutinin protein exclude PAmCherry-cofilin (merge, C).
Click the link below to watch a video recorded by the Journal of Visualized Experiments (JoVE), an online science video-journalism website:
Kim D.*, Curthoys N.*, Parent M.*, Hess S.T.*, “Bleed-through correction for rendering and correlation analysis in multi-color localization microscopy,” Journal of Optics, 15:094011, 2013.
Gudheti M.*, Curthoys N.*, Gould T.*, Kim D.*, Gabor K.*, Gunewardene M.*, Zimmerberg J., Gosse J., Kim C., Hess S.T.*, “Actin Mediates the Nanoscale Membrane Organization of the Clustered Membrane Protein Influenza Hemagglutinin,” Biophysical Journal 104:2182-92, 2013. PMCID: 3660645
Curthoys, N.M.*, Mlodzianoski, M.J.*, Kim, D.*, Hess, S.T.* “Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy.” Journal of Visual Experimentation Dec 9;(82): e50680, 2013. PMID: 24378721
* denotes former and current members of the Hess Lab