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In this Perspective the authors highlight and discuss the artifacts that can arise when using immunolabeling to examine protein localization in cell culture. They call for using both alternative fixation and permeabilization protocols and live-cell imaging of fluorescent protein fusions to reliably study subcellular protein localization.
In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes.
