Photoactivatable fluorescent proteins (PAFPs) represent a unique tool for monitoring cellular events. PAFPs change spectral properties in response to irradiation with specific light. In recent years PAFPs have been utilized for the development of novel methods for the optical labeling and tracking living cells, organelles, and intracellular molecules in a spatio-temporal manner. PAFPs became indispensible tools for super-resolution imaging techniques. Moreover, PAFPs opened new possibilities to study cell physiology, in particular, they allow careful determination of protein half-life.
KFP-Red (also referred to as KFP1) is a photoactivatable GFP-like protein generated on the basis of Anemonia sulcata chromoprotein, asFP595 [Lukyanov et al., 2000; Chudakov et al., 2003a; Chudakov et al., 2003b]. KFP-Red switches from a non-fluorescent to a red fluorescent form (with excitation/emission maxima at 580 nm and 600 nm, respectively) under the exposure to intense green light irradiation. A green light laser does not damage cells and tissues. Activated KFP-Red can be easily detected because its emission spectrum is beyond the region of cell autofluorescence.
KFP-Red can be used for in vivo monitoring cell and cellular organelle movement.
PA-TagRFP is a photoactivatable mutant of the bright monomeric red fluorescent protein TagRFP [Subach et al., 2010]. PA-TagRFP is capable of irreversible photoconversion from non-fluorescent to red fluorescent form (with excitation/emission maxima at 562 nm and 595 nm, respectively) in response to UV-violet light irradiation.
High brightness, photostability and monomeric nature of PA-TagRFP make it an excellent protein tag for both conventional microscopy and super-resolution PALM imaging techniques [Subach et al., 2010].
PA-TagRFP use in PALM imaging techniques
Tracking of PA-TagRFP-tagged epidermal growth factor receptor (EGFR-PATagRFP) and PAGFP-tagged vesicular stomatitus virus G protein tsO45 (VSVG-PAGFP) in live COS-7 cells by two-color single-particle tracking PALM.