Cyan-to-green photoswitchable fluorescent protein PS-CFP2

- Monomer, succesful perfomance in fusions
- Irreversible photoconversion from a cyan to a green fluorescent form
- High contrast of photoconversion
- High pH stability allowing labeling of acidic organelles
- Recommended for tracking cell, organelle, and protein movement, monitoring the protein turnover and superresolution imaging

Recommended antibodies, filter sets and laser lines

PS-CFP2 can be recognized using Anti-Tag(CGY)FP antibody (Cat.# AB121) or Anti-GFP antibody (Cat.# AB011) available from Evrogen.

Visualization before photoswitching: Before activation, PS-CFP2 produces cyan fluorescence with excitation and emission maxima at 400 and 468 nm, respectively. Standard levels of excitation do not cause significant photoswitching of cyan or photobleaching of green fluorescence.

PS-CFP2 excitation spectrum is absolutely different from that of common cyan fluorescent proteins, such as TagCFP, ECFP or Cerulean. Because of the shorter excitation wavelength, cyan fluorescence of PS-CFP2 can be easily separated from green fluorescence of its photoactivated state as well as from any green fluorescent protein. Therefore, common CFP filter sets are not optimal for PS-CFP2 visualization and photoactivation.

Recommended filter sets are: XF119-2*, XF131, XF06, XF03, XF11, XF129-2, XF05-2 (Omega Optical); DAPI-5060B* and DAPI-1160A (Semrock); 31037, 31041, 31016, 31021, 31000v2, 1009v2, 31013v2, 11005v2, 31047 (Chroma Technology Corp.).
* – preferred filter sets

Photoswitching: PS-CFP2 undergoes irreversible photoconversion (in response to intense 405 nm light irradiation) expressed in a decrease in cyan fluorescence and appearance of a 490 nm excitation peak with emission maximum at 511 nm.

Visualization after photoswitching: For visualization of green fluorescence of photoactivated PS-CFP2, filter sets used for routine GFPs visualization are recommended with excitation about 470-500 nm and emission collected at about 500-550 nm (e.g. Omega Optical XF100-2). Importantly, excitation wavelength must not be below 450 nm to avoid cross-excitation of non-photoactivated PS-CFP2.

Note: To avoid undesirable photoactivation of PS-CFP2 and photobleaching of its photoactivated form it is recommended that excitation light intensities and exposition times be minimized during visualization of both protein forms.

PS-CFP2 visualization, photoactivation and tracking in a confocal microscope: The following parameters are recommended for Leica microscope DMIRE2, confocal TCS-SP2, equipped with 25 mW 405 nm diode laser, 125 mW Ar laser, objective HCX-PL-APO-63x/1.40-0.60/OIL (parameters may vary for different experimental systems):

For visualization of PS-CFP2, we recommend the use of 405 nm laser excitation. In a scanning mode, 1-5% power of 405 nm laser causes only negligible PS-CFP2 photoactivation, and can thus be used for preliminary visualization. Fluorescence emission of nonphotoactivated PS-CFP2 can be collected from about 420 nm to about 510-530 nm to gain maximum signal.

For the fast PS-CFP2 photoactivation (which is required to track dynamics of rapidly moving protein of interest), we recommend using 50-100% power of 405 nm laser, 50-2000 ms irradiation in a point, using "point-bleach" mode in Leica confocal software. From our experience, "point-bleach" mode allows much faster photoactivation than any scanning modes, probably because of the continuity of irradiation. Tracking of a protein characterized by slow dynamics does not require fast photoactivation. In this case, high-power scanning of the ROI works perfectly well for PS-CFP2 photoactivation.

For photoactivated PS-CFP2, you can use 488 nm laser excitation and collect fluorescence emission between 500 nm to 550 nm. To avoid crosstalk, we recommend the use of sequential mode if you need to visualize both initial and photoactivated PS-CFP2. Use low intensity excitation light to avoid photobleaching.

Please refer also to the detailed protocol is published in Chudakov et al., 2007.