Photoactivatable Proteins

Coloration of cells reflects excitation (or absorbance), fluorescnce and photoconversion light for an FP, with the gray color corresponding to dark (nonfluorescent) state. The upper row for each FP describes its parameters before photoconversion and the lower row - after photoconversion. Complex photoswitching behavior of mIrisFP and NijiFP is described in the text. Maturation half-times are indicated for purified FPs. Abbreviations: ND, not determined; PALM, photoactivated localization microscopy. ^aA brightness in (mM.cm)^-1 is determined as the product of extinction coefficient in (M.cm)^-1 and quantum yield. ^bAn increase (fold) of fluorescence signal of the photoconverted state. ^c Maturation process includes both protein folding and chromophore formation. ^d Values are based on comparison with tsEosFP and mEos2. ^e Values are based on comparison with Dendra2 and mEos2. ^f Values are based on data for residual fluorescence in the off state reported in (29; 31; 32). ^g Although monomeric at 37°C, Padron was shown to form dimers at 4°C (29). ^h Data from (33). ⁱ The numbers were averaged to account for possible variability in biological samples expressing FPs, their preparation and imaging conditions.

Table 2. Specific properties of reversibly switchable FPs related to their applicability to RESOLFT and PALM microscopy.

The fatigue resistance and residual fluorescence may vary depending on the sample preparation and experimental conditions, particularly the power of irradiating light. For example, the background for Dreiklang is 5-10% in bacterial streaks, whereas it is 1.4% in wide-field images of mammalian cells (30). Equilibrium state indicates the relative fluorescenece intensity achieved by an FP after the relaxation in the darkness. Abbreviation: ND, not determined; PALM, photoactivated localization microscopy; RESOLFT, reversible saturatable optical fluorescence transition. ^a The wavelengths of switching light correspond to shown in Table 1, except for ^d 491 nm, ^g 390 nm, and ^h 480 nm. The filter widths are ^b 488/10 nm, ^b 405/10 nm, ^f 365/25 nm, ^g 390/40 nm, ^h 480/40 nm, ⁱ 550/20 nm, ^j 450/20 nm, ^k 570/30 nm, ^l 436/20 nm, ^m 570/5 nm, and ⁿ 440/5 nm. ND, not determined or not demonstrated. ^e Temperature dependent value, decreases with the temperature increase (30).