Fluorescent Protein Resource Center

Photoactivatable Proteins

Protein	Excitation (Absorbance) max, nm	Emission max, nm	Extinction Coeff., M^-1 cm^-1	Quantum yield	Brightⁿ	pKa	Photo- stability	Photo- activation contrast^b	Photo light, nm	Maturation	Localization, nm	Ref.	Source
Photoactivatable (PAFPs)
PA-GFP	400 504	515 517	20,700 17,400	0.13 0.79	2.6 13.7	ND 6.3	ND Low	200	405	ND	~40 (1)	(2)	Addgene
PAmCherry1	(404) 504	ND 595	6,500 18,000	ND 0.46	ND 8.3	ND 6.3	ND Medium	4,000	405	23	~10-15 (3); ~20 (4)	(4)	Addgene
PATagRFP	(278) 562	ND 595	ND 66,000	ND 0.38	ND 25	ND 5.3	ND High	>5,000	405	75	~20 (5)	(5)	Addgene
Photoswitchable (PSFPs)
PSCFP2	400 490	470 511	43,000 47,000	0.20 0.23	8.6 11	4.3 6.1	ND Low	400	405	ND	~30 (6)	(7;8)	Evrogen, Addgene
Dendra2	400 490	470 511	43,000 47,000	0.20 0.23	8.6 11	4.3 6.1	ND Low	400	405 488	90	~10 (9); ~20 (10)	(8;11)	Evrogen, Addgene
tdEosFP (tandem dimer)	506 569	516 581	84,000 33,000	0.66 0.60	55 20	5.7 ND	Medium High	200	405	72	~10 (6); ~20-25 (4;12)	(13)	Authors
mEos2	506 573	519 584	56,000 46,000	0.84 0.66	47 30	5.6 6.4	Medium High	~200^d	405	<120	~10 (10)	(10)	Addgene
mEos3.2	507 572	516 580	63,400 32,200	0.84 0.55	53 18	5.4 5.8	Medium High	ND	405	<90^e	~10 (14)	(14)	Authors
mKikGR	505 580	515 591	49,000 28,000	0.69 0.63	34 18	6.6 5.2	Low Medium	400	405	ND	<15 (15)	(16)	Authors
mClavGR2	488 566	504 583	19,000 32,000	0.77 0.53	15 17	8.0 7.3	Low Medium	~200^d	405	90^e	<30 (14)	(17)	Authors
mMaple	489 566	505 583	15,000 30,000	0.74 0.56	11 17	8.2 7.3	High High	~400^d	405	90^e	ND	(18)	Authors
mIrisFP	(386) 486 (446) 546	ND 516 ND 578	12,000 47,000 21,000 33,000	ND 0.54 ND 0.59	ND 25 ND 19	ND 5.4 ND 7.6	ND ND	ND	405 488 473 488 561	>14	~30-50 (19) ~20-40 (19)	(19)	Authors
NijiFP	(~375) 469 (~440) 526	ND 507 ND 569	ND 41,000 ND 42,000	ND 0.64 ND 0.65	ND 26 ND 27	ND 7.0 ND 7.3	ND HIGH	ND	405 405 488 446 561	ND	~25 (20) ~35 (20)	(20)	Authors
PSmOrange	548 636	565 662	113,300 32,700	0.51 0.28	58 9.2	6.2 5.6	High High	560	480 540	90	~45 (21)	(22)	Addgene
PSmOrange2	546 619	561 651	51,000 18,900	0.61 0.38	31 7.2	6.6 5.4	Medium High	680	480 540	210	~10-20 (23)	(24)	Addgene
Reversibly Switchable (rsFPs)
Dronpa	(392) 503	ND 518	ND 95,000	ND 0.85	ND 81	ND 5.0	ND Low	20	405 488	~20 (25); ~30 (3)	~10-20 (23)	(26)	MBL Intl
Dronpa3	(ND) 487	ND 514	ND 58,000	ND 0.33	ND 19	ND ND	ND ND	ND	405 488	ND	ND	(27)	Authors
rsFastLine	(384), (496) 496	ND 518	ND 39,100	ND 0.77	ND 30	ND ND	ND Medium	~30-50^f	405 488	ND	~40 (25)	(28)	Authors
Padron^g	(505) (396}, 503	ND 522	ND 43,000	ND 0.64	ND 28	ND ND	ND Medium	~20^f	488 405	ND	ND	(29)	Authors
bsDronpa	(385) 460	ND 504	ND 45,000	ND 0.50	ND 23	ND ND	ND Medium	~20^f	405 488	ND	ND	(29)	Authors
mGeosM	(390) 503	ND 514	ND 51,600	ND 0.85	ND 44	ND 5.0	ND ND	20	405 488	ND	~15 (3)	(3)	Authors
Dreiklang	(340), (511) (412), 511	ND 529	ND 83,000	ND 0.41	ND 34	ND 7.2	ND Medium	>20	365 405	120	~15 (30)	(30)	Authors
rsEGFP	(396) 493	ND 519	ND 47,000	ND 0.36	ND 17	ND 6.5	ND High	~30-50^f	405 488	180	ND	(31)	Authors
rsEGFP2	(408) 478	ND 503	ND 61,300	ND 0.30	ND 18	ND 5.8	ND High	~12-15^f	405 488	~20	ND	(32)	Authors
rsTagRFP	440 567	585 585	15,300 36,800	0.0013 0.11	0.02 4	ND 6.6	ND Medium	20	440 570	43	ND	(33)	Addgene

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.

Protein	Residual signal in the Off state, %	Fatigue resistance (number of switching cycles to bleach to 50% of initial fluorescence)	Photoswitching kinetics: On-to-Off / Off-to-On switching half-times, (s) and light power (W/cm²)^a	Relaxation half-time at 22°C, min	Equilibrium % of maximal fluorescence at 22°C	Reference
Dronpa	~10	4	15.0 (45 x 10^-3 ^b) / 0.12 (48 x 10^-3 ^c) (29) 16 x 10^-3 (0.6 x 103 ^d) / 0.05 x 10^-3 (2 x 103) (31) 0.8 (0.4 ^g) / 0.6 (40 ^h) (33)	840	100	(29; 31; 33)
rsFastlime	1-2	35	2.6 (45 x 10^-3 ^b) / 0.03 (48 x 10^-3 ^c)	8	93	(29; 31)
Padron	15	16	0.06 (48 x 10^-3 ^b) / 5.6 (45 x 10^-3 ^c)	150	5	(29; 31)
bsDronpa	10	~100	1.25 (45 x 10^-3 ^b) / 0.04 (48 x 10^-3 ^c)	54	95	(29)
Dreiklang	1.4-15	~160^e	< 1 (70^b) / ~3 (0.7^f)	4	100	(30)
rsEGFP	1-2 (31) ~3-4 (32)	>500 ~1,100	1x10^-3 (0.6x10³ ^d) / 0.02x10^-3 (2x10³)	23	100	(31; 32)
rsEGFP2	~7-8	~2,100	0.4x10^-3 (5.5x10³ ^d) / <0.04x10^-3 (2x10³)	ND	100	(32)
rsEGFP2	~7-8	~2,100	0.4x10^-3 (5.5x10³ ^d) / <0.04x10^-3 (2x10³)	ND	100	(32)
rsTagRFP	~5	>140	1.3 (5x10^k / 1.7x10^-3 (10^l) .18 (4.9x10³ ^m / 2.8x10^-3 (0.3x10³ ⁿ)	65	30	(33)

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).