Photophysical Properties of Photoactivatable Fluorescent Proteins

Super resolution microscopy is a robust method of determining biological structure well below the diffraction limit. The question of how quickly a full image can be acquired has been estimated but has not been answered. The absolute limit of this speed is determined by the photophysical properties of the fluorescent probe being used. Presented is a quantitative measure of three fluorescent proteins, two of which are commonly used in Fluorescence Photoactivatable Localization Microscopy (FPALM), Dendra2 and PAmCherry, and a relatively new photoswitchable protein PSmOrange. In order to acquire an FPALM image more quickly, a high molecular count rate (photons per second) is required. These proteins are evaluated using Fluorescence Correlation Spectroscopy (FCS). Common logic states that increasing laser power is an easy way to increase molecular count rate, however there is a limit to this increase. As excitation intensity is increased, eventually molecular count rate will plateau and then fall off as intensity is increased further due to triplet effects becoming more prevalent. The active form Dendra2 plateaus at excitation intensity of 12.5±6kW/cm2 at 561 nm with a peak molecular count rate of (170±60)kHz, active form PSmOrange plateaus at excitation intensity of 690kW/cm2 at 633 nm with a peak molecular count rate of (206±4)kHz, while PAmCherry plateaus at 150-400kW/cm2 with a peak molecular count rate of (26±14)kHz. These count rates are lower than reported molecular count rates for organic dyes; PSmOrange is about 20% lower than the reported 250kHz molecular count rate for Alexa 555.The photostability of these proteins was also evaluated. The probability that a fluorescent molecule will permanently go into a dark state is called the photobleaching yield, active Dendra2 has a photobleaching yield o f(2 .6± .8 )x 10-5, active PSmOrange has a photobleaching yield of (1.4 ± . 1) x 10-5, and PAmCherry has a photobleaching yield of (11.0±1.6)x10-5. Flicker yield is a similar concept; it is the probability that a molecule will enter into a temporary dark state given the absorption of an excitation photon. Measured flicker yields are as follows: active Dendra2 has a flicker yield of (1.2 + .6)x 10-3, active PSmOrange has a flicker yield o f(2 .0± .4 )x 10-3, while PAmCherry has a flicker yield of (2.7 ± .4) x 10-3. Thus it can conclusively be stated that (per excitation photon at the wavelengths used here), Dendra2 is the least likely of these molecule to flicker, while PSmOrange is the least likely to bleach.