Fluorescence Photobleaching Recovery Method with Pulse-Position Modulation of Bleaching/Probing Irradiation

Abstract

The fluorescence photobleaching recovery method with pulse-position modulation (PPM) of bleaching/probing irradiation is presented. This approach is modification of previously announced photobleaching recovery under decaying photobleaching (FRDP) approach, which employs the amplitude control of irradiation intensity. Underlying idea was to solve the problem of setup nonlinearity by methodical modification that was successfully done. The irradiation intensity is series of equal bleach-probe pulses with increasing distance. The more sophisticated series was proposed as a convolution of subseries, which one characterizing its own time scale. It allows to detect the fluorescence kinetics in the wide range of time and of different nature simultaneously. The method was applied on model systems as FITC-HSA in the water/glycerol mixture and FITC-DMPE in the POPC multibilayers. The method allowed to detect and investigate diffusion processes, presence immobile fraction, effect of limited area of liposomes and reversible fluorescence kinetics.

Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

“…To expand the time range, these series were repeated three times, resulting in a total kinetics time of 60 s (figure 2). Such a method allowed us to analyze the kinetics simultaneously in the wide range of kinetics rates, from dozens of milliseconds to dozens of seconds [19].…”

“…It uses bleaching/probing irradiation with a constant pulse width and increasing pulse distance. Such an approach was earlier demonstrated for measurement of lateral diffusion in a cell membrane [19] as alternative to the pump-and-probe approach. It has some advantages, such as a better signal-to-noise ratio, absence of 'unwanted' photobleaching, and being able to determine the direct and reverse rate constants in a wide range of time scale.…”