Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments.

In fluorescence fluctuation polarization sensitive experiments, the limitations associated with detecting the rotational timescale are usually eliminated by applying fluorescence correlation spectroscopy analysis. In this paper, the variance of the time-averaged fluorescence intensity extracted from the second moment of the measured fluorescence intensity is analyzed in the short time limit, before fluctuations resulting from rotational diffusion average out. Since rotational correlation times of fluorescence molecules are typically much lower than the temporal resolution of the system, independently of the time bins used, averaging over an ensemble of time-averaged trajectories was performed in order to construct the time-averaged intensity distribution, thus improving the signal-to-noise ratio.

The influence of dead time related distortions on live cell fluorescence lifetime imaging (FLIM) experiments.

Recent developments in the field of fluorescence lifetime imaging microscopy (FLIM) techniques allow the use of high repetition rate light sources in live cell experiments. For light sources with a repetition rate of 20-100 MHz, the time-correlated single photon counting (TCSPC) FLIM systems suffer serious dead time related distortions, known as "inter-pulse pile-up". The objective of this paper is to present a new method to quantify the level of signal distortion in TCSPC FLIM experiments, in order to determine the most efficient laser repetition rate for different FLT ranges.

Short time behavior of fluorescence intensity fluctuations in single molecule polarization sensitive experiments.

Recent developments in the field of single molecule orientation imaging have led us to devise a simple framework for analyzing fluorescence intensity fluctuations in single molecule polarization sensitive experiments. Based on the new framework, rotational dynamics of individual molecules are quantified, in this paper, from the short time behavior of the time averaged fluorescence intensity fluctuation trajectories. The suggested model can be applied in single molecule fluorescence fluctuations experiments to extract accurate expectation values of photon counts during very short integration time in which rotational diffusion is likely not to be averaged out.