Search

Fluorescence lifetime imaging microscopy (FLIM) is an essential tool in many scientific fields such as biology and medicine thanks to the known advantages of the fluorescence lifetime (FLT) over the classical fluorescence intensity (FI). However, the frequency domain (FD) FLIM technique suffers from its strong dependence on the reference and its compliance to the sample. In this paper, we suggest a new way to calculate the FLT by using the crossing point (CRPO) between the modulation and phase FLTs measured over several light emitting diode (LED) DC currents values instead of either method alone. This new technique was validated by measuring homogeneous substances with known FLT, where the CRPO appears to be the optimal measuring point. Furthermore, the CRPO method was applied in heterogeneous samples. It was found that the CRPO in known mixed solutions is the weighted average of the used solutions. While measuring B16 and lymphocyte cells, the CRPO of the DAPI compound in single FLT regions was measured at 3.5 ± 0.06 ns and at 2.83 ± 0.07 ns, respectively, both of which match previous reports and multi-frequency analyses. This paper suggests the CRPO as a new method to extract the FLT in problematic cases such as high MCP gains and heterogeneous environments. In traditional FD FLIM measurements, the variation in phase angle and modulation are measured. By measuring over varying DC currents, another variation is detected in the FLT determined through the phase and modulation methods, with the CRPO indicating the true FLT.

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. Rotational correlation times of fluorescein molecules in different viscosities of the medium within the range of the anti-bunching time (1-10 ns) were then extracted using this method.

In the present study we introduce a Whole-Object Fluorescence Life Time (wo-FLT) measurement approach for ease and a relatively inexpensive method of tracing alterations in intracellular fluorophore distribution and in the physical-chemical features of the microenvironments hosting the fluorophore. Two common fluorophores, Rhodamine 123 and Acridine Orange, were used to stain U937 cells which were incubated, with and without either Carbonyl cyanide 3-chlorphenylhydrazon or the apoptosis inducer H(2)O(2). The wo-FLT, which is a non-imaging quantitative measurement, was able to detect several fluorescence decay components and corresponding weights in a single cell resolution. Following cell treatment, both decay time and weight were altered. Results suggest that the prominent factor responsible for these alterations and in some cases to a shift in emission spectrum as well, is the intracellular fluorophore local concentration. In this study it was demonstrated that the proposed wo-FLT method is superior to color fluorescence based imaging in cases where the emission spectrum of a fluorophore remains unchanged during the investigated process. The proposed wo-FLT approach may be of particular importance when direct imaging is impossible.