Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein, Quinine Sulfate and Green Fluorescent Protein.

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10- 3 - 10- 5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.

Enhancing EPA Content in an Arctic Diatom: A Factorial Design Study to Evaluate Interactive Effects of Growth Factors.

Microalgae with a high content of the omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are of great demand for microalgae-based technologies. An Arctic strain of the diatom Attheya septentrionalis was shown in previous experiments to increase its EPA content from 3.0 to 4.6% of dry weight (DW) in the nutrient-replete exponential phase and nutrient-depleted stationary phase, respectively. In the present study, a factorial-design experiment was used, to investigate this effect in more detail and in combination with varying salinities and irradiances. A mathematical model revealed that both growth phase and salinity, alone and in combination, influenced the EPA content significantly. Maximum EPA values of 7.1% DW were obtained at a salinity of 22 and after 5 days in stationary phase, and might be related to a decreased silica content, an accumulation of storage lipids containing EPA, or both. However, growth rates were lower for low salinity (0.54 and 0.57 d-1) than high salinity (0.77 and 0.98 d-1) cultures.

Chlorophyll a fluorescence lifetime reveals reversible UV-induced photosynthetic activity in the green algae Tetraselmis.

The fluorescence lifetime is a very useful parameter for investigating biological materials on the molecular level as it is mostly independent of the fluorophore concentration. The green alga Tetraselmis blooms in summer, and therefore its response to UV irradiation is of particular interest. In vivo fluorescence lifetimes of chlorophyll a were measured under both normal and UV-stressed conditions of Tetraselmis. Fluorescence was induced by two-photon excitation using a femtosecond laser and laser scanning microscope. The lifetimes were measured in the time domain by time-correlated single-photon counting. Under normal conditions, the fluorescence lifetime was 262 ps, while after 2 h of exposure to UV radiation the lifetime increased to 389 ps, indicating decreased photochemical quenching, likely caused by a damaged and down-regulated photosynthetic apparatus. This was supported by a similar increase in the lifetime to 425 ps when inhibiting photosynthesis chemically using DCMU. Furthermore, the UV-stressed sample was dark-adapted overnight, resulting in a return of the lifetime to 280 ps, revealing that the damage caused by UV radiation is repairable on a relatively short time scale. This reversal of photosynthetic activity was also confirmed by [Formula: see text] measurements.

Testing fluorescence lifetime standards using two-photon excitation and time-domain instrumentation: rhodamine B, coumarin 6 and lucifer yellow.

Having good information about fluorescence lifetime standards is essential for anyone performing lifetime experiments. Using lifetime standards in fluorescence spectroscopy is often regarded as a straightforward process, however, many earlier reports are limited in terms of lifetime concentration dependency, solvents and other technical aspects. We have investigated the suitability of the fluorescent dyes rhodamine B, coumarin 6, and lucifer yellow as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine which wavelengths we should use for the excitation and an appropriate detector range. We also measured lifetimes for different concentrations, ranging from 10(-2)- 10(-6) M, in both water, ethanol and methanol solutions. We observed that rhodamine B lifetimes depend strongly on concentration. Coumarin 6 provided the most stable lifetimes, with a negligible dependency on concentration and solvent. Lucifer yellow lifetimes were also found to depend little with concentration. Finally, we found that a mix of two fluorophores (rhodamine B/coumarin 6, rhodamine B/lucifer yellow, and coumarin 6/lucifer yellow) all yielded very similar lifetimes from a double-exponential decay as the separate lifetimes measured from a single-exponential decay. All lifetime measurements were made using two-photon excitation and obtaining lifetime data in the time-domain using time-correlated single-photon counting.

Chlorophyll a and NADPH fluorescence lifetimes in the microalgae Haematococcus pluvialis (Chlorophyceae) under normal and astaxanthin-accumulating conditions.

In vivo fluorescence lifetimes of chlorophyll-a (chl-a) and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from the green microalgae Haematococcus pluvialis under normal and nutrient-stressed conditions (green stage and red stage, respectively), using two-photon excitation provided by a laser generating pulses in the femtosecond range, and a Leica microscope setup. Analysis of the fluorescence lifetime decay curve revealed two separate lifetime components in all our measurements. A short-lifetime component for chl-a of ~250 ps was completely dominant, contributing more than 90% of overall intensity in both green-stage and red-stage cells. Green-stage cells inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU) displayed a significant chl-a lifetime increase for the short component. However, this was not the case for red-stage cells, in which DCMU inhibition did not significantly affect the lifetime. For green-stage cells, we found a short NADPH (free) lifetime component at ~150 ps to be completely dominating, but for red-stage cells, a longer component (protein bound) at ~3 ns contributed as much as 35% of the total intensity. We hypothesize that the long lifetime component of NADPH is connected to photoprotection in the cells and coupled to production of astaxanthin. DCMU does not seem to affect the fluorescence lifetimes of NADPH.