A Real-Time and Non-Invasive Procedure for estimating Phosphate Concentration in Living Cells
This is a procedure for quantifying soluble phosphate in the cytoplasm of living cells that involves measuring the fluorescence decay times and fluorescence-lifetime imaging microscopy (FLIM).
The proton-transfer reaction in the excited state that occurs between the neutral and anionic species of the compound 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG), only occurs at a relatively low concentration of phosphate buffer at a pH close to neutral. This means that the overall fluorescence decay of the system is characterised by two individual decay times. One of these is very short, of the order of a few picoseconds, and is essentially non-existent at phosphate concentrations higher than 0.05 M. The other decay time is much longer (between 2.5 and 3.7 ns) and is dependent on the phosphate concentration in the medium in which the fluorophore is found. The advantageous spectroscopic characteristics of 2-Me-4-OMe TG resulted in the photophysical study of this dye being broadened to include individual molecules. The most interesting quantitative finding was that an increase in phosphate concentration from 0 to 0.3 M decreased the decay time from 3.7 ns to 3.0 ns at pH 7.
In light of this, a procedure for estimating the phosphate concentration in the cytoplasm of living cells involving recovery of the fluorescence decay times for 2-Me-4-OMe TG (previously excited with a pulsed laser) and conversion of these decay times into microscope images has been developed. As a result, the penetration of phosphate ions into the cytoplasm of pre-osteoblasts in their cell-differentiation phase has been followed in real time.
An aqueous solution of the xanthene dye, preferably with a concentration of 10-7 mol·L-1, is added to the cell sample beforehand. As the dye used is apolar, it can cross the cell membrane spontaneously, remaining inside the cells for a longer time than required to collect the fluorescence decays.
A new fluoresceine-based xanthene fluorophore, 9-(4-tert-butyl-2-methoxyphenyl)-6-hydroxy-3H-xanthen-3-one, which we have termed 2-OMe-4-tBu GG and which possess better spectroscopic properties than that used previously, thus making it highly suitable for use in the procedure described, has also been synthesised.
The phosphate and pyrophosphate concentration of aqueous solutions can be calculated using previously known techniques. Although a few methods for determining these concentrations in living cells using radioactive phosphorus are known, such techniques have major inherent drawbacks. However, calculation of the phosphate concentration inside living cells in real time, using non-invasive methods, has not been possible until now.
The main advantage of the present invention is the ability to estimate the (mono- and dibasic) phosphate ion concentration in living cells in real time which, to the best of our knowledge, was not previously possible.
To this end, the fluorescence decay times for a xanthene dye added to the cells must be obtained. These decay times are directly related to the phosphate ion concentration in the medium.
A fluorescence microscope with pulsed laser light excitation is required.