Microfluorimetry

calcium waveCalcium is one of the most important intracellular messengers in biological systems (I might say the most important except that I might get hassle from phosphoinositide biochemists or fans of cyclic nucleotides). One of the things that makes the study of calcium signalling so popular is that it is really pretty easy to do. All you need is a microfluorimetry system; load the cells with a fluorescent dye and then stand back and watch. The really hard part of designing a suitable fluorescent probe has already been done for you. Ta Roger.

oscillationsFrom an experimental point of view, the beauty of both patch-clamp and microfluorimetric measurements is that they are so fast. It is possible to measure changes in intracellular calcium or ion currents on a millisecond timescale.

photometry rigFluorimetric measurements have been around for a long time, and are not restricted to calcium studies. In essence, if you have a dye that fluoresces in the presence of a target substance you can detect the presence of the target by determining whether or not your dye is fluorescing. If the intensity of fluorescence increases with the concentration of the target then you can measure changes in the concentration of the target. What is really, really clever and not that obvious is that if the dye undergoes a spectral shift in the presence of the target substance then you can determine the absolute concentration of the target without even knowing the concentration of the dye. Fura-2 is just such a fluorescent dye, the target of which is calcium. Microfluorimetry is just fluorimetry writ small. Stick a microscope into the system somewhere and measure the fluorescence in a single cell.

Fura-2

fura 2Fura-2, developed by Roger Tsien, is a second-generation calcium indicator based on BAPTA. Crudely speaking Fura-2 inherits its high affinity for calcium and selectivity over magnesiumfrom the chelator but along the way has also accumulated the ability to fluoresce when excited with ultraviolet light.

The excitation spectrum for fura-2 changes, depending whether or not it is bound to calcium. If fura-2 is bound to calcium then the excitation maximum is around 340 nm, if fura-2 is not bound to calcium then the excitation maximum is about 380 nm. The ratio of fluorescence measured at 340 nm to that measured at 380 nm is therefore directly proportional to the free calcium concentration. Because the fluorescence seen at either wavelength is also dependent on dye concentration, the dye concentration term cancels out in the ratio and thus calcium concentration may be determined independently of dye concentration.

Ratiometric calcium dyes make fluorimetry indecently easy because

  1. You don't have to worry about a loss of signal because of photo-bleaching (The signal to noise ratio may decrease but the ratio is unaffected)
  2. You can load cells using solute trapping techniques. Solute trapping is easy to do but gives a variable and difficult to estimate final concentration of dye in the cells. With a ratiometric dye, you don't care.
Solute Trapping

solute trappingSolute trapping is a bit like the greenhouse effect for cells. In the greenhouse effect, light gets into the atmosphere but radiated heat can't get out. In solute trapping, a substance gets into the cell but then for one reason or another can't get out. Fura-2 free acid cannot cross cell membranes but the acetoxy methyl ester or AM form can (because the AM groups shield the charge on the fura and an unchanged molecule is much more lipophyllic). Once inside the cell, esterases chop the AM groups from the fura-2 and trap the free acid inside the cell. Not only can the fura-2 free acid not get out of the cell, there is a continual gradient for fura-2 AM (because the esterases 'destroy' fura-2 AM in making fura-2 free acid). The net result is concentration of the free acid within the cell. This is a wonderful way of making best use of often-expensive dyes, however it does make it almost impossible to determine the final concentration of free acid within the cell. The good news is that if you use a ratiometric dye, such as fura-2 then you don't need to know the dye concentration in order to calibrate your results. Before going completely berserk and loading cells until they glow like little green light houses, it is worth bearing in mind that fura-2 is a calcium chelator and too much of it in the cell will simply damp out any calcium signal.

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