Figure 4
Ratiometric imaging of calcium elevation obtained by confocal microscopic measurement of fluorescence of coinjected tetramethylrhodamine, dextran 10,000 MW and Calcium Green-1, dextran 10,000 MW. Single z-sections constitute these images. These ratiometric images were obtained by calculating Δf/f at each pixel (fCaGreen-fRhodamine)/fRhodamine) which is the fractional increase in the fluorescence of the green channel (Calcium Green-1) over the red channel (tetramethylrhodamine); in all experiments, there was no measurable variation in the amplitude of the tetramethylrhodamine fluorescence (data not shown) making it useful for these ratiometric calculations). The inset is a plot of the average Δf/f within the area of the fertilized oocyte. The fertilized oocyte is denoted by the arrow. The empty spermatheca is denoted by the arrowhead (the redness of the spermatheca is an artifact of the ratio calculation; where there is no dye, green background fluorescence exceeds red background fluorescence, the area outside the worm was painted black for presentation purposes). At t = 3 min, the oocyte has completely entered the spermatheca and has been fertilized. The oocyte has entered the uterus at t = 9 min (the actual entry was missed between scan intervals). After ~ 12 min, cytosolic [Ca++] essentially reaches the level prior to fertilization. At t ~ 40 min, the first cell division takes place (data not shown), thus the calcium dynamic imaged here can be considered physiologically normal and not perturbative.