Fig. 6. Two-photon microscopy detects mitotic Ca²⁺ transients in fertilized embryos and reveals Ca²⁺-independent increases in indicator fluorescence within the nucleus at mitosis entry in parthenogenetic embryos. Ca²⁺ transients are readily detectable in a fertilized mitotic embryo using two-photon microscopy (A). (top) A series of images taken during the first transient plotted underneath. The mitotic Ca²⁺ transients cause a threefold increase in fluorescence. Ca²⁺-Green/dextran (B) and rhodamine-dextran (C) were monitored during NEBD in parthenotes. Bright-field optics were used to determine the timing of NEBD (Bi, top). In addition, the exclusion of the indicators from the nucleolus allowed mitosis entry to be established from the fluorescence images as the time at which the nucleolus disappeared (Bi, arrowheads indicate the position of the nucleolus). Notice the striking increase in fluorescence that occurs within the nucleus at the time of NEBD in both Ca²⁺-Green/dextran- and rhodamine/dextran-injected embryos (Bi,Ci). (Bii,Cii) The same images as in Bi and Ci but illustrating the regions of interest used for data analysis (grey, peripheral cytoplasm; black, nucleus). Analysis of fluorescence intensities using these regions of interest confirms that the fluorescence increase is restricted to the nucleus in both cases (Biii,Ciii). Lower-case letters indicate which image corresponds to given points on the graph. Notice also that ionomycin addition resulted in a dramatic fluorescence increase in Ca²⁺-Green/dextran-injected, but not rhodamine/dextran-injected, embryos.