|Figure 1: P. miniata (front view)|
|Figure 2: P. miniata (side view)|
The infrequent dots of contrasting color (orange in Fig 1, white in Fig 2) shows light emitted by from the fluorescent antibody. Last week my blog post discussed primary and secondary antibodies. The primary antibody in this case was phospho-histone.
|Picture credit: |
The phospho-histone antibody binds specifically to these activated histones, thereby tagging only the cells which are actively dividing or that have just finished dividing. The secondary antibody binds to the phospho-histone and then can be excited to fluoresce, which shows us where the dividing cells are.
The confocal microscope works by scanning one layer at a time and saving the information about light emitted by the sample specifically in that layer. By scanning thin slices through the sample and then compiling the data, we are able to see 3-D images such as the ones shown in Figures 1 & 2. Two lasers alternate light emission on the sample. One laser is set to excite the Hoescht dye and the other to excite the phospho-histone antibody. The confocal microscope "reads" the fluorescence emitted from the sample in each of the channels. The fluorescence information from each channel is compiled separately. The images can be overlapped to show both the Hoescht dye and the phospho-histone, thus illuminating all the nuclei as well as individual dividing nuclei on the same larva.