Keira- How does the confocal microscope take such pretty pictures? (Week 4)

This week I got to test some fluorescent antibodies with the confocal microscope. While many did not work or yielded inconclusive results, some worked very well with lovely results. These are photographs of Patiria miniata (sea star) taken on the confocal microscope.

Figure 1: P. miniata (front view)

Figure 2: P. miniata (side view)

The blue and orange colors are added to show the contrast; the images are actually in greyscale when scanning specimens. The colors represent the two different dyes used on these larvae. Most of the color you see (blue in Fig 1, orange in Fig 2) is fluorescence emitted from Hoescht 33342. This is a dye that intercalates with DNA, so it stains the nucleus of every cell. This helps discern the structure of the organism. In these Patiria, the two ciliated bands are shown by the areas of greatest color density. Patiria have cells that are monociliate, meaning that each cell can form only 1 cilium. Thus, in order to form a ciliated band, the larva must have a great density of cells in that area. The cells are comparatively sparse in the rest of the larval body.

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:
http://idv.sinica.edu.tw/ckao/Research%20interest.html

Histones are the "spools" around which DNA winds. Most of the time, DNA is loosely arranged in the nucleus, but when the cell is getting ready to divide, it starts organizing the DNA in order to form chromosomes. The simplified cartoon to the right demonstrates this idea. The "phospho-" prefix means that the histones have been activated by the addition of a phosphate group, an energy rich molecule comprised of four oxygen atoms in a tetrahedral arrangement around a phosphorus atom. The activated histones begin organizing the DNA in preparation for mitotic division.

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.