Fluorescence Microscopy and all the pretty colors

Hi guys,

So far I’ve been posting about the characterization of the rrf-3 mutant and I mentioned that fluorescence microscopy was used in analyzing cellular structures in comparison to the wildtype phenotype. We can better appreciate what the pictures reveal if we understand the basics of this amazing technique, so here’s an attempt at it.

All the way back in high school chemistry and physics, we learned that when molecules absorb light, their electrons go from the ground state to a higher energy level. Normally these electrons go back to their ground state via rotations and vibrations, and most of the energy is lost to the solvent and surroundings. In our analyses, we observe different cell structures (e.g. tubulin) by tagging the specimen with an antibody, which itself has a fluorophore attached, causing the tubulin to be fluorescent. Fluorescent molecules absorb light of one wavelength, follow a straight path down to the ground state and emit light of lower energy, releasing a photon. The energy difference is absorbed as heat.

In the fluorescence microscope, some of the main components are the excitation filter, the dichroic mirror, the objective lens, and the barrier filter. White light hits the excitation filter, which lets only the correct wavelength light to pass through, in this case blue. This blue light, the only wavelength capable of exciting the tubulin fluorophore , is deflected 90 degrees by the dichroic mirror and hits the specimen. Tubulin is most of the time labeled with FITC (fluorescein isothiocyanate), which has a green emission color. The specimen emits green exciting light (due to the change in energy levels); most of it is scattered and lost but some fits the “acceptance angle” of the objective lens, which captures it and sends it back up through the filter cube. The barrier filter only lets green light and blocks, hence its name, any other wavelengths that bounces off the specimen.

DAPI (4′,6′-diamidino-2-phenylindole), which enters the cell membrane and binds to the minor groove of the DNA, is the most  common stain for looking at the DNA and chromatin structure, which can reveal a lot about the mitotic and meiotic divisions, and the transcriptional state of the cell. In this picture below, one can see the temporal progression of a male wildtype C.elegans nematode gonad. Undifferentiated germ cells start at the distal tip (top right), become spermatocytes (bottom right) and eventually spermatids. Each blue circular structure is a nucleus. One can see some of the divisions toward the end, where microtubule spindles are assembled and chromosomes are pulled apart in anaphase.

 

Germline Spermatogenesis. DNA (blue), tubulin (green).

 

The spermatids crawl on to fertilize an oocyte. The “crawling” is done with the help of MSP (Major Sperm Protein) and this is the topic of the next post, along with some other updates. I hope this was somewhat informative; it certainly helps me appreciate the pictures more and actually aim for optimizing their preparations after knowing a bit more about the microscopy behind it.

Here’s a quote I thought was very cool; it’s from Daniel Mazia, a famous cell biolgist.

The gift of the great microscopist is the ability to think with they eyes and see with the brain. Deep revelations into the nature of living things continue to travel on beams of light. “