Chris - Week 3: Scanning Electron Microscopy




This past week I learned how to prepare specimens for scanning electron microscopy (SEM). This process requires the fixatives glutaraldahyde and osmium tetroxide, the latter of which is extremely volatile and toxic ( it can cause temporary blindness if not used under the fume hood). A myriad of other steps are required, which are largely intended to gradually remove the water from the sample. Eventually, the specimens are placed into a critical point dryer, a machine that dries the sample using the instantaneous evaporation of liquid carbon dioxide. After this, the specimens are placed in a sputter coater, a machine that coats the samples with a thin layer of gold. This metallic coat creates a surface by which electrons, emitted by a "gun", can be reflected off and be detected by the SEM. In addition to all the prep work, I also operated the SEM itself. The microscope is surprisingly easy to use, especially considering how powerful it is.

 

 For my project, I examined several life stages of M. alaskensis using confocal and scanning electron microscopy. My goal was to determine at what approximate age cilia began to develop, where, and on what cells. Specifically, I looked at the 6 hour, 10 hour, and 15 hour age groups using the confocal microscope. I used the SEM to examine 10 hour, 15 hour, and 2 day old pilidia. The confocal specimens were inconclusive (due to some errors on my part), so that part of the experiment will have to be redone. Fortunately, I had much better luck with the SEM. Svetlana and I found that cilia had clearly developed by the 15 hour stage, with some larvae possessing cilia only along the equatorial region, and with others apparently covered in them . The 10 hour stage had little to no cilia. With this information, I will prepare new specimens at slightly different ages to get a better understanding of the cilial development.

  

Fig 1:  A lateral view of a 10 hour old M. alaskensis larva

Fig 2: An anterior/posterior view of a 10 hour old M. alaskensis larva

In figure 1, note the cilia beginning to form at the top right and bottom left areas of the specimen. Figure 2 is apparently devoid of cilia. Both are covered in tiny cytoplasmic projections called microvilli.



Fig 3: An anterior/posterior view of  a
15 hour old M. alaskensis larva



 

Fig 4: An anterior/posterior view of a 15 hour old M. alaskensis larva


  

 Figures 3 and 4 both display relatively well-developed cilia. Note, however, that in figure 3 the cilia are largely restricted to the equatorial region, along the margin of the specimen. Conversely, the larva of figure 4 clearly possesses cilia near its center.

Fig 5: A lateral view of a M. alaskensis pilidium

Fig 6: A posterior view of a M. alaskensis pilidium

Note the dense, "furry" patches of cilia that run along the edge of the specimen in figure 5. These are the ciliary bands. Also note the apical tuft at the anterior end of the pilidium, another densely ciliated patch. Figure 6 show the interior side of the lappets (the "ear flaps"). The mouth of the pilidium is located in between these lappets.