Friday, August 1, 2014

Jezzi Reynolds Scanning Electron Microscopy

We dropped the blocks at their sites.
In the meantime I worked on a couple of other projects. I did Electron Scan Microscopy on larvae samples.
First we separated out the larvae from the 70% ethanol they were originally stored in. I used a pipette to place them in ½ inch capsules with mesh coverings and lids on either end. The mesh allows other liquids to penetrate the sample when needed.  
After we did this we placed them into jars of ethanol solutions at different concentrations. The first jar had a 70% ethanol 30% water solution. The following jars had 80, 90, 95,100 and 100 percent. We used 100% washes to be sure that the larvae were immersed in ethanol.
We left them in the different solutions for 10 minutes each then we moved them to the succeeding solution.  
We then put all of the capsules  (sometimes as many as 8) into 100% solution to keep them wet until the next step.
The second step in this process was to place the capsules into the critical point drier. This machine replaces the ethanol with Carbon Dioxide. The point of this machine is to use carbon dioxide to dry the samples completely. Though carbon dioxide is used in both a liquid and gas from when the process is complete it is neither liquid nor gas but somewhere in between.
Then we place some 100% ethanol and the capsules into the chamber.
We then “purge” the capsules by letting in gas and then releasing it. We turned it on the stirrer to mix the capsules in the 100% ethanol efficiently. You can tell that there is ethanol in the chamber if you hear a sputtering from the valve at the back. This should only release gas. The sputtering means it is releasing a liquid (ethanol) and a gas versus just the gas. We use Kim wipes (kind of like a paper towel) to double check this, if it is wet then there is liquid. If dry there is only gas.
We usually have to complete 3 purges to get only gas. We lower  the temperature to 50 degrees Celsius from about 19 degrees room temperature.
When we do the the purges we look at the meniscus (liquid Carbon Dioxide) level through a circular viewing window. This is how we watch to make sure the levels don’t get too high when we intake carbon dioxide or too low when we let it out. If the levels were to get too high the chamber would explode (luckily there are several safety measures in place to prevent this from happening). Next we increase the pressure to 1250 PSI and the temperature to 35 degrees. These are the “Critical Points”. This takes about 15 minutes.
Then we lower the psi to 0 and the temp to 5 degrees.
The third step is to mount the larvae and place them in the gold sputter coater.
We mount the larvae by using a eyelash brush (literally an eyelash taped to a piece of wood) to pick up the specimens and place each of the larvae from a capsule on a metal stub with tape on it.
We put the stubs into the sputter coater which covers them in liquid gold. They have to be covered in gold so the scanning electron microscope will be able to read the larvae.
Finally we place the stubs into the electron scan microscope. Then we can clearly see the larvae up close. The machine can magnify them up to 5000 percent and we can identify and take pictures to be analyzed later.
I have also been completing video analysis of some shipwrecks in the Caribbean. I look through the footage to find good views where there are a large quantity of diverse organisms and variation in species. Then I take snapshots of these views to be analyzed later.

These are the stubs with larvae on them. On the right are after they are coated in gold. On the left are before they are put in the sputtercoater.
  All images below are taken using the Electron Scan Microscope.
Cyprid or Barnacle Larva

Gastropod Veliger or Snail Larvae

Ophiuroid or Sea Star (commonly referred to as Starfish) Larvae This is one of my favorites.

Also Gastropod Veliger or snail larvae

A magnified image of the center of a Sea star larvae. This is the underside of the Sea star where the mouth will form. This is one of my favorites because it clearly shows the 5-part radial symmetry that all Sea stars as well as Sea Cucumbers, Sea Urchins and Sand Dollars have.
Another Sea Star Larvae with only 3 arms.

This shows the structure or Ossicles of the Sea Star. The Ossicles are the part of the endoskeleton around the holes. They are made out of Calcium Carbonate.

A Gastropod Veliger with spikes to protect itself from potential predators. These will fall off when it becomes an adult and they are not needed.

1 comment:

  1. Jezzi, these images are so amazing! Good description of the process too. I love the spiked veliger!