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Reviving long lost scientific techniques to make a new discovery


I spent 4 years in the basement of my university working in an electron microscopy lab. And although there were 30,000 students at the university, I was pretty much working down there by myself. 

During the time I was earning my degree I learned about biological development of all species which led into my research into anti-aging biology and eventually skin care biology. But it was my research on black widow spiders required me to become proficient in electron microscopy techniques and histology, which are slowly fading into the ether of classic research techniques and slowly becoming lost knowledge.

Once at the forefront of scientific innovation, electron microscopy was what allowed us to first visualize the interior components of the cell and gave us the original knowledge for which all future cellular research was based. 

And despite that this is the best technique we have for visualizing the interior of the cell, it is becoming an abandoned science. Why? Because it takes a lot of effort and a lot of time to get a result and modern-day scientists want to put in the least amount of effort into research and get an immediate result. 

Electron microscopy doesnt just involve looking at a specimen under the electron microscope, it is also the weeks of preparation to make you specimen ready to be viewed under the scope. Here was the protocol I used for my spiders:

1. For one important aspect of my research I had to determine exactly where eggs were in the spider's body while she was in the process of laying them. We didn't know how the eggs went from floating in the abdomen to inside the oviduct (tube system where eggs go) to outside the body. Therefore if you're able to get a specimen that was taken at the very moment of egg-laying you can see where the eggs are in the body at this point.

But the problem is that spider's don't give much warning when they are ready to lay eggs, so I had to sit in a spider growth chamber for hours a day, many days a week to wait for one to start popping out eggs. When that happened, I would have about 5 minutes to act. So I would take the spider (which was in a large petri-dish style habitat), run down the hallway to our other lab, pull the spider out of the dish and put it in a CO2 chamber. I would turn the CO2 on, which would cause the spider to go unconscious and then I would cut its head off. 

2. Once you have the specimen that you need for your research, you then need to begin the histology techniques needed to prepare it for viewing under the microscope. You cannot just take a specimen and put it under the scope, you have to go on a long and arduous process first. The first thing you have to do is prevent your specimen from decaying now that its dead. So you have to fixate it. The fixation step is the most important step because if you do not fixate your tissue just right, it will fall apart and you either wont be able to view it under the scope. Or the wrong fixative will change the appearance of your sample and you won't be viewing an accurate representation of the sample and your results will be false.

I first used a fixative called Glutaraldehyde because that was what everyone was using as their fixative. However I was running into an issue for a month where my sample would fall apart during later steps. After tweaking one thing at a time I determined the fixative I was using wasn't right here, so I changed to something called Bouin's fixative, which is rarely used in biology anymore but it worked here. I just put the whole spider abdomen in there and let it sit for a long time. 

3. The fixative worked so well that I was able to peel the spider's patent leather skin right off the abdomen and it just left me with a little semi-hardened ball of fat and interior organs and other tissue. The next step was to get all of the water out of the sample. I put the sample in increasing concentrations of alcohol over a day starting in the morning at 10% alcohol and working up to increasingly higher concentrations as the day progressed. 

4. Now that the water was out the sample was ready for the embedding steps. Electron microscopy uses a different embedding medium than light microscopy techniques. Electron microscopy used epoxy resin while all other embedding techniques typically use paraffin wax. However, whenever I tried to use paraffin embedding, the sample fell apart. I believe there were 6 months and perhaps 200 attempts to embed in paraffin and it never worked right, so regardless of whether I was using the electron or light microscope, I always used epoxy embedding techniques. 

5. Now that the sample was embedded, it is ready to be cut into sections. Sections are then put on a slide to be viewed under the scope. One of the things I love about electron microscopy is its "one stop shop" approach to research. In an electron microscopy lab, we make our tools. So to section our specimens, we make our knives. Our knives are glass knives that are made using a knife maker. The knives are sharp and exact enough to cut sections 1um thick which is 1/1000 of a mm.

Knifemaker to make glass knives

Glass knives

Once the knife was made, I would put it into the ultratome to section the specimen. 

6. Once the sections were made, I would stain them to help visualize the components. Samples are usually very light in color to almost clear, and the right stain will help all cellular components become visible.

7. Now that the sections were stained I would mount them on copper disks and they are ready for visualization.

8. While looking at the specimen under the electron microscope (TEM) if I saw something of evidentiary value I would take a photo of it. Electron microscopy is old school and we kept it that way because sometimes the first way of doing things is the best way. So when I took a photo, I mean I took a photo, on film. Because film has higher resolution than digital. And then when I got my photos, I would develop them in the dark room we had in the lab. When you do electron microscopy you do everything yourself. 

Transmission electron microscope. When in use, we would turn all the lights off in the room so we could see our specimens under the green glow of the phosphorus screen.

 

And this was my favorite result:

 

Now, what does this have to do with skin care? I would say, everything. One thing I have noticed is that skin care research is mostly for marketing purposes or it is a dead end. And I believe that the reason for the flawed results, the misleading results and the bad results is because of bad methodology. 

Skin care "researchers" are funded by cosmetic companies, so their results need to please their customers. Therefore those results are biased or false. The results are also rushed which corrupts the data.

Electron microscope labs are the last vestige of lost era of science. When you go into an electron microscope lab it is like stepping into the past. The equipment may be new, but it looks old, because the technology hasn't changed in 70 years. There is no need for it, because it was right 70 years ago and nothing that has emerged since has been better. It is just faster with less effort.

Electron microscopy techniques are slow, but they are important. And the best results that I achieved in my research were from doing things the old fashioned way, the way science used to be done. With artful techniques that may have taken forever to do (the above protocol would take weeks), but they gave a true and accurate depiction of the sample. 

Future studies will use human skin samples will be exposed to various skin care and put under various conditions to get an accurate understanding of how skin behaves. I will be exploring the following:

1. Changes in skin water content from exposure to skin care containing hyaluronic acid

2. Changes in collagen levels from exposure to vitamin C serums

3. Changes in skin extracellular matrix from exposure to daily exfoliation. 

I have added electron microscopy equiment to my lab and I will be using the above methods for my sample preparation. I believe the results will be important.