Transmitted Light versus Scanning Electron Microscopy

[Ecki]

Recently I have been working with a Scanning Electron Microscope (SEM). Identifying species is challenging - they look very different when compared to light microscopic images. But after I have seen a species in an SEM image I understand the light microscopic image much better.



This not not a pile of debris but a rather well-camouflaged amoeba - Gocevia. Unfortunately I have no light microscopic images available. But if you google Gocevia, there are plenty of images from Ferrys www.arcella.nl website. If you compare the images with the SEM image you will see that it is nearly impossible to understand the form of this amoeba from the light microscopic image.

This sample was fixed with osmium tetroxide and sputter coated with gold.

Best regards
Ecki

[René]

I would say, without the LM image it is near impossible to recognize this as a amoeba

The combination is of course best of both worlds!!

Best wishes, René

[Jacek]

wow, I love the high magnification

[Mitch640]

The first impression I get from seeing this is that the shells are porous. I thought they needed built in vents to get fresh water. Amazingly clear and sharp images.

[Bruce Taylor]

Dazzling image, Ecki.

For those of us who have never used SEM, it might be interesting to hear more about the process (especially the fixing of the specimen).

[Ecki]

Bruce,

the sample was on a coverslip that was inside a petri dish for a week. The amoebae I wanted to study adhered to the coverslip and I could prepare them for the SEM. Once everything is published I will show a series of images here, too. The Gocevia smuggled itself on that coverslip too but was not my subject of interest - still interesting to see.

The preparation takes approximately 5 hours and goes as follows:

Fix in a solution of 4% osmium tetroxide and 2 % mercuric chloride for 15 minutes
Rinse 4 x in aqua dest
15 minutes 30% Ethanol
15 minutes 50% Ethanol
15 minutes 70% Ethanol
15 minutes 90% Ethanol
15 minutes 100% Ethanol (4 x)

Critical Point Drying
Gold sputter coating

Beware, osmium tetroxide is as poisonous as it gets.

Best regards
Eckhard

[René]

4% OsO4, gee, you can kill the world with that!

You might want to check the use of HMDS (hexamethyldisalazane, or something like that) instead of critical point drying. It works quite well for me, and makes handling a lot easier.

Best wishes, René

[Bruce Taylor]

Thanks for describing the process, Ecki! I'm in no position to make use of that protocol, but it's interesting to know how it's done.

[Baley]

Why the exotic osmium fixation?
And what is critical point drying?

Whatever the answers, thanks for a remarkable image showing some unusually geometric features of an amoeba.

[discomorphella]

HgCl2 is not exactly safe either. In fact, it may be more poisonous than the OsO4. Just not as volatile. All those traditional coagulating fixatiives are very, very nasty. Ecki is correct that OsO4 will likely blind you before it kills you. But a VERY tiny quantiy of HgCl2 will also severly poison you.
That being said, they are very good at preserving morphology, although for the external features of tests or diatom shells you can usually get away with just fixing in acetone, vacuum drying and then coating with Au or Au/Pd or Rh. Its a lot simpler and for external test/shell details I don't think it will make a difference. If you're trying to see cytological details then you're pretty much going to have to use the usual nasty chemicals, although I often use glutaraldehyde/formaldehyde instead of osmic acid. Neither are particularly good for your health...

David

[Ecki]

what is critical point drying?

In a Scanning Electron Microscope there is an electron gun that produces a narrow beam of electrons. The beam is scanned in a regular pattern over the sample. As the electron beam hits the sample it produces secondary electrons that are captured by a detector. Of course the detector captures more electrons from the side of the sample closest to the detector. That is why my amoeba has a bright and a dark side.

As the chemicals that make most lifeforms have a pretty low atomic number, the number of secondary electrons emitted from the sample are low, too. Thus a thin coat of gold which has a high atomic number is applied to the sample. Now the electron beam has a higher chance to cause secondary electrons to be emitted. The result is better contrast.

If we want to sputter coat the sample we will need vacuum. If our sample contains any liquid matter f.e. in cells they would literally explode while the sputter coater is evacuated.

If we would simply heat the sample for water or ethanol to evaporate, the surface tension at the phase transition would destroy our sample. But under high pressure (218 atm) and high temperature (374 °C) there is a so called critical point in the phase diagram. At and above that critical point there is no distinction between liquid and gas - it is called a supercritical fluid. As 218 atm and 374°C is a bit hard to achieve, CO2 is used. The critical point for CO2 is 31°C and 73 atm.

So we have our sample in 100 ethanol. The ethanol bathes (30%, 50%, 70%,90% 4x 100%) are used to exchange water against ethanol. Then we put our sample in the pressure chamber of the Critical Point Dryer. We cool the sample to 10°C and then open a valve to release some liquid CO2 into the chamber. Now we wait a little while so ethanol and CO2 mixes. Then we open another valve to release some of the liquid. This is repeated about 30 times. This causes the entire ethanol to be replaced with CO2. Then the temperature is increased to 40°C. Around 31°C the CO2 becomes supercritical. The valve is opened again and slowly the CO2 evaporates without any damage to the sample.

No we have a totally dry sample that is ready to be sputter coated.

Sorry for this lengthy explanation.


@David,

I totally agree. I wish less nasty chemicals would do a good job

Of course this was done under a fume cupboard. OsO4 and HgCl2 kill so fast that cilia etc. are "frozen".

regards
Ecki

[Baley]

Ｔｈａｎｋ　ｙｏｕ　ｆｏｒ　ｔｈｅ　ｍｏｓｔ　ｉｎｔｅｒｅｓｔｉｎｇ　ｅｘｐｌａｎａｔｉｏｎ．　Ｉ　ｈｏｐｅ　ｙｏｕ　ｄｏｎ’ｔ　ｍｉｎｄ　ａ　ｆｏｌｌｏｗ－ｏｎ　ｑｕｅｓｔｉｏｎ．　Ｗｈｙ　ｇｏｌｄ　sputter ａｎｄ　ｎｏｔ　ｓｏｍｅ　ｏｔｈｅｒ　ｈｅａｖｙ　ｍｅｔａｌ　ｌｉｋｅ　ｌｅａｄ　ｏｒ　ｓｏｍｅ　ｓｕｃｈ？

[Charles Krebs]

The preparation takes approximately 5 hours and goes as follows:

Fix in a solution of 4% osmium tetroxide and 2 % mercuric chloride for 15 minutes
Rinse 4 x in aqua dest
15 minutes 30% Ethanol
15 minutes 50% Ethanol
15 minutes 70% Ethanol
15 minutes 90% Ethanol
15 minutes 100% Ethanol (4 x)

Critical Point Drying
Gold sputter coating

Beware, osmium tetroxide is as poisonous as it gets.

As the chemicals that make most lifeforms have a pretty low atomic number, the number of secondary electrons emitted from the sample are low, too. Thus a thin coat of gold which has a high atomic number is applied to the sample. Now the electron beam has a higher chance to cause secondary electrons to be emitted. The result is better contrast.

If we want to sputter coat the sample we will need vacuum. If our sample contains any liquid matter f.e. in cells they would literally explode while the sputter coater is evacuated.

If we would simply heat the sample for water or ethanol to evaporate, the surface tension at the phase transition would destroy our sample. But under high pressure (218 atm) and high temperature (374 °C) there is a so called critical point in the phase diagram. At and above that critical point there is no distinction between liquid and gas - it is called a supercritical fluid. As 218 atm and 374°C is a bit hard to achieve, CO2 is used. The critical point for CO2 is 31°C and 73 atm.

So we have our sample in 100 ethanol. The ethanol bathes (30%, 50%, 70%,90% 4x 100%) are used to exchange water against ethanol. Then we put our sample in the pressure chamber of the Critical Point Dryer. We cool the sample to 10°C and then open a valve to release some liquid CO2 into the chamber. Now we wait a little while so ethanol and CO2 mixes. Then we open another valve to release some of the liquid. This is repeated about 30 times. This causes the entire ethanol to be replaced with CO2. Then the temperature is increased to 40°C. Around 31°C the CO2 becomes supercritical. The valve is opened again and slowly the CO2 evaporates without any damage to the sample.

No we have a totally dry sample that is ready to be sputter coated.
Sorry for this lengthy explanation.

@David,

I totally agree. I wish less nasty chemicals would do a good job

Of course this was done under a fume cupboard. OsO4 and HgCl2 kill so fast that cilia etc. are "frozen".

Uhhh... let's see.... where's my old peanut butter jar and my eyedropper? The most dangerous part of my prep is cutting my finger while cleaning a cover slip! (Or perhaps falling into a warm pond while collecting, and getting a naegleria fowleri up my nose )

Seriously though, it is always fascinating to see the incredible detail of electron microscope shots. Thanks for the post and the details.

[johan]

Sorry for this lengthy explanation.

Are you kidding?! Fascinating stuff

[chrismower]

that was absolutely fascinating. I have often wondered how specimens were prepared for use in SEM. Thanks for sharing that info.