Hello everyone,
I am trying to troubleshoot my SEM procedure. I am trying to image gelatin gel microstructure using SEM. These gels extruded out of a small diameter nozzle (0.33 mm ID).
I have included to images that I took of the gels, and I have detailed my procedure below. I would appreciate any insight anyone could provide.
1. Extrude out gelled gelatin.
2. Place a small portion of the extruded gelatin into 2.5% glutaraldehyde in 1X PBS for 1 hour at 4 C.
3. In the subsequent steps, I remove the glutaraldehyde and slowly replace the liquid content with increasing concentration of ethanol (25%, 50%, 75%, 95%, 100%) with each step lasting 10 minutes.
4. Use a super critical drier with CO2 to replace ethanol and then evaporate CO2.
5. Coat sample with carbon.
6. Adhere to SEM sample chuck.
7. Image under SEM.
I am expecting to see more of a lattice/mesh in the gelatin rather than this rough morphology (A quick Google search for "Gelatin SEM" reveals the images I expect to get). Some questions I have about my procedure are: 1) Should I be including long wash steps and if so in 1X PBS or water? 2) Could the carbon coating step be applying too thick of a layer ? I try to follow the instructions on the machine I use and layer thickness indicator on the machine always gives me a number between 5 and 10 nm. 3) Should I be use carbon coating or some metal coating? I have gone with carbon coating as this was the first recommendation by one of the tech. However, I see many papers use Pt or Au coatings. 4) Should I be fracturing the sample any to reveal inner surfaces, or should I expect the outside surface of the extruded gelatin to also reveal a porous surface?
I would appreciate any feedback. Thank you very much.
What you see after Google search is mostly not gelatin per se, but gelatin in highly processed mode; scaffolds and fibers made from gelatin. Your pictures are much close to real gelatin than those you paid attention at.
Change of coating material can improve quality of picture (contrast, noise level), but not to change it in any other way.
Any coating can damage delicate specimen. Sometimes gels are delicate enough. You can check you specimens without any coating working at accelerating voltage close to 1 kV.
I agree with Vladimir Dusevich, it is NOT an issue of coating. I think the issue is related to the process that you followed, the process must ensure the followings:
1- Obtaining hydrogel.
2- Fixation of the hydrogel.
3- Converting the fixed hydrogel into alcogel.
4- Converting the alcogel into aerogels or xerogel.
I guess that there is a lack in the fixation step as your SEM images, as Vladimir said, are close to real gelatin images.
Additives, such as salts, acids, and formaldehyde, are commonly used for fixation. referring to some litterateurs and doing few experiments will be helpful to select the proper additive for your system.
The amount of GA and duration of fixation is too high to fix the gel in the primary structure which probably hide the fine structure of gelatin rather than providing the fixed secondary microstructure. Therefore, I recommend 0.5 to 1% GA and fixation time is less than one hour. I also prefer Au or Pt coating than the C coating. Coating layer thickness should not be more than 3 nm to observe the inner view of the sample. During handling SEM be more patient and try to adjust the sample stage with different axes (X, Y and Z) and appropriate voltage and proper resolution to get high quality images.
Consider imaging in an E-SEM with water vapor in chamber on cryostage, which avoids all processing steps. Second, I don't see a scale bar, and the image does not look sufficient hires so that you would see macromolecules.
Dear Freeman,
Kindly try this procedure. Dried Gelatin Hydrogels were sputter-coated with gold (Autoconductavac IV, See-Vac) for approximately 3 min to a thickness of approximately 20–30 nm, and imaged using scanning electron microscopy (SEM).
Dear Sebastian, prior to the SEM analysis, you can freeze the hydrogels by immersion in a block of dry ice in order to preserve their microstructure. Frozen hydrogels can be then fractured with a scalpel and immediately placed on a lyophilizer for freeze drying. Subsequently, the hydrogels are affixed to a metal stub with carbon film and they are sputter-coated with a layer of Au for 2 minutes in order to increase the conductivity of your scaffolds and obtain good SEM images.
If this is still relevant, then I would suggest to prolong the last time step in point 3 and use orbital shaker during these steps.
It is also good to have the flask (or whatever You are having the sample plunged in) on some ice to keep it cool.
Point 4. - I do not know what Your device is, but You can try this, just to make sure You have done it properly: let the gas in the device, wait for the level of the liquid to rise to an appropriate height (according to Your device), then stop the gas and let it out - use the fine valve. Do this TWICE! Proceed to the critical drying only after the third "gas in".
I suppose that Your SEM is Zeiss EVO, so skip point number 5. and use EP mode (easyVP aperture) with water vapor. Use VPSE or C2D / C2DX detector instead of SE.
If You go with the carbon coating and high vacuum observation choice, then it is a good idea to let the sample "sit" in the microscope for at least 30 minutes with beam off before observation.
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