Hi Everyone,
I am going to use Scanning Electronic Microscopy (SEM) to take some images from some wood biofilm samples. To prepare the samples for SEM, I am going to use the following procedure (Fischer et al, 2012), please let me know if the procedure is correct, any thought will be appreciated.
1- In a fume hood, add ~0.5 ml primary fixative, typically 2.5% GA in 0.1M CAC, by dispersing a slow stream of the liquid (Fix the specimen at room temperature for 30 – 60 minutes in GA)
2- Wash 3 × 2 minutes with rinsing buffer
3- Post-fix with 2% OsO4 in dH2O for 30 – 60 minutes.
4- Rinse 1 × 2 minutes with rinsing buffer, and then 2 × 2 minutes with dH2O
5- Dehydrate with a graded ethanol series:
25% ETOH, 1 × 5 minutes for delicate specimens
50% ETOH, 1 × 5 minutes
75% ETOH, 1 × 5 minutes
95% ETOH, 1 × 5 minutes
100% anhydrous ETOH* 3 × 10 minutes
6- Drying the samples with Vacuum dryer (in which temperature and pressure)? (vacuum dryer is the only available option in the lab)
7- Coating the samples
8- Use SEM
Thanks in advance,
The method you describe sounds more like a preparation step for optical microscopy or transmission electron microscopy (TEM) rather than SEM. A common reason for fixation and ethanol series dehydration is to produce a sample that can be embedded for sectioning with a microtome. Because SEM shows a surface image rather than a transmitted light image, sectioning is not necessary. The main goal is simply to avoid distortion from shrinkage during drying A common SEM method would be critical point drying, if you had access to the apparatus, but perhaps simple air drying would be worth a try. Another possibility would be to examine specimens in variable pressure mode, where the specimen does not need to be dried. This of course assumes the SEM you are using is capable of variable pressure operation.
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