Hi, Daniel!

Even with unknown EDS spectrum, could you give us a hint as to which elements you detect?

regards,

Daniel (also)

May be some contamination from your sputtering process.

From EDS you can confirm about which material is growing in such a way. It may be a gold or any chemical impurity which is growing. I have seen this type of structure in ZnO nanostructures.

Just think a bit what kind of salt you haven't removed from your cells (less likely, but it can be NaCl, for instance, from buffer, or excess of OsO4). You use critical point drying, so crystals might grow like that. Overall, just increase number of rinsing steps - I think it will help.

Hello, thank You for the answers - the problem is, that I get all the stuff involved in the preparation process in the spectrum - a lot of Os, some Na, lot of Au, some Phosphorus hidden behind the Au peak. To rule out sputtering or CPD, I may ask my colleagues if they have some unsputtered, or even some non-CPD samples from the same tissue pickup. But I think the sputtering should not be responsible because this problem also occured when using an older sputter coater. I will post some EDS spectra from the crystals in the afternoon. @Evgeny: I will forward this information to my colleagues, because I also think that the crystals are somtimes grouped-up in some places on the sample that is not very accessible to the chemical during rinsing.

//Hello, here are some spectra - it seems, that the crystals are somehow related to Na? (I decreased the resolution for image in spect2-4 because I was not able to get a "still" picture)

///posts merged

It is definitely not because sputtering. Most probably - insufficient washing. Or dirty solvent (alcohol/acetone). Contamination in CPD machine.

You may want to try to change or eliminate some steps.

- Use more water or more washing steps before dehydration.

- Skip Os treatment (you do not really need it for SEM, its for TEM).

- Replace CPD with HMDS (works fine with most specimens, sometimes its better than CPD).

And what about durcupan? Are you sure you really use it for SEM? It's embedding media, not fixation agent. Or am I missing something?

@vladimir: sorry, no durcupan. We use OsO4 for fat tissue post fixation - it was neccessary, for example, in human heart samples.

Good morning, fixation is by the 3% glutaraldehyde in phosphate buffer solution, not in durcupan (it was such a mistake of my collegue). Postfixation is not just for TEM, we tried it to bypass in other type of tissue (containing some fatty tissue in heart) and lipid-rich structures were totaly distroyed, so we standardly use it in SEM sample procedure to preserve cell membrane components and lipids.

I think the washing may help the most.

We dealt with this problem last year and adding washing before dehydration with decreasing the concentration of buffer solution solved the problem. We avoid also sputtering, it didn´t help.

Thank to all of you for your oppinion and ideas.

Paula

Hello,

I still do not know why the crystals are growing - what is the cause in the first place,

BUT more washing steps solved the problem - only a few crystals are present and their location is at the most inaccessible locations within the sample.

So the problem with contamination is gone although I have yet to check the sample without CPD and sputter coating.

The morphology of the crystals clearly indicates it to have precipitated and grown. Most likely it is achieved by oxidation of some solvent to grow crystals of oxide from a super-saturated solution. It could be OsO4.

Additionally doing point EDS on the crystal will not help. Typically beam diameter in a SEM is of the order of ~1 micron. The thickness of your flower petal is in the order of nm. Thus, you might be picking up other elements in SEM EDS.