Andrew Syred

The colleague’s concern regarding the use of xylene as an intermediate medium in the critical point drying process for SEM analysis is justified and reflects a responsible approach to working with delicate biological samples. Although a test run without the sample appeared to show that xylene and liquid CO₂ can mix, this is not reliable evidence of their full miscibility under actual operating conditions. According to available chemical data, xylene is not thermodynamically fully miscible with liquid CO₂, which poses a significant risk when replacing the medium during drying.

Immiscibility may lead to the formation of phase boundaries within the sample, potentially resulting in the collapse of cellular structures, the formation of artifacts, or permanent damage to fine morphology. Additionally, residual xylene in the system can negatively affect the drying chamber, increasing the risk of contamination and complicating maintenance. It should be noted that standard protocols have been developed precisely to avoid such problems—by using ethanol or acetone, which are proven to be miscible with LCO₂ and allow safe, controlled solvent exchange.

If returning the sample to ethanol or acetone is to be avoided, a gradual replacement of xylene with an intermediate solvent compatible with both xylene and LCO₂ (such as n-hexane) may be considered, although this too requires careful validation. Consulting the critical point dryer manufacturer or an experienced SEM technician can provide guidance on the feasibility of using alternative protocols.

In conclusion, while the observed mixing during a test run may suggest compatibility, it does not guarantee the safety of the method. The recommendation is to revert to validated and proven protocols involving ethanol or acetone in order to protect the sample and ensure reliable microscopy results without risking damage to the specimen or contamination of the equipment.

Thank you Dragan Ugrinov for your time and erudite answer. It's back to ethanol for the sample, with the hope it can handle the medium change.

Liquid CO2 and xylene are partially miscible but not fully miscible under all conditions. Using xylene as an intermediate in critical point drying may risk sample damage due to limited miscibility with liquid CO2.

Thanks Abdelhak Maghchiche for your input which is gratefully appreciated. Three people have now told me xylene is not fully miscible with liquid CO2 so I'll abandon the idea. The sample is going to have to tolerate going back into ethanol.