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Vitrification is a promising approach for ice-free cryopreservation ofbiological material, but progress is hindered by the limited set ofexperimental tools for studying processes in the interior of the vitrifiedmatter. Isochoric cryopreservation chambers are often metallic, and theiropacity prevents direct visual observation. In this study, we introduce photoncounting X-ray computed tomography (CT) to compare the effects of rigidisochoric and unconfined isobaric conditions on vitrification and ice formationduring cooling of two aqueous solutions: 50 wt% DMSO and a coral vitrificationsolution, CVS1. Previous studies have only compared vitrification in isochoricsystems with isobaric systems that have an exposed air-liquid interface. We usea movable piston to replicate the surface and thermal boundary conditions ofthe isochoric system yet maintain isobaric conditions. When controlling for theboundary conditions we find that similar ice and vapor volume fractions formduring cooling in isochoric and isobaric conditions. Interestingly, we observedistinct ice and vapor cavity morphology in the isochoric systems, possibly dueto vapor outgassing or cavitation as rapid cooling causes the pressure to dropin the confined systems. These observations highlight the array ofthermo-fluidic processes that occur during vitrification in confined aqueoussystems and motivate the further application of imaging techniques such asphoton counting X-ray CT in fundamental studies of vitrification.

Direct Comparison of Isobaric and Isochoric Vitrification of Two Aqueous Solutions with Photon Counting X-Ray Computed Tomography