  Vitrification is a promising approach for ice-free cryopreservation of
biological material, but progress is hindered by the limited set of
experimental tools for studying processes in the interior of the vitrified
matter. Isochoric cryopreservation chambers are often metallic, and their
opacity prevents direct visual observation. In this study, we introduce photon
counting X-ray computed tomography (CT) to compare the effects of rigid
isochoric and unconfined isobaric conditions on vitrification and ice formation
during cooling of two aqueous solutions: 50 wt% DMSO and a coral vitrification
solution, CVS1. Previous studies have only compared vitrification in isochoric
systems with isobaric systems that have an exposed air-liquid interface. We use
a movable piston to replicate the surface and thermal boundary conditions of
the isochoric system yet maintain isobaric conditions. When controlling for the
boundary conditions we find that similar ice and vapor volume fractions form
during cooling in isochoric and isobaric conditions. Interestingly, we observe
distinct ice and vapor cavity morphology in the isochoric systems, possibly due
to vapor outgassing or cavitation as rapid cooling causes the pressure to drop
in the confined systems. These observations highlight the array of
thermo-fluidic processes that occur during vitrification in confined aqueous
systems and motivate the further application of imaging techniques such as
photon counting X-ray CT in fundamental studies of vitrification.
