Phase Identification in Cryopreserved Systems via Thermal Conductivity and Crystal Symmetry Effects

Long-term preservation of biological tissue is a research goal amongst biomedical researchers and bioengineers for its potential impacts such as improving efficacy of organ transplants, a problem for which incremental advances correlate to thousands of lives saved each year. In particular, there is a concentrated research push towards achieving long-term organ storage via bulk vitrification of tissues, in which tissues are rapidly cooled while suppressing ice formation. Low-temperature storage of tissues in an amorphous phase prevents ischemic cell death and enables virtually limitless storage times. Among the barriers to this goal is the lack of a non-invasive diagnostic method to determine the phase of solid biological samples. Current quantitative techniques, including X-ray diffraction and direct measurement of cell death, are time- and cost-intensive and damaging to samples. Otherwise, imprecise measures such as visual examination for cracking are used. An optimal technique for probing phase in vitreous biological systems would be non-destructive to cells, distributable for research and clinical applications, and resolvable for times and sizes used in vitrification protocols.<br/><br/>To achieve this, we implement the 3ω method as an <i>in situ</i> thermal method to determine phases of representative samples, made possible by the high dependance of thermal conductivity on crystal structure. To demonstrate this, solutions of water and cryoprotective agents (CPAs) are used as representative thermal systems on a temperature-controlled cryogenic stage with electrical and optical feedthroughs. By manipulating CPA concentration and cooling rate, the sample can be selectively solidified into a vitrified (amorphous) or crystalline state. This solid phase is stable and allows for exploration of a variety of potential characterization methods, including a modified 3ω method for thermal conductivity. Today, we demonstrate the viability of an alternative scheme in which a ‘buried substrate’ is measured allows for simplified experimentation and fabrication, which was preferred for dissemination of the technique.<br/><br/>A parallel study utilizes the refractive properties of crystals as an indication of phase. Ordinary ice (ice I_h) has a hexagonal crystal structure which exhibits birefringence. This causes the double refraction of incident polar excitations. As amorphous solid water, or a vitreous organ, is isotropic and therefore not birefringent, polarized light macroscopy can be used as an independent metric for the phase of representative samples. Here, we demonstrate the use of optical techniques as a means for phase identification in representative systems and explore the potential of polarized acoustics as a high-speed, volumetric probe for crystal symmetries in opaque systems.