005 Integration of polarized light into scanning cryomacroscopy

This study presents the integration of polarized light into the scanning cryomacroscope—a recently developed device to visualize physical effects in large-scale cryopreservation [Y. Rabin, J.S.G. Feig, A.C. Williams, C.C. Lin, C. Thaokar, Cryomacroscopy in 3D: a device prototype for the study of cryopreservation, ASME-SBC 2012, Fajardo, Puerto Rico]. The cryomacroscope is the only means available for visualization and recording physical events along the path-dependent process of cryopreservation in large samples. The application of polarized light enhances the ability to trace ice nucleation, thermo-mechanical stress formation, fracturing, and contamination in the cryopreserved material. Several versions of the cryomacroscope have been presented in recent years, all of which share the same key features: (i) a cryogenic setup to cool a specimen along cryopreservation protocols of interest, (ii) a data acquisition system to monitor and record the thermal history of the specimen, (iii) a borescope to transfer sample images from the cryogenic environment to, (iv) a camera held at room temperature, and (v) image post-processing capabilities. The scanning cryomacroscope was recently developed to analyze samples bigger than the field of view of the borescope. The current study is focused on enhancing visualization capabilities by integrating, for the first time, polarized light with cryomacroscopy. The implementation of polarized light uses two parallel and concentric polarizing filters, rotated 90° to one another around their common axis. As a result, only refracted light refracted by an object placed intermediate to the first filter (polarizer) and second filter (analyzer) may pass the analyzer and be captured by the cryomacroscope camera. The filters used in the new setup are 0.3-mm thick dichroic polarizing film sheets sandwiched (epoxied) between protective glass windows. These glass windows are coated by an antireflective film, which is optimized for the visible light spectrum that in the range from 400 to 700 nm. The analyzer is secured to the borescope tip with a 3D-printed housing. The polarizer is secured in a housing attached to a beam-rotating mirror. Light is directed from a halogen source (150 W at 3200 K) via fiber optics to the mirror. Alternating diffuse light is directed from an independent LED source (5 W at 6000 K) to the sample by a second set of fiber optic bundles. Vitrification experiments on Me2SO solutions at various concentrations and subject to varying cooling protocols have been conducted. The following effects are demonstrated with the new polarized light setup: (1) display of contaminants otherwise unobservable with diffuse light, some of which are longer than 5 mm; (2) observation of ice nuclei larger than 15 μm in characteristic diameter within a 4.5 ml domain; (3) improved contrast in fractured sites; and, (4) visualization of mechanically strained vitrified material (cryoprotective agent and container), where the strained material is coded with the visible light spectrum. Source of funding: This project has been supported by Award Number 5R21RR026210 from the National Center for Research Resources, and Award Number 8R21GM103407 from the National Institute of General Medical Sciences. Conflict of interest: None declared. rabin@cmu.edu