capabilities of such systems have advanced a great deal. The breadth of applicationshas also expanded tremendously. While the spatial resolution of light microscopy islimited relative to electron microscopy, its advantage is that real-time, continuousoptical interrogation of biomaterials and the solutions they are in is possible duringcooling and warming, freezing and thawing. This has proven to be a powerfulapproach to understanding the response of biomaterials exposed to low tempera-tures, including cryogenic temperatures. In this talk a brief historical developmentis presented, along with some examples of the types of devices and applicationsthat have helped improve our understanding of cryobiology.

Source of funding: None declared.Conflict of interest: None declared.Email address: mcgra2jj@cmich.edu

http://dx.doi.org/10.1016/j.cryobiol.2013.09.019

014The use of cryo techniques for correlative light electron microscopy. Paul Verkade,University of Bristol, UK

Correlative Light Electron Microscopy (CLEM) combines the advantages of bothLight Microscopy (LM) and Electron Microscopy (EM) and analyses a single samplewith both modalities. LM is for instance able to provide the history of an eventthrough live imaging, whereas EM can provide higher resolution and surroundingultrastructural information. CLEM is usually a compromise between the 2 modali-ties and often the ultrastructural preservation is sacrificed by chemical fixation.When one studies structures that are susceptible to chemical fixation we have toresort to cryofixation. We and others have developed techniques to use cryofixationin a CLEM set up. I will discuss a number of these techniques and will highlight thelatest developments.

Source of funding: This work was funded by Wellcome Trust, British Heart Foun-dation, MRC, and BBSRC grants.

Conflict of interest: None declared.Email address: p.verkade@bristol.ac.uk

http://dx.doi.org/10.1016/j.cryobiol.2013.09.020

015Cryo-electron microscopy and tomography of molecular machines, proteinaggregates and cells. Wah Chui, National Center for Macromolecular Imaging, BaylorCollege of Medicine, Houston, TX, USA

Electron cryo-microscopy and tomography has become a critical structural toolthat brings structural insights that cannot be obtained by X-ray crystallography.The advances in cryo-specimen preparation, data collection and data processinghave allowed us to reconstruct 3D images of molecular machines, amyloid proteinsand cells at unprecedented resolutions. I will present multiple examples to demon-strate how this emerging technology reveals protein folds in virus, multiple confor-mations during protein aggregations in neuro-degenerate disease and virusassembly inside a bacterium.

Source of funding: NIH and Welch Foundation.Conflict of interest: None declared.Email address: wah@bcm.edu

http://dx.doi.org/10.1016/j.cryobiol.2013.09.021

016Integration of transient hot-wire method into scanning cryomacroscopy in thestudy of thermal conductivity of dimethyl sulfoxide. L.E. Ehrlich, J.S.G. Feig, J.A.Malen, S.N. Schiffres, Y. Rabin, Carnegie Mellon University, Pittsburgh, PA, USA

A pilot study was conducted to investigate the dependency of thermal conduc-tivity on temperature, phase of state, and solution concentration in the range of 2 M(classical cryopreservation) and up to 10 M Me2SO (simulative of highly concen-trated cocktails used for vitrification).

Cryoprotective agents (CPAs), such as dimethyl sulfoxide (Me2SO) are used tocontrol ice formation—the cornerstone of cryoinjury. When cooled, the CPA may crys-tallize in low concentrations and relatively low cooling rates, or vitrify (form glass—betrapped in an amorphous state) in high concentrations and relatively high coolingrates. Analysis of cryopreservation protocols and explanation of related physicalevents may be assisted by computer simulations of the thermal process. Unfortu-nately, the physical properties of CPAs necessary for thermal analysis represent a lar-gely uncharted area. Furthermore, the difference in thermal conductivity between an

amorphous and crystalline material of the same composition can vary by orders ofmagnitude.

A transient hot-wire technique was used to measure thermal conductivity ofMe2SO solutions in a controlled-rate cooler. Physical events in the sample along thecryopreservation protocol were recorded with the application of the scanning cry-omacroscope to confirm whether the sample was vitrified (transparent) or crystal-lized (opaque). Selected cooling and rewarming rates were chosen to promoteeither vitrification or crystallization, based on literature data. Thermal conductivitymeasurements were made continuously during the rewarming phase of the protocol,in the temperature range of �100 to +20 �C.

Thermal conductivity of crystalline Me2SO was found to be five-fold higher thanthat of amorphous Me2SO, with relatively little dependency on the concentration. Inthe amorphous state, thermal conductivity decreases with the increasing concentra-tion: at 15�C ranging from 0.24 to 0.31 W/m K for 10 M and 7.05 M Me2SO, respec-tively, and at �100 �C ranging from 0.22 to 0.26 W/m K for 10 M and 7.05 MMe2SO, respectively. The decrease in thermal conductivity with decreasing tempera-ture is consistent with literature data for other amorphous materials such as SiO2, Se,and PMMA. The measured thermal conductivity of 2 M Me2SO above �4 �C is consis-tent with the dependency of thermal conductivity on concentration for the othersolutions in the liquid phase. The onset of crystallization based on a water–Me2SOphase diagram, �4 �C, correlates well with the measured increase in thermal conduc-tivity of the mixture around this temperature. The increased thermal conductivityupon phase transition is consistent with literature data on pure water, although ther-mal conductivity of pure water increases by a factor of four (from 0.566 to 2.25 W/m K), whereas the thermal conductivity of 2 M Me2SO increases by a factor of three,from 0.47 W/m K to higher than 1.4 W/m K. The integration of the transient hot-wiremethod into scanning cryomacroscopy developed in this study represents the onlyavailable method for measuring thermal conductivity in cryogenic temperature whilevisually verifying the phase of state.

Source of funding: This study is supported, in part by, Award NumberR21RR026210, National Center for Research Resources (NCRR) and R21GM103407,National Institute of General Medical Sciences (NIGMS).

Conflict of interest: None declared.Email address: rabin@cmu.edu

http://dx.doi.org/10.1016/j.cryobiol.2013.09.022

017Cryomicroscopic analysis of damage mechanisms during freezing and thawing ofa human diploid fibroblast cell line. Sean J. McManus, Jens O.M. Karlsson, VillanovaUniversity, Villanova, PA, USA

Human diploid cell strains are useful for commercial production of viral vac-cines. For example, the MRC-5 cell line (lung fibroblast) is used to produce vaccinesagainst chickenpox and other childhood diseases. To support the safe mass produc-tion of such vaccines, it is necessary to cryopreserve standardized batches of con-centrated cell suspension, thus ensuring a consistent supply of cells formanufacturing. However, the cryopreservation process can result in deleteriousintracellular ice formation (IIF), or trigger other mechanisms that also lead to celldamage. Therefore, towards improving the efficiency of MRC-5 cryopreservationtechniques, the IIF phenomenon was investigated in cell suspensions using ahigh-speed video cryomicroscopy system. Previous high-speed imaging studieshave visualized the IIF process in adherent cells, but not in suspended cells. Inthe present study, the effect of cooling rate on IIF in MRC-5 cells in suspensionwas determined. High-speed videos were captured at recording rates of 540–1200 frames/s during freezing to �60 �C at various rates of cooling (1–120 �C/min). In the suspended cells, intracellular ice formation manifested as an advancingsolidification front, with minimal change in cell transparency; the median durationof such IIF events was 1 ms during rapid cooling (120 �C/min), demonstrating theimportance of high-speed imaging techniques to detect intracellular freezing. Thecumulative incidence of IIF increased with increasing cooling rate, from 0% at1 �C/min to 96% at 120 �C/min. The mean temperature of IIF was �21.7 �C (n = 75)and �33.0 �C (n = 853) during cooling at 15 �C/min and 120 �C/min, respectively.For MRC-5 cells cooled at 120 �C/min in the presence of 5% Me2SO, the mean IIFtemperature was depressed to �46.1 �C. Whereas the majority of cells did not dar-ken significantly during cooling, prominent cell darkening was often observed uponwarming of the frozen cells. Video recordings were acquired during warming from�60 �C to 4 �C at a warming rate of 120 �C/min. In cells with intracellular ice, theprobability of darkening during warming was greater than 50%, and darkening dur-ing warming appeared to be more likely for cells that had been frozen at a highercooling rate. In slowly cooled samples, another phenomenon was observed duringrapid warming: prior to melting of extracellular ice, a transient swelling was seen inmany cells. Because the magnitude of these volume excursions was often large (e.g.,doubling the original cell volume), it is likely that such events are damaging, andmay represent a new form of slow-cooling injury.

Source of funding: Funded in part by a gift from Merck & Co., Inc. to VillanovaUniversity.

402 Abstracts / Cryobiology 67 (2013) 398–442