All biological samples for cryogenic electron microscopy are prepared in a vitreous state, meaning a non-crystalline “glassy” ice. This vitreous ice is critical as crystalline ice formation disrupts biological structures and leads to diffraction of the transmitted electron beam, preventing high-quality electron microscopy. Vitrification is achieved through plunge freezing, a process involving rapid (>10 K/ms) cooling rates in thin (1 K/ms) cooling rates in thicker samples (<200 μm) under high-pressure (∼2,100 bar). While commercialized instruments have been developed to achieve this rapid freezing, there are still many challenges and the process frequently goes wrong. Further, confirming that your sample is vitreous is currently accomplished by transmission electron microscopy, potentially involving arduous workflows and requiring expensive instrument time. An optical method of assessing sample vitrification would greatly improve the experimental workflow. Pursuing this, I have been using a temperature sensitive fluorescent dye, which rapidly converts between two spectral species, the abundance of which changes as a function of temperature. Ratiometric measurements of the two spectral species allow temperature to be determined, but when frozen out of equilibrium, this ratio of spectral species instead informs on the cooling rates that were achieved during freezing. Preliminary results suggest that while the method itself has potential, in its current state, this method cannot be applied to plunge frozen samples due to their rapid cooling rates. Among plunge frozen samples, the rapid cooling rates cause both crystalline and vitreous samples to exhibit similar spectral ratios. We are currently exploring the use of this dye for high-pressure freezing applications, where cooling rates are slower.
Ecklund et al. (Sun,) studied this question.