Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame

[Image: see text] Cryomicroscopy is commonly hampered by frost accumulation, reducing the visual clarity of the specimen. Pulling a vacuum or purging with nitrogen gas can greatly reduce the sample chamber’s humidity, but at cryogenic temperatures, even minute concentrations of water vapor can still result in frost deposition. Here, a hygroscopic ice frame was created around the specimen to suppress frost growth during cryomicroscopy. Specifically, fluorescently tagged rat brain vessels were frozen on a silicon nitride window with an ice frame, and the luminescence of the fluorescent tag was improved by a factor of 6 compared to a similar specimen in only a nitrogen purge environment. These findings suggest that the simple implementation of a hygroscopic ice frame surrounding the specimen can substantially improve the visual clarity for cryomicroscopy, beyond that of a vacuum or nitrogen purge system.