High-throughput imaging of fresh-frozen plant reproductive samples in a variable pressure SEM

Conventional light and electron microscopy are the most widely used techniques for examining plant reproductive tissues; however, they are time-consuming or expensive. The anther is the male part of the plant reproductive system. Structural changes drive development, and any structural defect may lead to an increase in fertility or cause sterility; thus, quick detection of structural changes is crucial in reproductive biology. We optimized an existing low-temperature SEM alternative to examine the internal structure of hydrated, fresh-frozen anthers. In contrast with the original technique, our method does not require precooling adhesion (ethanol to fix the specimen), and the cryo-sectioning can be conducted at atmospheric pressure. In addition to enabling the differentiation between aerial and liquid-filled intercellular spaces, this method is expected to facilitate the detection of quick (during a day) developmental changes in plant reproductive tissues, which is a current challenge using conventional approaches. • This method allows the high-throughput imaging of fresh-frozen plant reproductive samples collected every 10 min, which is important for developmental studies. • The cryo-images of samples with thickness ranging from 0.2 to 3 mm can be well-preserved at 800X magnification. • This method does not require chemical processing, critical point drying, customized cryo-accessories, controlled temperature cold stages, or metal coating. This simplified method does not require highly skilled personnel, and it is suitable in most microscopy laboratories.