Abstract
Cryo-FIB/SEM has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling specimens with preferred orientation, low throughput when milling small specimens, cellular specimens that concentrate poorly in grid squares, and thick specimens that do not vitrify well. Here we present a general approach we call the ‘waffle method’ which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the waffle method to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations by cryo-ET, which was previously not possible due to preferred orientation of the spores on the grid. We also present a unique and critical stress-relief gap design specifically for waffled lamellae. Additionally, we describe applications of the waffle method which are currently being explored. We propose the waffle method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, technically-demanding process that cryo-liftout requires.
Competing Interest Statement
The authors have declared no competing interest.