Compared to the divide-and-conquer approach employed in single particle analysis by cryogenic electron microscopy, cryo-electron tomography (cryo-ET) offers the advantage of directly visualizing the 3D architecture of cellular structures and their molecular organization with unparalleled detail, while avoiding the potentially disruptive purification process that can interrupt fragile interactions (Beck and Baumeister, 2016). However, in order to achieve high-contrast imaging, the thickness of the sample must be less than the inelastic mean free path of the electrons, which is typically between 300–400 nm for vitrified biological materials (Rice et al., 2018), whereas most cells exceed this range. Therefore, methods are required to render samples electron-transparent. Among these methods, focused ion beam (FIB) instruments, which were originally developed for material science, have demonstrated potential for high-resolution cryo-ET sample preparation (Marko et al., 2007, Rigort et al., 2012). However, due to time constraints and the limited depth of focus of the ion beam, the typical cryo-FIB workflow is only suitable for small samples, such as single cells, that can be vitrified by plunging.

The study of multicellular models is essential for understanding critical aspects of life, which cannot be investigated in isolated cells. Preserving subcellular architecture is crucial for studying cellular constituents without introducing significant structural alterations. Vitrification, which is the only method capable of achieving this goal, requires the use of high-pressure freezing to efficiently vitrify multicellular samples up to a size of 200 μm (Moor, 1987). Therefore, it is crucial to develop a cryo-FIB-based pipeline that can be adapted to high-pressure frozen samples for in situ study of tissue samples. The first step involves loading the sample into the cryo-FIB. Previous studies have successfully achieved high-pressure freezing of samples directly on a standard EM grid (Harapin et al., 2015, Kelley et al., 2022). Adequate high-pressure freezing vitrification requires replacing air with a cryo-filler fluid in the specimen carrier. The most commonly used cryo-filler for high-pressure freezing is 1-hexadecene, which has the advantages of being chemically inert and non-penetrating (Studer et al., 1989). However, the drawback is that the sample is embedded in a thick slab formed by the cryo-protectant/filler, which interferes somewhat with the recognition and exposure of the region of interest. To address this issue, a cryo-ultramicrotome is needed to carefully trim the high-pressure freezing carrier before cryo-FIB milling (Hsieh et al., 2014, Zhang et al., 2021). Non-embedded sample preparation using high-pressure freezing has also been reported before, in which 2-methylpentane was used as the cryo-filler and sublimated in a high-vacuum chamber (Harapin et al., 2015). However, this sublimation step required a long time (∼30 min), and any residual buffer or medium reduced the performance of cryo-filler removal and grid separation.

For the following sample thinning, although various conceptual methods have been previously described, their application is limited due to requirements for prototype instruments or skilled operators (Mahamid et al., 2015, Parmenter and Nizamudeen, 2021, Rubino et al., 2012, Schaffer et al., 2019, Wang et al., 2023, Zhang et al., 2021). Recently, a method called the Waffle has shown significant advances in relatively large sample preparation; however, its application to tissue has not yet been reported (Kelley et al., 2022).

Our study presents an optimized and, importantly, practical pipeline for the preparation of multicellular samples for cryo-electron tomography imaging, which includes sample isolation, vitrification, and lamellae preparation steps using commercially available instruments. By applying our developed pipeline to pancreatic islets, we demonstrated the significance of tissue usage for gaining insights into critical biological processes.