The 3-Dimensional Architecture of Platelets in the Native State by Electron Cryotomography.

Conventional electron microscopy requires that samples be chemically fixed, embedded in plastic and stained prior to examination. This technique always raises uncertainty as to whether the observations are faithful to the native cellular organization. These limitations are overcome with the technique of electron cryomicroscopy, in which samples are prepared and observed in the frozen, hydrated state. A modification of this technique, electron cryotomography, is a method capable of recording images from the same specimen area at different tilt angles under low electron dose conditions, revealing the spatial relationships of organelles and molecular assemblies within a cell that has not been subjected to chemical fixation or metal staining.

Here, we used electron cryotomography to examine the 3-dimensional architecture of platelets in the resting state and in various stages of activation. Platelets in citrated plasma isolated from healthy human subjects were allowed to settle onto a perforated carbon grid and frozen immediately in liquid ethane or were allowed to settle for different time periods on a grid coated with fibrillar type 1 collagen before being frozen. The platelets suspended across holes were embedded in a thin layer of vitreous ice. Successive images of these frozen, hydrated platelets were then acquired as the stage was tilted through a range of ±70° using a JEM2010F electron cryomicroscope. Each image was recorded using a 4kx4k CCD camera. The digital images were computationally aligned and merged to reconstruct a 3-dimensional density map with an 4.0 gigapixel volume. Multiple tilt-series were reconstructed and analyzed using segmentation and visualization tools. The platelet reconstructions were annotated in terms of the sizes, shapes and relative locations of the subcellular components, including mitochondria, α and dense granules, dense clusters, the dense tubular system, microtubules, and the surface-connected canalicular system. We observed great variability in the extent of ordered structure in the α granules, and in the filopodia adhering to the grid surface. The tomograms resolve the fibrous actin bundles driving filopod extension and the individual actin cross-links within the bundles. In some platelets, we observed membrane tethers that attached to the surface but were devoid of actin. This represents the first 3-D visualization of human platelets under native conditions. These observations and the ease of generating such data suggest that electron cryotomography can become a useful tool for dissecting the molecular sequence of cytoskeletal rearrangements occurring during platelet activation and granule secretion and for understanding the structural basis of platelet diseases.