Applications of Cryogenic Electron Microscopy in Biomineralization Research

Abstract

Biological mineralization is a natural process manifested by living organisms in which inorganic minerals crystallize under the scrupulous control of biomolecules, producing hierarchical organic-inorganic composite structures with physical properties and design that galvanize even the most ardent structural engineer and architect. Understanding the mechanisms that control the formation of biominerals is challenging in the biomimetic engineering of hard tissues. In this regard, the contribution of cryogenic electron microscopy (cryo-EM) has been nothing short of phenomenal. By preserving materials in their native hydrated status and reducing damage caused by ion beam radiation, cryo-EM outperforms conventional transmission electron microscopy in its ability to directly observe the morphologic evolution of mineral precursor phases at different stages of biomineralization with nanoscale spatial resolution and subsecond temporal resolution in 2 or 3 dimensions. In the present review, the development and applications of cryo-EM are discussed to support the use of this powerful technique in dental research. Because of the rapid development of cryogenic sample preparation techniques, direct electron detection, and image-processing algorithms, the last decade has witnessed an exponential increase in the use of cryo-EM in structural biology and materials research. By amalgamating with other analytic techniques, cryo-EM may be used for qualitative and quantitative analyses of the kinetics and thermodynamic mechanisms in which organic macromolecules participate in the transformation of mineral precursors from their original liquid state to amorphous and ultimately crystalline phases. The present review concentrates on the biomineralization of calcium phosphate mineral phases, while that of calcium carbonate, silica, and magnetite is only briefly mentioned. Bioinspired organic matrix–mediated inorganic crystallization strategies are discussed from the perspective of tissue regeneration engineering.

Attaching organic fibers to mineral: The case of the avian eggshell

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Figure 4 Attachment of eggshell membrane fibers to the shell at the nanoscale: Multiscale assessment of fiber mineral granules and spikes Transient amorphous mineral phases at fine dimensions are sensitive to aqueous-based conventional sample-preparation techniques. 37 After acquiring several FIB-SEM volumes that were conventionally processed and stained (A, D, F, and J) – versus cryo-prepared (B, C, E, and G–J) – BSE differences in contrast suggested that within fiber mantles that adopt the more porous “void-like” morphology (D, black arrows, also see Video S8 ), there was in fact propagation of small mineral spots/streaks (D, white arrows in inset). Diffuse electron diffraction reflections after SAED of fiber mantle from a FIB-prepared lamella of the same sample corroborated that these were mostly voids with small amounts of mineral (F).

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Mechanisms of Biomolecular Self‐Assembly Investigated Through In Situ Observations of Structures and Dynamics

“…Graphene-encapsulated hydrated biological samples can be imaged for several minutes without any visible structural damage to the samples. [101,102] Another way around the problem of radiolytic damage to biomolecules is to use cryogenic electron microscopy (Cryo-EM), [103] which enables imaging biomolecules in their native state and obtaining high-resolution structural information. The basic principle of cryo-EM is to fix the samples in amorphous ice and obtain 2D images of the sample over an extensive range of orientations.…”

Matrix Vesicle–Mediated Mineralization and Potential Applications

“…Moreover, cryo-electron microscopy outperforms conventional electron microscopy in its ability to directly observe the morphological evolution of mineral precursor phases at different stages of biomineralization with nanoscale spatial resolution and subsecond temporal resolution in 2 or 3 dimensions. The use of cryo-electron microscopy for mineralization research has been recently reviewed (Lei et al 2022). Accordingly, these novel imaging tools could be beneficial for studying matrix vesicles, especially in terms of organelle colocalization.…”