![]() ![]() 2B, D exhibit identical d-spacings and angles between reflections. This crystal had most likely begun to nucleate in the bile some time after time zero, because upon aspiration, samples are centrifuged and any existing crystals removed. Figure 2C shows a representative cryo-TEM image of a crystal seen in gallbladder bile number 2 ( Table 1), 3 days after removal from the patient, and Fig. The crystal habits were long, rectangular or classical, plate-like rhombus-shaped. Thus, a single representative image is shown. Several crystals were observed, and all exhibited identical diffraction patterns. The compositions and characteristics of these biles are shown in Table 1. Three native bile samples were studied by cryo-ED. Selected-area electron diffraction patterns were obtained from several crystals identified by imaging at low and medium magnifications of 3,400× and 40,000×, respectively. Images were recorded at nominal underfocus of 2–4 μm to enhance phase contrast. #Singlecrystal electron diffraction pattern example softwareA Gatan MultiScan791 cooled CCD camera operated with the Digital Micrograph 3.1 software package (Gatan), was used to acquire the images and diffraction patterns. Images and electron diffraction patterns were recorded in the low-dose mode to minimize electron beam radiation damage to the very radiation-sensitive samples. An Oxford CT3500 cooling-holder (Gatan Pleasanton, CA) was used to load the specimens into the TEM. Standard crystals (see above) that served as controls were prepared at room temperature and then cooled to liquid nitrogen temperature for sample stability. Thus, large crystals are always found flat on the grid. Structures larger than ∼300 nm in diameter are blotted off or may be distorted or positioned flat within the liquid film. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers ( Biophysical J., In press) in cholesterol monolayers compressed on the air–water interface. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The exact structure of early-forming crystals is still controversial. With more and more laboratories setting up 3D ED techniques, we envision that it will not only continue providing critical structural information, but also establish a wide impact on chemistry, materials science and life science.Cholesterol crystals are the building blocks of cholesterol gallstones. We focus on metal–organic frameworks (MOFs) and small-molecule-based organic crystals, and highlight the insights provided by 3D ED such as structure–property relationships, polymorphism, hydrogen bonding, and crystal chirality, which are crucial subjects in crystal engineering. ![]() Herein, we provide an overview on 3D ED, including its development, data collection protocols, and their applications for investigating crystal structures. Recent advancement in three-dimensional electron diffraction (3D ED) and its application on structural characterizations have expanded single-crystal analysis into nano-sized materials. Understanding crystal structures and behaviors is crucial for constructing and engineering crystalline materials with various properties and functions. ![]()
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