PubMed · 7552700
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H Frauenfelder. 1995. Complexity in proteins.. https://doi.org/10.1038/nsb1095-821
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When biological macromolecules are imaged in the transmission electron microscope (TEM), their inherent handedness is lost because the three-dimensional (3D) structure is projected onto a two-dimensional (2D) plane, and identical 2D projections can be made from either 3D enantiomer. Nevertheless, tilt experiments in the TEM can be used to determine handedness. These experiments have been performed successfully on negatively stained specimens. More recently, the method was applied to unstained, frozen-hydrated specimens imaged by means of cryoelectron microscopy (cryoTEM) methods. Tilt experiments involve recording two micrographs of the same particles at different tilt angles, computing enantiomeric reconstructions from particle images in one micrograph, predicting orientations of corresponding particles in the second micrograph, and comparing model projections with particle images in the second micrograph. In principle, this procedure can be used to determine the handedness of any biological macromolecule imaged by cryoTEM, provided the enantiomeric reconstructions are distinguishable.
Flowers of blackcurrant (Ribes nigrum L.) were subjected to freezing stress in vivo, and the resulting damage examined in three dimensions using a spin-echo nuclear magnetic resonance imaging sequence. Increased signal intensity was detected in the damaged flowers, particularly at the base of the style, in T2-weighted images. This is thought to be the result of intracellular freezing, which causes membrane damage and leakage of cellular contents. It is proposed that this represents the main site of damage within the flowers. The imaging of flowers of differing developmental ages showed larger increases in signal from fully open flowers after freezing damage compared with those in the initial stages of bud, suggesting that the enclosed nature of the flower buds may have a protective effect on the sensitive stylar base. The use of three-dimensional nuclear magnetic resonance imaging provides a rapid and effective means for the visualisation of freezing events within floral tissues; the effective resolution of the images enables greater accuracy and clarity in interpretation than hitherto possible in two dimensions.