Quality evaluation of quick-frozen biological specimens by simultaneous microbeam SAXS/WAXS recordings

The quality of small-angle X-ray scattering (SAXS) patterns from quick-frozen hydrated biological specimens was correlated with the extent of ice crystal formation by simultaneously recording wide-angle X-ray scattering (WAXS) of ice, at a micrometer-order spatial resolution by using X-ray microbeams. Flight muscle fibers from a giant waterbug, Lethocerus, known to generate well defined small-angle reflection spots originating from the hexagonal lattices of myofilaments, were quick-frozen in the presence or absence of various cryoprotectants. Freezing without a cryoprotectant resulted in massive ice-crystal formation at almost all depths of the specimen, and the occurrence of reflection spots was limited to the region close to the specimen surface. Inclusion of 20% dimethyl sulfoxide or methylpentanediol ensured ideal vitreous ice formation and good diffraction qualities for up to 100 microm from the specimen surface. Glycerol and sucrose were found to be inferior at a 20% concentration, but left the reflection spots observable at depths of up to 100 microm. Thus, the microbeam SAXS/WAXS recording offers a high-spatial-resolution means of evaluating the extent of structure preservation of quick-frozen biological specimens. The technique presented here may also provide useful information in cryoelectron microscopy.