Quantitative dark-field mass analysis of ultrathin cryosections in the field-emission scanning transmission electron microscope.
The availability of a cryotransfer stage, highly efficient electron energy loss spectrometers, and ultra-thin-window energy-dispersive x-ray spectrometers for the VG Microscopes HB501 field-emission scanning transmission electron microscope (STEM) provides this instrument with the potential for high resolution biological microanalysis. Recent technical advances offer cryosections that are thin enough to take advantage of the analytical capabilities of this microscope. This paper first discusses the quantitative characterization of freeze-dried, ultrathin cryosections of directly frozen liver and brain by low-dose dark-field STEM imaging. Such images reveal high-quality sections with good structural detail, mainly due to reduced preparation artifacts and electron beam damage. These sections are thin enough for dark-field mass analysis, so that the mass of individual organelles can be measured in situ, and their water content deduced. This permits the measurement of mass loss-corrected subcellular elemental concentrations. The results suggest several new applications for cryosections as illustrated by data on synaptic activity-dependent calcium regulation in Purkinje cells of mouse cerebellum. Low-dose mass analysis of cryosections in combination with x-ray and electron spectroscopy is a promising approach to quantitating physiological changes in mass distribution and elemental composition.
Andrews
S B
SB
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-4062, USA.
Buchanan
R A
RA
Leapman
R D
RD
D000818:Animals; D002531:Cerebellum / Q000648:ultrastructure; D003593:Cytoplasm / Q000648:ultrastructure; D003712:Dendrites / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D005615:Freezing; D006652:Histological Techniques; D008099:Liver / Q000648:ultrastructure*; D051379:Mice; D017348:Microscopy, Electron, Scanning Transmission / Q000295:instrumentation / Q000379:methods*; D008930:Mitochondria, Liver / Q000648:ultrastructure; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure*; D011689:Purkinje Cells / Q000648:ultrastructure*; D014131:Trace Elements / Q000032:analysis
D014131:Trace Elements