Measurement of low calcium concentrations in cryosectioned cells by parallel-EELS mapping.
Electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope provides a high sensitivity for microanalysis of certain important biological elements such as calcium whose physiological concentrations in cells are rather low. Application of parallel-EELS mapping to the analysis of freeze-dried cryosections of rapidly frozen tissue provides a means of detecting small amounts of calcium in structures with diameter approximately 50 nm. Detector pattern noise due to channel gain variations can be reduced by acquiring difference spectra at each pixel. By segmenting nitrogen maps that reflect the structure through the protein distribution it is possible to sum spectra from specific compartments. These are then processed by fitting reference spectra for the Ca L23-edge and the carbon background. It has been found that useful data can be collected at 100 keV beam energy from freeze-dried cryosections of cerebellar cortex cut to nominal thickness of 100 nm. The analysis results in a sensitivity of +/- 0.4 mmol Ca/kg dry weight with a total acquisition time of 400 s, a significant improvement over that achievable with energy-dispersive X-ray spectroscopy.
Leapman
R D
RD
Biomedical Engineering and Instrumentation Program, NCRR, National Institutes of Health, Bethesda, MD 20892.
Hunt
J A
JA
Buchanan
R A
RA
Andrews
S B
SB
D000818:Animals; D002118:Calcium / Q000032:analysis*; D002525:Cerebellar Cortex / Q000737:chemistry* / Q000166:cytology; D015925:Cryopreservation; D007091:Image Processing, Computer-Assisted; D051379:Mice; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods*
D002118:Calcium