We evaluated factors affected the accuracy and precision of quantitating trace concentration of Ca with electron energy loss spectroscopy (EELS). These factors include internal
reflection in the spectrometer, precision of correlation between
standards and experimental spectra, and radiation damage-induced
spectral changes. We present methods of correcting for these effects
and improving the reliability of trace Ca quantitation. A two-step
fitting procedure is described that improves the retrieval of small Ca
signals from the large background common to biological specimens. After
optimizing the experimental conditions and data processing procedures,
our current system can detect about 2.2 mmol/kg Ca in a 730-Å thick
specimen at a total dose of about 410 nA . sec at 95%
confidence level by fitting the first difference spectra. Because of
the 0.1% residual gain variation after gain normalization, the
first difference spectrum fitting is still the preferred method for
trace Ca quantitation. Our study also demonstrates the clear advantage
of using a 200 keV system, instead of 120 keV or lower accelerating
voltages, for EELS analysis of relatively thick biological
cryosections.