Calcium Quantitation with a Parallel Electron Energy Loss Spectroscopy/Cooled Charge-Coupled Device/200 keV System Ruoya Ho, Jianglin Feng, Zhifeng Shao, and Andrew P. Somlyo* Department of Molecular Physiology and Biological Physics, University of Virginia, HSC, Box 449, Charlottesville, VA 22908 Abstract: 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 quanti- tation. 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 process- ing 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. Key words: electron energy loss spectroscopy (EELS), Ca quantitation, internal reflection, precise correlation, radiation damage–induced spectral changes I NTRODUCTION The large collection efficiency of electron spectrometers for inelastically scattered electrons recommends electron en- ergy-loss spectroscopy (EELS) as a valuable method for compositional imaging with high detection sensitivity and at high spatial resolution (Isaacson and Johnson, 1975). Given the importance of calcium in biological signal trans- duction, its detection and imaging has become a high- priority objective for analytical electron microscopy in bi- ology and it has been verified experimentally that EELS can detect low concentrations of Ca with a 3- to 5-fold higher sensitivity than that attainable with available X-ray detec- tors used for electron probe microanalysis (Shuman and Somlyo, 1987; Leapman et al., 1993; Leapman and New- bury, 1993). Detection of Ca by EELS has been optimized through the use of parallel detectors and implementation of multiple least-squares fitting to extract the characteristic Ca L-edge signal from the background (Shuman and Somlyo, 1987; Leapman and Swyt, 1988; Leapman and Andrews, 1991; Leapman and Hunt, 1991; Wang et al., 1992; Leap- man et al., 1993; Leapman and Newbury, 1993; Door and Ga¨ngler, 1995; Egerton, 1996), while computer modeling Received December 17, 1998; accepted January 19, 1999. *Corresponding author Microsc. Microanal. 5, 17–28, 1999 MicroscopyAND Microanalysis © MICROSCOPY SOCIETY OF AMERICA 1999