Applications of energy filtered imaging in biology

We describe the use of a magnetic sector spectrometer positioned below the projection chamber of an electron microscope for energy filtered transmission imaging. The spectrometer used has circular pole face edges and is corrected for second order aberrations. A round EM lens is placed after the sector to form a real image of the virtual achromatic image produced by the spectrometer. A slit placed in the dispersion plane allows the passage of electrons in a selected energy range. The filtered image is projected onto a transmission phosphor and acquired with a silicon intensified TV camera and stored in digital form on computer disk. Filtered images are taken at two energies, one immediately preceding (pre-edge) and one on the characteristic energy loss (edge). To obtain images showing the distribution of elements, background subtraction is performed by either subtraction or division of edge and pre-edge images. The optical properties of the imaging system are described and the results are illustrated by energy filtered images of single ferritin molecules (Fe M2,3 and C k), the phosphorus distribution in ribosomes (PL2,3) and the localization of calcium in muscle (Ca L2, 3). The major advantage of the system, compared to other energy filtered imaging methods, is that it can be readily adapted to existing high vacuum microscopes without the necessity of modifying the column to insert a spectrometer.