Visualization of subsurface structures in cells and tissues by backscattered electron imaging.

A fraction of the beam electrons which interact with a specimen scatter back. The number of backscattered electrons (BE's) increases with the atomic number of the elements encountered. Cell and tissue structures lacking a heavy metal content yield few BE's compared to structures affixed with heavy metals, either vitally or by means of staining methods applied after fixation. The BE imaging mode of a scanning electron microscope (SEM) provides an intensity map of the BE yield from the specimen. BE imaging of selectively stained structures in cells and tissues renders these structures visible in contrast to the unstained surround. Since BE's can emerge from a significant depth within the material, BE imaging can be used to view such heavy metal stained structures beneath intact cell surfaces. The microcontours of the overlying surface can be viewed concurrently by using the surface scanning (i.e., the secondary electron imaging; SEI) mode of the microscope. Methods for selectively contrasting subsurface structures can be adapted from existing light microscope (LM) and transmission electron microscope (TEM) methods. Staining methods have been devised for subsurface viewing of cell organelles, including nuclei, mitochondria, peroxisomes, lysosomes, and phagosomes. A physical model is presented which describes these observations and suggests future possible trends in this subject. Specifically the image contrast and resolution are described in terms of the physical properties of the stain and specimen and of the SEM operating conditions of energy and current. Finally a summary of instrumentation considerations describes present and potential BE detectors, their ancillary electronics, and image processing.