Video-enhanced microscopy with a computer frame memory.

Abstract

Video-enhanced microscopy combined with the use of a computer frame memory extends considerably the useful range of our video enhanced contrast (AVEC) methods for polarizing, double-beam interference and differential interference contrast microscopy. Increased visual contrast is achieved by two stages of amplifications: the first optical, by using high bias retardation settings, and the second electronic. These steps are followed by a reduction of background brightness by means of a clamp voltage applied to a DC restoration circuit of the video camera. One of the limitations of the AVEC method alone is the inevitable appearance under high gain conditions of a pattern of mottle due to inaccessible dirt and defects in the lenses even of high quality. This limitation has been circumvented by storing the mottle pattern in the frame memory (frame store) and continuously subtracting it from each succeeding frame to clear the image. A major gain in image quality has resulted. In polarizing microscopy, the frame memory can be used also to subtract the image at one compensator setting from that at the equivalent setting of opposite sign, thus removing from the final image not only most of the mottle pattern but also the contrast due to the bright-field contrast. In the polarizing microscope, these manipulations of the raw video image make it possible to observe and measure the birefringence of various organelles and elements such as microtubules, intermediate filaments and bundles of as few as a half dozen actin filaments. Since scattered light is also removed from the image, features hidden from view in the unprocessed image become visible. In differential interference microscopy, the AVEC method makes visible (i.e. detectable) many linear elements and particles that are an order of magnitude smaller than the resolution limit and not visible in the optical image. Such features are inflated by diffraction, however, to Airy disk size.