REDUCED-CARRIER SINGLE-SIDE-BAND MICROSCOPY - A POWERFUL METHOD FOR THE OBSERVATION OF TRANSPARENT MICROSCOPIC OBJECTS

The theoretical and practical properties of different forms of contrast formation in the microscope based on anaxial illumination are investigated: so-called single-sideband (SSB) techniques. The use of anaxial illumination in transmitted-light microscopy is by itself a form of phase contrast (asymmetric illumination contrast, or AIC), but needs enhancement via a video circuit coupled to the microscope. The addition of a partially absorbing mask, known as a carrier-attenuation filter (CAF), in a proper, conjugate plane in the microscope improves contrast substantially. The imaging properties of this reduced-carrier, single-sideband imaging method (RC-SSB)were tested using the transparent parts of a compact disc (CD); the tracks may be treated as small objects with a controllable phase shift. The results were compared both theoretically and experimentally with Zernike phase contrast and with Nomarski differential interference contrast. The SSB technique has been shown to reveal transparent, submicrometre parts of living, unstained tissue, such as microvilli on sensory receptor cells of the transparent catfish, Kryptopterus, The high resolving power, together with the variable spatial-frequency contrast enhancement, makes it a powerful technique for the imaging of in-vivo subcellular details.