Resolving Low-Contrast Microstructure Using Transmitted and Reflected Circular Oblique Lighting (COL)

Abstract

A little-known illumination method for light microscopy goes by several names, the most prominent being “circular oblique lighting” (COL) and “hollow-cone illumination”. Matthews notes that hollow-cone or annular bright field illumination can give contrast and resolution superior to that obtainable with narrow-pencil illumination and under favorable conditions comparable to that obtained with phase optics. He demonstrates this with photomicrographs of the same unstained epithelial cell from the mouth mounted in saliva, imaged with a 0.65 numerical aperture (NA) 40× objective. Matthews also notes that the dot pattern of Pleurosigmaangulatum can be resolved with a 0.50 NA objective using circular oblique lighting. Leitz previously marketed the Heine illuminator for transmitted annular (hollow cone) illumination. The NA of the Heine condenser's annular illumination can be adjusted to match the phase annuli in phase contrast objectives. The NA can be increased to provide dark field illumination or circular oblique illumination in bright field. The instructions for the Heine condenser call for the annular illumination just falling within the NA of the objective, what Paul James calls COL and Frithjof A. S. Sterrenberg calls extreme annular illumination, “bright field with very rich contrast.” H. J. Dethloff published a more recent article describing the need for the increased contrast of hollow cone bright field to help resolve the striae of pores in the diatom Amplipleurapellucida. This diatom has been the traditional test of the resolution limit of the light microscope; it is considered a low-contrast subject because the visibility of pores in the transparent amorphous silica frustules is determined by the refractive index difference between the mountant and the frustules. The low contrast makes this a challenging, perhaps even unsuitable, test object for resolution. Resolution tests of modern objectives are done with high-contrast but costly patterns of chrome on glass obtained by electron lithography.