What is it about?
This article reports and explains the results of measurements of the resolving power for Olympus research microscope equipped with a homemade illumination system. The measurements of the resolution were performed using the calibrated microscopic slide. For certainty, the sizes of test patterns on this slide were tested and confirmed by scanning electron microscopy. The effective resolution was measured as the size of the smallest clearly visible features in microscope images of different shape objects on the test slide. These shapes include line gratings, bar sets, circles, stars, and complex shape patterns shown in the article. Both transparent and opaque fragments of below 90 nm size are clearly resolved in the images observed visually or recorded by video camera. When a color filter was used, the resolution power increases with the decrease of the light wavelength, lambda, and is evaluated to be better than lambda divided by five. Therefore, the documented resolution of a microscope equipped with homemade illuminator is 90 nm. This is above two times better compared to typical 240 nm resolution for research microscopes. Even for modern confocal models, the resolution is limited by 180 nm. The theoretical part of the article is focused on the understanding why and how the homemade illumination system provides the substantial resolution enhancement. The strategy used is straightforward: the same small object, an opaque disk with the radius 50 nanometers, is imaged using the same microscope, but two different illumination systems, homemade or standard bright-field Olympus condenser. In both cases, the same classical diffraction theory of optical images and actual parameters of the two illumination systems are used to calculate the theoretical image. Comparison of theoretical and experimental images shows accurate accord of theory and experiment. Importantly, the accord is obtained without using in calculation any fitting parameters. The theory successfully accounts for the shape of the central spot in the disk’s image as well as for the position and intensity of first two diffraction fringes around. Therefore, the general conclusion is that the enhanced resolution with our homemade is in complete accord with the diffraction theory of light and by no means breaks the limitations on microscope resolution imposed by the wave nature of light. Indeed, the enhanced resolution appears as a result of two features of homemade design compared to the standard circular condenser. First, in the homemade system, the object is illuminated by the focused hollow cone of light. The effect of the annular illumination is the narrowing of the central spot of the diffraction pattern and an increase of the intensity of the diffraction fringes. The similar effect of annular aperture is known and used in telescopes for the resolving power increase. Second, homemade illumination system produces a coherent illumination of small (compared to the wavelength) object and thus the coherent image. Hence, a partial cancellation of the diffraction amplitude occurs when the antiphased subsequent fringes of diffraction are added in the image. This makes the image edge more sharp and thus increases the resolution.
The following have contributed to this page: Professor Vitaly Vodyanoy