Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy

Abstract

Oil-immersion microscope objective lenses have been designed and optimized for the study of thin, two-dimensional object sections that are mounted immediately below the coverslip in a medium that is index matched to the immersion oil. It has been demonstrated both experimentally and through geometrical- and physical-optics theory that, when the microscope is not used with the correct coverslip or immersion oil, when the detector is not located at the optimal plane in image space, or when the object does not satisfy specific conditions, aberration will degrade both the contrast and the resolution of the image. In biology the most severe aberration is introduced when an oil-immersion objective lens is used to study thick specimens, such as living cells and tissues, whose refractive indices are significantly different from that of the immersion oil. We present a model of the three-dimensional imaging properties of a fluorescence light microscope subject to such aberration and compare the imaging properties predicted by the model with those measured experimentally. The model can be used to understand and compensate for aberration introduced to a microscope system under nondesign optical conditions so that both confocal laser scanning microscopy and optical serial sectioning microscopy can be optimized.