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.

Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

“…It should be noted that for high NA oil immersion objectives, the PSF approximates the theoretical PSF only very close to the coverslip surface. Spherical aberrations due to the mismatch in the refractive indices of glass and aqueous media rapidly distort the PSF away from the coverslip surface (Gibson and Lanni, 1992) and reduces the overall intensity (see the description later) (Fig. 4A -C).…”

The colored revolution of bioimaging

“…A common and often unavoidable source of aberrations is the imaging depth in situations where the refractive indices of the specimen and immersion layers are mismatched. In the case where this mismatch is significant, it may result in the PSF becoming nonstationary, especially along the axial direction z [9].…”

Estimation Methods of the Point Spread Function Axial Position: A Comparative Computational Study

“…The model developed by Gibson and Lanni [13] provides an accurate way to determine the 3D PSF for a fluorescence microscope. This is based on the calculation of phase aberration (or aberration function) W [1,22,23], from the optical path difference (OPD) between two rays, originating from different conditions.…”

“…From an optimization point of view, SE exhibits convexity, symmetry and differentiability properties, and its solutions usually have analytic closed forms; when not, there exist numerical solutions that are easy to formulate [29]. Kirshner et al [12] developed and evaluated a fitting method based on the SE optimization between data and the Gibson and Lanni model [13]. They used the Levenberg-Marquardt algorithm to solve the non-linear least square problem, obtaining low computation times, as well as accurate and precise approximation to the point source localization.…”