Measured and computed point spread functions for an indirect water immersion objective used in three-dimensional fluorescence microscopy

Living cells and other non-embedded biological specimens present a challenge in high- resolution microscopy because of their relatively low refractive index. It is well known that when oil-immersion objectives are used to look into these specimens, spherical aberration resulting from the index mismatch causes axial as well as transverse broadening of the 3D point spread function. Both effects reduce 3D resolution significantly, and cause the microscope to be axially shift-variant, thus complicating 3D deconvolution-based computational refinement. Axial shift variance should be much less a problem when water immersion objectives are used. With the aim of improving image data and our ability to refine it computationally, we have evaluated an apochromatic 40X/1.2 NA indirect water- immersion objective having an adjustable corrector for coverslip thickness. This work involves comparison of measured and computed 3D point spread functions, and comparison to the data acquired with an oil-immersion lens system. We show that focus-dependent spherical aberration is greatly reduced in the water-immersion system.