Active management of spherical aberration in live cell microscopy

Light microscopy of living cells requires a compromise between conditions that are on the one hand as viable as possible and on the other hand optically well defined. Especially the mismatch of refractive indices of immersion liquid and cell culture medium often creates spherical aberration, that also depends on how deep the cells are embedded in the medium. These effects not only degrade image quality and resolution but also result in a significant signal loss in confocal microscopy. Using objectives with correction collar is an established approach to overcome these problems, that has its own drawbacks. Correction objectives are usually expensive and the internal movement of optical elements can introduce coma. An alternative concept to overcome the spherical aberration is the active use of Herschel’s condition. Microscopy violates Herschel’s condition very severely. This results in spherical aberration if the positions of object and image are shifted along the optical axis. By optimally balancing the shifts of object and image position, both kinds of spherical aberration cancel each other and the image quality becomes perfect.