Spherical Aberration | Spherical Aberration

Key Words: confocal, N.A., oil immersion, resolution, water immersion, focus, Chromatic aberration axial, chromatic aberration lateral, correction collar

Definition:Spherical aberration occurs when light passing through the periphery of the lens is not brought into focus at the same plane as that travelling through the centre of the lens

TECHNOLOGY:

When light passes through a lens, rays passing near to the center of the lens are refracted to a lesser degree than waves passing through the periphery of the lens. This results in the production of different focal points along the optical axis and results in loss of resolution. Spherical aberrations increase with numerical aperture. Spherical aberration can seriously compromise the sharpness and clarity of an image and a number of solutions have been devised including the use of diaphragms to reduce the outer limits of the lens, aspherical lens surfaces, specialized lens-grinding methods, new glass formulations and methods to restrict or control the optical path. Spherical aberration can be a particular nuisance in confocal microscopy because as a confocal pinhole rejects out-of-focus light, it also eliminates out-of-focus fluorescence due to spherical aberration.

Spherical aberration is not solely a function of lens design but is influenced by the media through which light travels. Spherical aberration is minimized when the entire light path has the same refractive index and increases with distance into a medium with a different refractive index. In microscopy, light usually travels though water (the specimen) and glass (the coverslip). Oil immersion objectives were historically considered the best way of correcting spherical aberration because oil has the same refractive index as glass. However, aberration still occurs with distance into the aqueous specimen. Water immersion objectives are matched in the refractive index of the immersion medium and the specimen and thus making spherical aberration independent of imaging depth. This enables imaging to the limit of the objective's working distance (frequently hundreds of micrometers into a sample) (see figure 1).

APPLICATIONS:

Spherical aberration can be a particular problem in confocal imaging. The design of high numerical aperture, Plan Apochromat water immersion objectives has significantly helped confocal microscopy realize its potential in three-dimensional biological imaging.

Comparison of oil immersion 100x Plan Apochromat objective used to collect an image of cells labeled with F-Tf and mounted at a depth of either zero micrometers (a) or 35 micrometers into an aqueous medium (b) and a water immersion 60x Plan Apochromat objective collected either zero (c) or 66 micrometers into an aqueous medium (d). In (d) the fluorescent signal is unaffected by the light path through the aqueous sample medium.
IMAGE: Courtesy of MicroscopyU

MICROSCOPE CONFIGURATION:

Nikon supplies a number of oil immersion, water immersion and multi-immersion objectives (water/glycerin/ oil). The CFI Apochromat TIRF oil immersion objectives provide the highest N.A. of all Nikon objectives and have been designed specifically for TIRF applications (CFI Apochromat TIRF 60x oil, N.A. 1.49; CFI Apochromat TIRF 100x oil, N.A. 1.49).

The TIRF Apochromat 60x and 100x NA 1.49 oil objectives incorporate a correction ring to compensate for spherical aberrations caused by temperature, distance into the mounting medium and other refractive index differences. Nikon's CFI Plan Apochromat VC (violet corrected) 60x oil (N.A. 1.40) and 100x oil (N.A. 1.40) are the deal choice for multi-stained, fluorescence confocal applications.

The Plan 100xW objective (NA 1.1, W.D. 2.5mm) is the first water dipping lens with a correction ring. This ring corrects spherical aberration induced by imaging deep in tissue or by working at physiological temperatures - providing outstanding z-axis resolution in IR-DIC imaging, as well as a tight point spread function for confocal applications. With excellent IR transmission, this lens is the choice for multiphoton imaging.

RECOMMENDED SYSTEM:

Please consult you local Nikon representative for advice suited to your imaging applications.

LINKS:

Optical aberrations and objective choice in multicolour confocal microscopy

Introduction to microscope objectives