This foundational knowledge article explores the effects of the numerical aperture (NA) of an objective lens on the resolution of images produced by a microscope. It explains the diffraction pattern produced by an objective lens and how increasing the NA results in higher resolution images. The tutorial demonstrates the changes in image structure as the NA is adjusted.

Key Learnings:

• The numerical aperture (NA) of an objective lens affects the resolution of images produced by a microscope.
• Increasing the NA results in higher resolution images.
• The light from a point-like structure of a sample passing through the objective pupil is imaged as a three-dimensional diffraction pattern in the intermediate image. The cross section through this three-dimensional diffraction pattern visible in the intermediate image plane consists of a central disc surrounded by bright and dark higher order diffraction rings which together form the Airy disc
  

The image formed by a perfect, aberration-free objective lens at the intermediate image plane of a microscope is a diffraction pattern with a very specific intensity distribution. This tutorial explores the effects of the objective´s numerical aperture (NA) on the diffraction pattern and the resolution of a microscope. The three-dimensional representation of the diffraction pattern is the Point-Spread-Function (PSF) which, in a coma- and/or astigmatism-free system, is symmetrically periodic both along the optical axis, and radially across the image plane. This diffraction pattern can be sectioned in the focal plane to produce a two-dimensional diffraction pattern, having a bright circular disk surrounded by an alternating series of bright and dark higher-order diffraction rings whose intensity decreases with distance from the central disk, the so-called Airy disk. Under visual microscopical observation, only two or three of the circular luminous rings are usually visible in the intermediate image plane.

The tutorial starts with a pattern of Airy disks appearing in the focal plane of the microscope and the point-spread function / three dimensional of a corresponding, single Airy disk pattern shown on the right. To operate the tutorial, use the Numerical Aperture slider to change the objective´s numerical aperture and the resolution of the Airy patterns. The left position of the slider shows the pattern at the lowest objective numerical aperture (= 0.20), and the right position illustrates the highest degree of resolution (numerical aperture = 1.30). As the slider is moved from left to right, the objective’s numerical aperture increases and the complex Airy pattern, as visible in the image, results in a progressively increased resolution of image detail. Correspondingly, the central peak and higher-order diffraction rings in the three-dimensional Airy pattern drawing grow smaller in diameter.

The resolving power of an objective determines the size of the formed Airy diffraction pattern: The radius of the central disk is determined by the combined numerical apertures of the objective and condenser. When condenser and objective have equivalent numerical apertures or the objective acts also as the condenser like in an inverted fluorescence microscope, the Airy pattern radius from the central peak to the first minimum is given by the equation:

r(Airy) = 1.22λ/2NA(Obj)(1)

r(Airy) is the Airy radius, λ is the wavelength of the illuminating light, and NA(Obj) is the objective´s numerical aperture (objective aperture = condenser aperture). The numerical aperture depends on the aperture angle of the illumination entering the objective aperture, as well as the refractive index of the imaging medium:

NA(Obj) = n(sin(θ))(2)

θ is the objective’s angular aperture and n is the refractive index of the medium (air, water, or oil) between the objective and the specimen.

The image resolution (D) is defined by this equation and hence corresponds to the Airy radius:

D = 0.61λ/NA(3)

Resolution is clearly influenced by the objective’s numerical aperture. Note that lower values of D indicate higher resolution. In the tutorial, the Numerical Aperture slider is used to control how the image structure evolves as the objective’s numerical aperture is increased. At the lowest numerical aperture value (0.20), the image details visible in the microscope are poorly defined and surrounded by diffraction fringes. As the slider is moved to higher numerical aperture values (0.50-0.80), the structural outline of the image becomes sharper and higher-order diffraction rings begin to emerge. At the highest numerical apertures (1.00-1.30), the diffraction disks are resolved individually as discrete luminous points surrounded by alternating series of bright and dark higher-order diffraction rings of decreasing intensity.