Oblique Illumination Light Pathways

Oblique Illumination Light Pathways - Java Tutorial

Achieving conditions necessary for oblique illumination can be accomplished by a variety of techniques with a simple transmitted light optical microscope. The easiest methods are to offset a partially closed condenser iris diaphragm, insert an opaque sector stop near the condenser aperture, or de-center the image of the light source. Regardless of the mechanism utilized to establish oblique illumination, the conditions required for image formation remain the same.

This interactive tutorial explores changes in microscope light paths and demonstrates events at the objective rear focal plane as illumination progresses from axial to highly oblique. The tutorial initializes with a schematic microscope diagram in the window that is configured for axial brightfield illumination. Above the microscope diagram is a view (black circle) of the virtual objective rear focal plane illustrating the zeroth-order (undiffracted light; represented by a yellow dot labeled 0 in the tutorial) light passing through the specimen, and two first-order diffracted sidebands (-1 and +1). To offset the illumination source from the microscope optical axis, use the mouse cursor to translate the Illumination Angle slider from left to right. In the far left-hand position, the virtual microscope exists in brightfield (axial) mode with illumination ray traces originating at the condenser front focal plane and centered on the microscope optical axis. The specimen is assumed to be a diffraction grating with a periodic structure that is very close to the limit of optical resolution of the microscope objective.

As the Illumination Angle slider is slowly translated to the right, the origin of ray traces through the microscope shifts to the left, demonstrating new pathways for oblique illumination through the microscope. Simultaneously, the diffraction spots in the virtual objective rear focal plane shift until the -1 sideband drops out completely, and oblique illumination is established. The incident angle of oblique illumination is indicated above the slider and achieves a maximum value of 72 degrees, the approximate one-half angular aperture (numerical aperture of 0.95) of dry microscope objectives. The upper value of this angle represents the limit of resolution available with the oblique illumination technique.

When the azimuth of illumination is shifted to the left in the condenser front focal plane, the images visible in the objective rear focal plane also undergo lateral displacements until one of the first-order maxima (-1 reaches an oblique angle sufficient to no longer be collected by the objective. The other maximum (+1) of the same order proceeds at a lower angle of inclination and can still enter the objective front lens, in addition to the zeroth order maximum (0). Because two adjacent maxima are collected by the objective when the specimen is illuminated obliquely, they can undergo constructive and destructive interference at the intermediate image plane to form a relatively high-contrast image.

Oblique illumination is capable of resolving very fine specimen detail that is difficult to distinguish using conventional brightfield techniques. When oblique light rays strike the objective front lens at angle "ob" to the microscope optical axis, and the refractive index of the medium between the condenser front lens and the objective is n, the relationship between resolution, illuminating wavelength (λ), and refractive index can be described as:

Resolution (D) = λ/(n • sin (ob) + Numerical Aperture (NA))

Some authors equate the term n • sin (ob) with the numerical aperture of illumination, but this is misleading because the equation is not intended to signify a full cone of light emanating from all azimuths, but is restricted to a beam of light produced by anaxial illumination from a single azimuth. In cases where specimen detail is so fine that the zeroth order undiffracted and first order diffracted sideband light are separated by a distance equal to the diameter of the objective aperture stop, the resolving power is twice as high as observed for axial transmission illumination and is expressed by the equation:

Resolution (D) = λ/2NA

The optical conditions required for the microscope to conform with the equation above (when utilizing oblique illumination) are such that the fine details of specimen periodicity are limited by the resolving power of the objective. In many cases, specimen details that are resolved utilizing brightfield illumination are so severely lacking in contrast that they can scarcely be visualized or imaged. The net result of observing specimens with oblique illumination is often an increase in resolution (over that obtained in brightfield illumination with a closed condenser aperture diaphragm) and also the production of a shadowed, relief-like pseudo three-dimensional appearance in the image of the specimen.