Wednesday, 17 October 2018

Basics of Light Microscopy: Incident Light Microscopy for opaque samples (II) - Dark Field

In the first part of our Incident Light Microscopy post we already explained the double function of the microscope objective in this kind of application field.

Playing an essential part in the imaging process is the task of every microscope objective. The manufacturer pays a lot of attention and spends remarkable efforts in creating a glass hardware for reliable and “close-to-truth” image results.

In incident light applications, the objective is additionally part of the illumination system. The lamp house sends light horizontally through the illumination axis. A semi-transparent mirror with a 45° orientation deflects the light and sends it through the objective. After reflection from the specimen surface, the light brings back sample information to the eyepieces and/or camera.

For the Darkfield method in an Incident light setup, this description is also valid. We just should know one more fact about optical reflection: The angle of Incidence is equivalent to the angle of Reflection.

This law of physics is implemented in the construction of a Darkfield (DF) objective for Incident light. In contrary to DF in Transmitted light, here we need a special objective. Imagine a standard objective for Brightfield, complemented by an illumination ring which encloses the Brightfield (BF) optics. That kind of objective by construction has a larger diameter and therefore requires a different thread. While Motic’s BF objectives do have the traditional RMS thread (W 0,8" × 1/36”; DIN/EN/ISO 8038-1), the DF objectives of AE2000MET and PANTHERA TEC MAT show a metric thread (M32).

This picture of a DF objective displays an inner part, responsible for the imaging process, and an outer ring being part of the illumination system. A side-by-side presentation of PANTHERA TEC MAT and a schematic diagram shows how such an objective is implemented in the illumination system.

The illumination axis of an industrial microscope carries both Field and aperture diaphragm for adjusting the illumination according the demands of the sample. Additionally, in a DF setup a Central Stop is implemented to block all central light, just keeping the periphery (= illumination ring) ready to illuminate the sample.

The lower part of the objective carries a mirror system to send light in a clearly defined angle onto the specimen surface. A perfect specular reflection will bypass the imaging part of the DF objective, following

A realistic metallurgical sample will show (like in Transmitted light DF) border structures abrupt changes in reflection properties.

Source: Wikipedia

Curious and adventurous people may try out the Darkfield illumination with a Brightfield objective. You may use a cold light source with a 2-arm light guide, responsible for the illumination within a compound microscope in an Incident Brightfield setup. The angle of illumination has to be adjusted properly, so that perfectly reflected light from the sample misses the front lens of the BF objective. Take a mirror as a sample to adjust the illumination. An uncommon experiment, but a nice way to understand the principle.

In Inverted microscopes, the described physics are verified in an equivalent way. The advantages of this microscope construction are valid as in Brightfield: We need only one flat surface to start the investigation, and we do enjoy the freedom of an enlarged sample size. As in an Inverted metallurgical microscope mostly the focus mechanism is done by moving the nosepiece with its objectives, samples with reduced weight limitations can be handled.

In comparison to transparent samples, Darkfield for opaque samples is a far more established contrast method. Grain boundaries in complex steel mixtures can be detected easily. Quality control in automotive industries, technical education on compound materials can hardly be effective without Darkfield.

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