Question

Select a contrast mode in microscopy. In the space below, describe and illustrate it in detail (hint: phase, dark-field, bright-field)
Please answer in detail.(2 page limit)

Answer 1

1)

Phase contrast microscopy was first introduced in the 1934. It is the contract enhancinh optical technique which is used to for the production of high contrast images of transparent specimens like living cells in the culture.

In effect, the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. One of the major advantages of phase contrast microscopy is that living cells can be examined in their natural state without previously being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail.

2)

Bright-field microscopy is one of the simplest optical microscopy. In bright-field microscopy, illumination light is transmitted through the sample and the contrast is generated by the absorption of light in dense areas of the specimen. The limitations of bright-field microscopy include low contrast for weakly absorbing samples and low resolution due to the blurry appearance of out-of-focus material.

Colloidal gold nanoparticles can serve as labels in bright-field microscopy due to their large absorption and scattering cross sections. When applied in live cells, they usually appear as dark spots on a blurred, bright image of the host cell.

In the 1980s, with the development of video-enhancement techniques, small gold nanoparticles (20–40 nm) became visible and can be imaged at video rates (∼ 30 Hz) through digital enhancement of the image contrast (Saxton and Jacobson, 1997). This advancement resulted in a wave of live-cell single particle tracking (SPT) studies of cellular activities including receptor-mediated endocytosis, motion of cell surface molecules, intracellular transport, etc. Bright-field microscopy is still being used frequently today, despite its high background level.

3)

In darkfield microscopy the condenser is designed to form a hollow cone of light (see illustration below), as opposed to brightfield microscopy that illuminates the sample with a full cone of light. In darkfield microscopy, the objective lens sits in the dark hollow of this cone and light travels around the objective lens, but does not enter the cone shaped area. The entire field of view appears dark when there is no sample on the microscope stage. However, when a sample is placed on the stage it appears bright against a dark background. It is similar to back-lighting an object that may be the same color as the background it sits against - in order to make it stand out.

Darkfield Microscope Applications

1. Viewing blood cells (biological darkfield microscope, combined with phase contrast)
2. Viewing bacteria (biological darkfield microscope, often combined with phase contrast)
3. Viewing different types of algae (biological darkfield microscope)
4. Viewing hairline metal fractures (metallurgical darkfield microscope)
5. Viewing diamonds and other precious stones (gemological microscope or stereo darkfield microscope)
6. Viewing shrimp or other invertebrates (stereo darkfield microscope)