Optical Microscopy

Bright Field Microscopy

Variation in the light intensity (contrast) in the image occurs because different parts of the specimen absorb or reflect light to differing degrees.

• 2% contrast is needed by the human eye to distinguish differences between the image and its background.
• This may well be different for an image sensor, e.g., digital camera.

This contrasting mechanism is known as “amplitude” contrasting.

$$R=\frac{(n-1{)}^2+k^2}{(n+1{)}^2+k^2}$$

$R$ – the reflectance – is the ratio of the intensities of the incident and reflected rays;

In the case of strongly absorbing materials, e.g., metals, the absorption coefficient $k$ dominates, and $R$ approaches unity – i.e. metals are strong reflectors, approaching 100% for aluminum and silver. For these metals $R$ is constant over the whole spectral range, and, for example, aluminum and silver appear white.

Phase-contrast Microscopy

Normanski Differential-Interference-Contract (DIC) Microscopy

Nomarski microscopy, is an optical microscopy technique used to enhance the contrast in unstained, transparent samples.

It works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features.

The illumination is split into two coherent beams, which are then altered in phase with respect to one another after interaction with the specimen.

The image is formed when the two beams interfere upon recombination at the primary image plane. The differential interference contrast microscope converts changes in optical path $(n\Delta z)$ difference occurring in an invisible ‘phase specimen’ into visible amplitude changes.

Dark Field Microscopy

This has much better contrast than Bright-Field, it uses a very bright light behind the sample and catches the light that diffracts around. This bright light can however damage biological samples

A key goal of dark-field imaging is to prevent the specularly- reflected illumination – which carries no information about the structural detail – from entering the objective. As in transmitted- light microscopy, only the diffracted information-carrying light contributes to the image.