60. Methods that can be used for unstained (transparent) specimen observation.

Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample.

Principle. To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye. Everything is visible regardless of color, usually bright white against a dark background. Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens.

Phase contrast microscopy 

The phase shifts themselves are invisible to the human eye, but become visible when they are shown as brightness changes.

Phase contrast microscopy is particularly important in biology, as it reveals many biological structures that are not visible with a simpler bright field microscope. These structures were often made visible to earlier microscopists by staining the slide. This requires additional preparation and it also kills the cell. Phase contrast microscopy of live cells without staining allowed for the in vivo study of the cell cycle.

When light travels through a medium other than vacuum, interaction with this medium causes itsamplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude arise from absorption of light, which is often wavelength dependent and may give rise to colours. The human eye measures only the energy of light arriving on the retina, so changes in phase are not easily observed under optimal bright field illumination, yet often these changes in phase carry much important information.

63. Comparison of fluorescence and bright field microscopes construction.

Schematic of a fluorescence microscope.

Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused to the detector by the same objective that is used for the excitation which for greatest sensitivity will have a very high numerical aperture. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence method therefore gives a high signal to noise ratio. An additional barrier filter between the objective and the detector can filter out the remaining excitation light from fluorescent light.

BRIGHT FIELD MICROSCOPY

With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece.

A good quality microscope has a built-in illuminator, adjustable condenser with aperture diaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification.