55. Light microscopy

Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally.

Limitations of standard optical microscopy (bright field microscopy) lie in three areas;

1. The technique can only image dark or strongly refracting objects effectively.

2. Diffraction limits resolution to approximately 0.2 micrometres.

3. Out of focus light from points outside the focal plane reduces image clarity.

Types of light microscopy:bright field, dark field, phase contrast, differential interference contrast, Nomarski contrast, Hoffman modulation contrast, fluorescence, confocal

56. 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. We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections.

57. Dark field

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.

58. Сравнительная характеристика светлого и темного поля

For bright field microscopy We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation.

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. 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. Instead of coming up through the specimen, the light is reflected by particles on the slide.