04 the microscope
- 2. Types of Compound Light Microscopy • A brightfield microscope uses visible light as a source of illumination. – The specimen appears against a bright background. – The main purpose is to observe various stained specimens. • A stereoscope or dissecting microscope has two objective lenses that allow three-dimensional view. – Because specimens are usually viewed in a Petri dish rather than on a slide, manipulation of the specimen is possible. • A darkfield microscope uses a special condenser with an opaque disc that blocks light from entering the objective lens directly. – The specimen appears light against a black background. – The main purpose is to observe specimens that are invisible in brightfield microscopy, do not stain easily, or are distorted by staining.
- 3. Types of Compound Light Microscopy • A fluorescence microscope reveals transparent specimens when treated with special fluorescent dyes known as fluorochromes and illuminated with ultraviolet light. – The principal use is immunofluorescence to detect the locations of certain proteins in specimens. • A phase-contrast microscope uses a special condenser containing an annular diaphragm to convert normally invisible differences in the optical path of light into visible light-dark differences, which facilitates detailed examination of the internal structures of living specimens. – No staining is required. – The objective lens have a diffraction plate. – Both direct light and diffracted light are brought together to produce the image.
- 4. Types of Compound Light Microscopy • A differential interference contrast microscope uses two beams of light separated by prisms to produce colored three-dimensional images without staining. • A confocal microscope uses laser light to illuminate one plane of a specimen at a time. – The microscope also uses fluorochromes. – The principal use is to obtain two- and three- dimensional images with the help of computer software.
- 5. Preparation of a Slide for Compound Light Microscope 1. The specimen is preserved in a fluid known as fixative using a process known as fixation. 2. The specimen is dehydrated using alcohols of progressively higher concentrations. 3. The specimen is embedded in paraffin wax and cut into extremely thin slices by a microtome. 4. The specimen is cleared and mounted in a transparent medium between a slide and cover glass. 5. The specimen is immersed in colorful stains and dyes to differentiate the components.
- 6. Technique: Immunofluorescence • Antibodies are molecules produced by the human body and many animals in response to a foreign substance known as antigen for defense. 1. After mounting the specimen onto a slide, the specimen is blocked to prevent antibodies from sticking to locations where the protein of interest is not found. 2. The specimen is treated with a primary antibody to locate the protein of interest. 3. The specimen is washed to remove any excess primary antibody. 4. The specimen is treated with a secondary antibody attached to a fluorochrome that will detect the primary antibody. 5. The specimen is washed to remove any excess secondary antibody. 6. The specimen is viewed under a fluorescence microscope.
- 7. Electron Microscopy • An electron microscope uses electron beams reflected on electromagnetic lenses to magnify objects up to 100,000 times. 1. A scanning electron microscope (SEM) is used to produce detailed pictures of the specimen’s surface features. – The electron beam scans the surface of the sample. – The dislodged electrons from the specimen enter a detector, producing the image. 2. A transmission electron microscope (TEM) gives similar image as in compound microscope, but can reach magnifications up to 100,000 times.
- 8. Preparation of a Slide for Transmission Electron Microscopy 1. The specimen is fixed with a primary fixative. 2. The specimen is rinsed in appropriate buffer. 3. The specimen is fixed again with osmium tetroxide. 4. The specimen is rinsed to remove osmium. 5. The specimen is dehydrated by alcohols of progressively higher concentration. 6. The specimen is embedded in plastic resin.
- 9. Preparation of a Slide for Transmission Electron Microscopy 7. The resin is polymerized to the specimen by heat, catalysts, or UV light. 8. The specimen is cut into very thin slices by an ultramicrotome. 9. The specimen is cleared and mounted in a transparent medium between a slide and cover glass. 10. The specimen is immersed in heavy metal stains to differentiate the components.
- 10. Scanned-Probe Microscopy • A scanned-probe microscope uses probes to examine the surface of a specimen. • No special preparation required. • Resolving power is much greater than that of an electron microscope. 1. A scanning tunneling microscope uses a thin metal probe that scans a specimen and produces an image revealing the bumps and depressions of the atoms on the surface of the specimen. – The principal use is to provide very detailed views of molecules inside cells. 2. An atomic force microscope uses a metal-and-diamond probe gently forced down along the surface of the specimen. – The image produced is three-dimensional. – The principal use is to provide images of molecular processes and biological molecules in nearly atomic detail.
- 11. Types of Cells • Prokaryotic cells – Mainly bacterial cells – Lack a nucleus and instead contain a concentrated region of DNA (the nucleoid region) – Ribosomes and vacuoles are the only organelles present – Contain cell wall • Eukaryotic cells – Include plant cells, animal cells, fungal cells, and protist cells – Contain a nucleus – All organelles present – Only plant cells and fungal cells contain cell wall
- 12. Parts of a Cell • Cytosome or cytoplasm is a jelly-like fluid that is enclosed by the plasma membrane. • Organelles are organ-like structures found in the cytoplasm capable of performing specific functions. • Inclusions are byproducts of cell activity. 1. Plasma membrane – Composed of a fluid lipid bilayer made of phospholipids that serves as a selective barrier – Contains proteins that are either embedded in or attached to the lipid bilayer