The Electron Microscope

With an ordinary light microscope, objects down to approximately a micron (a millionth of a meter) in size can be seen. Typical bacteria are a micron or two long by about half a micron wide. Although bacteria are visible under a light microscope, their internal details are too small to see. The resolving power of a microscope depends on the wavelength of the light. In a light microscope, if two dots are less than about half a wavelength apart, they cannot be distinguished. Visible light has wavelengths in the range of 0.3 (blue) to 1.0 (red) micron, so bacteria are just at the limits of visible detection and most viruses cannot be seen.

A beam of electrons has a much smaller wavelength than visible light and so can distinguish detail far beyond the limits of resolution by light. Electron beams may be focused like visible light except that the lenses used for electron beams are not physical (glass absorbs electrons) but electromagnetic fields that alter the direction in which the electrons move. Using an electron microscope allows visualization of the layers of the bacterial cell wall and of the folded-up bacterial chromosome, which appears as a light patch against a dark background. When an electron beam is fired through a sample, materials that absorb electrons more efficiently appear darker. Because electrons are easily absorbed, even by air, an electron beam must be used inside a vacuum chamber and the sample must be sliced extremely thin (Fig. 21.26).

To improve contrast, cell components are usually stained with compounds of heavy metals such as uranium, osmium or lead, all of which strongly absorb electrons.

alkaline phosphatase An enzyme that cleaves phosphate groups from a wide range of molecules chemiluminescence Production of light by a chemical reaction chromogenic substrate Substrate that yields a colored product when processed by an enzyme lumi-phos Substrate for alkaline phosphatase that releases light upon cleavage X-phos Substrate for alkaline phosphatase that is cleaved to release a blue dye

FIGURE 21.24 Labeling DNA with Biotin

Uracil can be incorporated into a strand of DNA if the nucleotide has a deoxyribose sugar. Prior to incorporation, the uracil is tagged with a biotin molecule attached via a linker. This mode of attachment allows the biotin to stick out from the DNA helix without disrupting its structure.

Colored or luminescent products may be released when enzymes split specially designed artificial substrates.

Bacteria are just visible under a light microscope.

Electron microscopes allow viruses, subcellular components and even single macromolecules to be visualized.

The Electron Microscope 589

FIGURE 21.25 Detection Systems for Biotin

DNA that has biotin attached via uracil can be detected with a two-step process. First, avidin is bound to the biotin. The avidin is conjugated to an enzyme called alkaline phosphatase, which cleaves phosphate groups from various substrates. Second, a substrate such as X-phos (shown) or lumi-phos (not shown) is added. Alkaline phosphatase removes the phosphate group from either substrate. In the case of X-phos, cleavage releases a precursor that reacts with oxygen to form a blue dye. If the substrate is lumi-phos, cleavage allows the unstable luminescent group to emit light.

FIGURE 21.26 Principle of the Electron Microscope

Electron microscopy can reveal the substructures of animal cells, viruses, and bacteria. A beam of electrons is emitted from a source and is focused on the sample using electromagnetic lenses. When the electrons hit the sample, components such as cell walls, membranes, etc, absorb electrons and appear dark. The image is viewed on a screen or may be transferred to film for a permanent record. Since air molecules also absorb electrons, the entire process must be done in a vacuum chamber.

Vacuum chamber

Sample

Screen

Vacuum chamber

Sample

Screen

Electron source

Beam of electrons

Electromagnetic lenses

Electron source

Beam of electrons

Electromagnetic lenses

Individual, uncoiled DNA molecules can be seen if they are shadowed with metal atoms to increase electron absorption (Fig. 21.27). Shadowing is done by spreading the DNA out on a grid and then rotating it in front of a hot metal filament. Metal atoms evaporate and cover the DNA. Gold, platinum, or tungsten are typically used for shadowing.

Replicating plasmid DNA, showing the replication forks has been visualized under the electron microscope, as have a variety of other DNA and RNA molecules. A more recent example of this approach was the direct visualization of the introns found in eukaryotic genes (see Ch. 12). The messenger RNA and the DNA both contain the exons that comprise the coding sequence, but the final mRNA lacks the introns (non-coding regions). If mRNA is hybridized to single stranded DNA from the corresponding gene, the results are regions of base pairing (the exons) interrupted by loops due

FIGURE 21.27 Metal Shadowed DNA Molecules are Visible under an Electron Microscope

A hot metal filament releases vaporized metal atoms into the chamber containing a sample of DNA. The sample is rotated around the filament and metal ions attach to the exposed surface of the DNA. Once the DNA has a coat of metal atoms, it can be visualized by electron microscopy.

Vaporized metal atoms

Vaporized metal atoms

FIGURE 21.27 Metal Shadowed DNA Molecules are Visible under an Electron Microscope

A hot metal filament releases vaporized metal atoms into the chamber containing a sample of DNA. The sample is rotated around the filament and metal ions attach to the exposed surface of the DNA. Once the DNA has a coat of metal atoms, it can be visualized by electron microscopy.

DNA molecules to be shadowed

ds DNA

Exon Intron Exon Intron Exon

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