1.7: Microscopy Reveals Life's Diversity of Structure and Form

Broadly speaking, there are two main categories of microscopy. In Light Microscopy, the slide is viewed through optical glass lenses that see visible light reflected from or passing through the specimens on the slide. In Electron Microscopy, the viewer is looking at an image on a screen created by electrons passing through or reflected from the specimen, usually mounted on a copper grid. For a sampling of light and electron micrographs, check out this Micrograph Gallery. Here we compare and contrast different microscopic techniques.

1.7.1. Light Microscopy

Historically one or another version of light microscopy has revealed much of what we know of the structural diversity of cells, especially eukaryotic cells. Check out the Mitosis Drawings for a reminder of how eukaryotic cells divide, and then check out The Optical Microscope for descriptions of different variations of light microscopy (e.g., bright-field, dark field, phase-contrast, and fluorescence.). Limits of magnification and resolution of 1200X and 2m, (respectively) are common to all forms of light microscopy. Some variations of light microscopy are briefly described here:

• Bright-Field microscopy is the most common kind of light microscopy, in which the specimen is illuminated from below. Contrast between regions of the specimen comes from the difference between light absorbed by the sample and light passing through it. Live specimens lack contrast in conventional bright-field microscopy because differences in refractive index between components of the specimen (e.g., organelles and cytoplasm in cells) diffuse the resolution of the magnified image. This is why Bright-Field microscopy is best suited to fixed and stained specimens.
• In Dark-field illumination, light passing through the center of the specimen is blocked and the light passing through the periphery of the beam is diffracted (“scattered”) by the sample. The result is enhanced contrast for certain kinds of specimens, including live, unfixed and unstained ones.
• In Polarized light microscopy, light is polarized before passing through the specimen, allowing the investigator to achieve the highest contrast by rotating the plane of polarized light passing through the sample. Samples can be unfixed, unstained or even live.
• Phase-Contrast or Interference microscopy enhances contrast between parts of a specimen with higher refractive indices (e.g., cell organelles) and lower refractive indices (e.g., cytoplasm). Phase–Contrast microscopy optics shift the phase of the light entering the specimen from below by a half a wavelength to capture small differences in refractive index and thereby increase contrast. Phase–Contrast microscopy is a most cost-effective tool for examining live, unfixed and unstained specimens.
• In a fluorescence microscope, short wavelength, high-energy (usually UV) light is passed through a specimen that has been treated with a fluorescing chemical covalently attached to other molecules (e.g., antibodies) that fluoresces when struck by the light source. This fluorescent tag was chosen to recognize and bind specific molecules or structures in a cell. Thus, in fluorescence microscopy, the visible color of fluorescence marks the location of the target molecule/structure in the cell.
• Confocal microscopy is a variant of fluorescence microscopy that enables imaging through thick samples and sections. The result is often 3D-like, with much greater depth of focus than other light microscope methods. Click at Gallery of Confocal Microscopy Images to see a variety of confocal micrographs and related images; look mainly at the specimens.
• Lattice Light-Sheet Microscopy is a 100 year old variant of light microscopy that allows us to follow subcellular structures and macromolecules moving about in living cells. It was recently applied to follow the movement and sub-cellular cellular location of RNA molecules associated with proteins in structures called RNA granules (check it out at RNA Organization in a New Light).

1.7.2. Electron Microscopy

Electron microscopy generates an image by passing electrons through, or reflecting electrons from a specimen, and capturing the electron image on a screen. Transmission Electron Microscopy (TEM) can achieve much higher magnification (up to \(10^6X\)) and resolution (2.0 nm) than any form of optical microscopy. The higher voltage of High Voltage Electron microscopy allows TEM through thicker sections than regular (low voltage) TEM. The result is micrographs with greater resolution, depth, and contrast. Scanning Electron Microscopy (SEM)can magnify up to \(10^5X\) with are solution of 3.0-20.0 nm and allows us to examine the surfaces of tissues, small organisms like insects, and even of cells and organelles. Objects of SEM must be conductive, so that biological samples are usually spray-coated with a thin layer of metal (e.g., palladium, platinum) (check the link to Scanning Electron Microscopy for more on SEM and look at the gallery of SEM images at the end of the entry). Helium Ion Microscopy is a form of SEM that substitutes helium ions for the vacuum in which SEM samples are normally viewed, eliminating the need for metal spray coating.Thus, HIM enables investigators to examine e.g., cells and viruses in a more natural state (you can google this on your own to see some examples of this). Electron microscopy, together with biochemical and molecular biological studies have revealed how interacting cellular and molecular components work with each other, and continue to do so, shedding light on all manner of biological processes and interactions.

121-2 Electron Microscopy