1.7: Microscopy Reveals Life’s Diversity of Structure and Form
Broadly speaking, there are two kinds of microscopy. In Light Microscopy , the specimen on the slide is viewed through optical glass lenses. In Electron Microscopy , the viewer is looking at an image on a screen created by electrons passing through, or reflected from the specimen. For a sampling of light and electron micrographs, check out this Gallery of Micrographs . Here we compare and contrast different microscopic techniques.
A. Light Microscopy
Historically one form or other of light microscopy has revealed much of what we know of cellular diversity. Check out the Drawings of Mitosis 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 , fluorescence , etc.). Limits of magnification and resolution of 1200X and 2 mm, (respectively) are common to all forms of light microscopy. The main variations of light microscopy are briefly described below.
1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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).
B. Electron Microscopy
Unlike light (optical) 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 106X) and resolution (2.0 nm) than any form of optical microscopy! Scanning Electron Microscopy (SEM) can magnify up to 105X with a resolution of 3.0-20.0 nm. TEM, together with biochemical and molecular biological studies, continues to reveal how different cell components work with each other. The higher voltage in High Voltage Electron microscopy is an adaptation that allows TEM through thicker sections than regular (low voltage) TEM. The result is micrographs with greater resolution, depth and contrast. SEM allows us to examine the surfaces of tissues, small organisms like insects, and even of cells and organelles. Check this link to Scanning Electron Microscopy for a description of scanning EM, and look at the gallery of SEM images at the end of the entry.