Visualizing Cell Architecture
■ The limit of resolution of a light microscope is about 200 nm; of a scanning electron microscope, about 10 nm; and of a transmission electron microscope, about 0.1 nm.
■ Because cells and tissues are almost transparent, various types of stains and optical techniques are used to generate sufficient contrast for imaging.
■ Phase-contrast and differential interference contrast (DIC) microscopy are used to view the details of live, unstained cells and to monitor cell movement.
■ In immunofluorescence microscopy, specific proteins and organelles in fixed cells are stained with fluorescence-labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes.
■ When proteins tagged with naturally occurring green fluorescent protein (GFP) or its variants are expressed in live cells, they can be visualized in a fluorescence microscope.
■ With the use of dyes whose fluorescence is proportional to the concentration of Ca2+ or H+ ions, fluorescence microscopy can measure the local concentration of Ca2+ ions and intracellular pH in living cells.
■ Confocal microscopy and deconvolution microscopy use different methods to optically section a specimen, thereby reducing the blurring due to out-of-focus fluorescence light. Both methods provide much sharper images, particularly of thick specimens, than does standard fluorescence microscopy.
■ Specimens for electron microscopy generally must be fixed, sectioned, dehydrated, and then stained with electron-dense heavy metals.
■ Surface details of objects can be revealed by transmission electron microscopy of metal-coated specimens. Scanning electron microscopy of metal-coated unsectioned cells or tissues produces images that appear to be three-dimensional.
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