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Confocal microscopes, sometimes referred to as laser scanning confocal microscopes, take advantage of the properties of light to produce images at a single optical plane through thick specimens. To accomplish this, confocal microscopes rely on the property of certain molecules to produce fluorescence, and on the ability to collect this fluorescence from the plane of focus while rejecting all other fluorescence.
Many molecules have the ability to absorb certain wavelengths of light. For example, plant leaves appear green because the chlorophyll molecules in the leaf absorb wavelengths of light in the blue and red region of the spectrum. After subtracting the blue and red light from sunlight, much of the visible light that remains is green. Once absorbed, the light energy can be converted into chemical energy (photosynthesis) or into heat. Some molecules have the ability to re-emit light energy, and this process is called fluorescence. The light that is emitted always contains less energy than the light that was absorbed (remember thermodynamics?), and is thus always at a longer wavelength.
This property of certain molecules to produce fluorescence at a very particular wavelength (called “emission”) after absorbing light of a specific wavelength (called “excitation”) has been exploited for decades by traditional fluorescence microscopy. There is a mind-boggling assortment of dyes that localize to specific cellular structures or organelles, can respond to changes in ion concentrations, or can be attached to almost any antibody. Since the mid-1990’s, it has become possible to cause a cell to manufacture its own fluorescent molecule. By introducing a jellyfish gene coding for a molecule called Green Fluorescent Protein (GFP), researchers produced a tool capable of “reporting” on the location and timing of expression of any gene of interest.
While traditional fluorescence microscopy provides useful information about the localization of fluorescent compounds, the images produced can look blurry or out of focus. This is because the emitted light that is collected to form the image originate from any optical plane within the specimen. To overcome this limitation, confocal microscopy was developed. Confocal microscopy uses light from a laser through the objective of a standard light microscope to excite a specimen within a narrow plane of focus. Any emission of light from out-of-focus planes is rejected by the pinhole, or confocal aperture. A simplified lightpath for a confocal microscope is illustrated below. Only light that passes through the aperture contributes to the image formed by the photomultiplier tube. A photomultiplier tube is an extremely sensitive device for converting photons into an electrical signal.
In addition to scanning the specimen in the X and Y dimensions, confocal microscopes can control the focal plane by raising and lowering the microscope stage. Using a stepper motor, the stage can be stepped in tiny increments (0.1 microns) through a sample. The software controlling the microscope can store the image information as it steps through a sample, allowing a true three-dimensional analysis of specimens.