1 Principles of Confocal Scanning
For biological applications laser scanning confocal microscopy is used mostly as a form of high resolution epi-fluorescence microscopy, and allows the acquisition of thin optical sections of finite, but controlled thickness. A confocal microscope consists, basically, of a high quality compound microscope with a laser illuminator, electronic image detector and computer for image storage and processing, with an adjustable confocal pin hole in the imaging plane. In order to describe how a laser scanning confocal microscope works let us first consider a standard compound microscope (1.0.1), evolve it into an epi-fluorescent compound microscope (1.0.2) and then a confocal microscope (1.0.3).
1.0.1 Transmitted Wide Field Microscope: A conventional wide field (transmitted light) microscope consists of an illuminator (light source and condenser), a collecting lens (the objective), an eye piece lens and a detector (the eye usually). This type of microscope is good for looking at thin, mostly transparent samples which have the structures of interest stained to increase contrast relative to the surrounding material. At the imaging plane is an optical reconstruction (technically called a real image), which is what the viewer sees through the eye piece. This image appears to come from a a wider source than the original object, hence it appears magnified.
1.0.2 Epi Illumination Fluorescence Microscope: This is a form of wide field, conventional, microscopy. A light source which transmits light through the viewing path replaces the illuminator and condenser of the conventional wide field microscope. For this mode to function the structures of interest are labeled with a fluorescent compound called a fluorophore. Light from the source is collimated and directed into the tube of the microscope to a 45 degree mirror with an interference coating that lets light at particular wavelengths and is transparent at other wavelengths (a dichroic mirror). This light is directed to the back of the objective and is focused into the sample. The objective functions as a condenser. Exciting light is absorbed by the fluorophore in the sample. The energy of the absorbed photon causes an electron in the dye molecule to jump to a higher energy orbital state. The electron rapidly jumps back to its ground orbital state, the energy released becoming a photon, of less energy compared to the exciting photon (nothing is perfectly efficient in the real world). Quantum mechanics dictates that photons of less energy have a longer wavelength (E=hn). Hence the emitted photon has a color shifted towards red. The image is collected by the eye piece in an identical manner as is transmitted wide field microscopy.
1.0.3 Confocal Microscope: A Confocal microscope is like a convention epi-fluorescence microscope with a pinhole aperture at the imaging plane, and a laser beam light source. In focus light converges to a point at the imaging plane. Out of focus light converges either below or above the image plane. The pin hole present at the imaging plane blocks the out of focus light, which falls mostly onto the pinhole barrier. Most of the in focus light will pass through the pin hole. Hence the image observed is from in focus light. One problem with such a system is that only a point of the sample is imaged. To view a plane of the sample the spot must be made to scan the sample, conventionally in an x-y direction. To scan a 3 dimension image volume, not only must the sample be scanned in the x-y directions but it must also be rescanned at different position in the z-axis, which is achieved by scanning and refocusing.
Last updated 10/24/06
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