Among the most popular tools to conduct optical sectioning is the spot-scanning laser confocal microscope where a single spot of focused light is rapidly transitioned along the lateral axes of the specimen to excite fluorescence from labeled components. Fluorescence emission gathered from the specimen is filtered through a confocal pinhole aperture to reject light that originates from regions removed from the focal plane. The emission light that passes through the pinhole is detected by a photomultiplier to form the image in a serial manner. Although the spot-scanning approach is much slower than parallel scanning techniques (such as a spinning disk), it offers great flexibility in terms of image size and acquisition strategies. In addition, laser scanning confocal microscopy is capable of producing the highest out-of-focus discrimination of all routine optical sectioning techniques. This interactive tutorial explores optical sectioning with confocal microscopy and compares these sections to the results obtained with widefield fluorescence.
The tutorial initializes with the fluorescence image of a pollen grain appearing in the Widefield window and a corresponding optical section at the respective focal plane shown in the Confocal window. In order to operate the tutorial, use the Axial Focal Plane slider to transition along the z axis and observe changes to the confocal and widefield images. The optical section position is indicated in a box located beneath the slider. The scanning speed can be altered with the Scan Line Speed slider and the gain of each individual channel is adjustable with the PMT Channel Gain sliders, one each for the red, blue, and green channels. A new specimen can be loaded using the Choose a Specimen pull down menu.
In laser scanning confocal microscopy (LSCM), it is possible to exclusively image a thin optical slice out of a thick specimen (ranging in physical section thickness up to 100 micrometers), a technique known as optical sectioning. Under suitable conditions, the thickness (z-dimension) of such a optical slice can be less than 500 nanometers. The fundamental advantage of the confocal versus a traditional widefield microscope arises due to the restricted manner in which light reaches the photomultiplier through a pinhole. In widefield fluorescence, the image of a thick biological specimen will only be in focus if its axial dimension is less than the wave-optical depth of focus specified by the objective parameters. In cases where this condition is satisfied, the in-focus image information from the specimen plane of interest is mixed with out-of-focus image information arising from regions outside the focal plane. This reduces image contrast and increases the share of stray light detected. If multiple fluorophores are being observed, there will also be a color mix of the image information obtained from all of the channels involved.
Contributing Authors
Alex B. Coker and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.