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Comparing Confocal and Widefield Fluorescence Microscopy

Comparing Confocal and Widefield Fluorescence Microscopy - Java Tutorial

Confocal microscopy offers several distinct advantages over traditional widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the dimensions of the focal plane. This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope.

The tutorial initializes with a randomly-selected specimen from Set 1 appearing in the widefield (Widefield Image) and confocal (Confocal Image) specimen image windows. In order to operate the tutorial, use the Focus, Brightness, Z-Axis Position and PMT Channel Gain sliders to examine the specimen at different focal planes and intensity levels. When the Focus Lock checkbox is enabled, both the widefield and confocal focus sliders are simultaneously engaged to present a comparison of the images gathered at similar focal planes by each technique. The Scan Line Speed slider controls the scanning speed of the confocal image, while the Pinhole Aperture Size radio buttons can be utilized to toggle between small (1 Airy unit), medium (4 Airy units), and large (20 Airy units) pinhole sizes. Specimens from three sets can be observed using the Specimen Set radio buttons and the Choose A Specimen pull-down menu.

In a conventional widefield optical epi-fluorescence microscope, secondary fluorescence emitted by the specimen often occurs through the excited volume and obscures resolution of features that lie in the objective focal plane. The problem is compounded by thicker specimens (greater than 2 micrometers), which usually exhibit such a high degree of fluorescence emission that most of the fine detail is lost. Confocal microscopy provides only a marginal improvement in both axial (z; along the optical axis) and lateral (x and y; in the specimen plane) optical resolution, but is able to exclude secondary fluorescence in areas removed from the focal plane from resulting images. Even though resolution is somewhat enhanced with confocal microscopy over conventional widefield techniques, it is still considerably less than that of the transmission electron microscope. In this regard, confocal microscopy can be considered a bridge between these two classical methodologies.

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