Confocal microscopy in mycological research

Confocal microscopy in mycological research. Experimental Mycology 18, 275-293. Confocal microscopy is a revolutionary advance in light microscopy. Utilizing the latest laser, computer, and imaging technologies, the technique offers biologists a unique form of cell and/or subcellular visualization. The principal feature of confocal microscopy is that it permits “optical sectioning” rather than mechanical sectioning of both thin and relatively thick microscope specimens in fluorescence and reflection imaging modes; the resulting images have far better resolution and contrast than can be obtained with a conventional light microscope. This capability is achieved by using a monochromatic laser light in a scanning raster across a specimen. When a confocal pinhole is placed in the light path between the sample and the detector, most out-of-focus light is removed. Digitized confocal images may be further analyzed or improved by image processing and 3-D reconstruction. Laser scanning confocal microscopy can be successfully applied to a variety of fungal problems including cell dynamics, fungus-host interactions, organelle structure and function, or cytoskeletal localization and cytochemistry using an array of various reporters, in virtually any sample that can be viewed by conventional light microscopy. It is particularly advantageous for examining thick fungal samples or in host-pathogen interactions where the fungus is embedded deep within the host tissue. As with any technique, some artifacts and difficulties are inherently associated with laser scanning microscopy and computer processing of the resulting images. The advantages and disadvantages of confocal microscopy must be considered when determining its potential applications for specific mycological samples