Confocal Microscopy

Thursday, June 9, 2011

Confocal microscopy is an optical imaging technique which is used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of three-dimensional structures from the obtained images. This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and material science.

A number of different imaging modes are used in the application of confocal microscopy to a vast variety of specimen types. They all rely on the ability of the technique to produce high-resolution images, termed optical sections, in sequence through relatively thick sections or whole-mount specimens. Based on the optical section as the basic image unit, data can be collected from fixed and stained specimens in single, double, triple, or multiple-wavelength illumination modes, and the images collected with the various illumination and labeling strategies will be in register with each other. Live cell imaging and time-lapse sequences are possible, and digital image processing methods applied to sequences of images allow z-series and three-dimensional representation of specimens, as well as the time-sequence presentation of 3D data as four-dimensional imaging. Reflected light imaging was the mode used in early confocal instruments, but any of the transmitted light imaging modes commonly employed in microscopy can be utilized in the laser scanning confocal microscope.

Confocal microscopy offers several advantages over conventional widefield optical 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 immediate plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional fluorescence microscopy, and the growing number of applications in cell biology that rely on imaging both fixed and living cells and tissues. In fact, confocal technology is proving to be one of the most important advances ever achieved in optical microscopy.