Peter DeVries

Publisher Summary This chapter introduces the principle of confocal microscopy. The different types of confocal microscopes currently available and the various applications of confocal microscopy are discussed. Methods of specimen preparation for confocal microscopy are provided as guidelines and should be generally applicable to most cell types. The chapter also describes three-dimensional reconstruction, four-dimensional imaging, and the methodology for producing color prints and slides of confocal data. In conventional microscopy, much of the depth or volume of the specimen is uniformly and simultaneously illuminated in addition to the plane in which the objective lens is focused. This leads to out-of-focus blur from areas above and below the focal plane of interest. Out-of-focus light reduces contrast and decreases resolution, making it difficult to discern various cellular structures. In contrast, the illumination in a confocal microscope is not simultaneous, but sequential. The illumination is focused as a spot on one volume element of the specimen at a time.

A simple method for constructing two and three-color merged images from grayscale confocal fluorescence images using Adobe PhotoshopTM is outlined. Various computer methods for manipulating and displaying the images are discussed in light of several recent biomedical applications of multi-label confocal microscopy.

Publisher Summary This chapter introduces the principle of confocal microscopy. The different types of confocal microscopes currently available and the various applications of confocal microscopy are discussed. Methods of specimen preparation for confocal microscopy are provided as guidelines and should be generally applicable to most cell types. The chapter also describes three-dimensional reconstruction, four-dimensional imaging, and the methodology for producing color prints and slides of confocal data. In conventional microscopy, much of the depth or volume of the specimen is uniformly and simultaneously illuminated in addition to the plane in which the objective lens is focused. This leads to out-of-focus blur from areas above and below the focal plane of interest. Out-of-focus light reduces contrast and decreases resolution, making it difficult to discern various cellular structures. In contrast, the illumination in a confocal microscope is not simultaneous, but sequential. The illumination is focused as a spot on one volume element of the specimen at a time.