3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy

Imaging in confocal microscopy is characterized by the ability to make a selective image of just one plane inside a specimen, virtually unaffected -within certain limits- by the out-of-focus regions above and below it. This property, called optical sectioning, is accompanied by improved imaging transverse to the optical axis. We have coupled a confocal microscope to a computer system, making the combination of both an excellent instrument for mapping the 3-dimensional structure of extended specimens into a computer memory/data array. We measured that the volume element contributing to each data point has, under typical fluorescence conditions, a size of 0.2 X 0.2 X 0.72 micron. The data can be analysed and represented in various ways, i.e., stereoscopical views from any desired angle. After a description of the experimental arrangement, we show various examples of biological and food-structural studies. The microscope can be operated either in reflection or in fluorescence. In the latter mode a spectral element allows selection of the wavelength band of fluorescence light contributing to the image. In this way, we can distinguish various structures inside the cell and study their 3-dimensional relationships. Various applications in biology and the study of food structure are presented.