Practical Considerations for Collecting Confocal Images

Conventional microscopy delivers two-dimensional images in real time and real color to the eye of the user. Confocal microscopy adds a third dimension by imaging only one plane within the sample at a time so that variations in depth can be quantified (1 ). This has both positive and negative aspects. The advantage is that a series of such slices can be reconstructed to give 3D views and enable volume analysis of the sample, and that any one slice is crisper and clearer than a full-field fluorescence image. The disadvantage is that the portion of the sample visible at any one time is so small that finding the most interesting parts of the specimen may no longer be possible (Fig. 1 ).

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