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Light Microscopy Image Collection: Confocal, Widefield and Deconvolution

Light microscopy instrumentation and biological applications continue to expand rapidly, and an important aspect of this expansion is three-dimensional (3-D) imaging and image analysis. For many biological specimens, optical sectioning, i.e. collecting images at a sequence of depths under controlled conditions, provides true 3-D data through the entire specimen. This is especially important for specimens whose thickness exceeds the depth-of-field of the microscope objective lens. This sequence of optical sections is the basis for 3-D image reconstruction providing information from all three specimen dimensions instead of just the traditional two in the image plane of the microscope. The analysis of images in 3-D provides insights into the structure and function of biological specimens that are not available through other means. However, 3-D microscopy also presents additional choices for image collection. The first of which is whether to use widefield or confocal microscopy and the second is whether to utilize digital deblurring or deconvolution methods.