The basic concept behind the ZEISS ApoTome is the use of an evenly spaced grid in the aperture plane to serve as a mask through which the specimen is illuminated. The grid is inserted into the light path of the microscope and uses the epi-illuminator lens system to project a shadow of the grid lines into sharp focus, superimposed on the specimen, in the objective focal plane. In cases where the specimen is relatively thick (such as occurs a tissue section that is greater than 10 micrometers in size), then superimposed on the focal plane will be a composite blurred image from remote parts of the specimen positioned in planes that are remote with respect to the point of focus. During acquisition, three separate images of the thick specimen are sequentially gathered by shifting the grid projection by one-third between each image capture. The resulting image set contains the sum of contributions from the in-focus plane, which are shadowed by sharply defined stripes, plus blurred planes (not focused) that do not contain the distinct striping pattern from the grid.
The tutorial initializes with the focused light from the microscope epi-illuminator passing through the ApoTome grid and being projected onto a thick specimen in the Raw Image Window. To operate the tutorial, click on the Acquire Image button to sequentially capture a series of three images using ultraviolet, blue, and green excitation light (each displayed in the individual Channel windows). After processing, the final three-color image is displayed in the Composite Image window. To view individual channels, use the Channel Viewer Options buttons. Images from other regions of the specimen can be gathered by relocating the focal plane using the Focal Plane Z-depth slider.
The raw image set gathered by the ZEISS ApoTome is treated with a simple algebraic function by the application software to produce a single sharp, crisp derivative image that is free of the blur arising from remote focal planes. The simple sum of these three raw images is identical to the normal widefield image of a thin specimen without the use of a grid. In practice, the grid can be shifted rapidly and precisely (driven by a piezo-electric element), and the calculation is performed very rapidly. The rate-limiting step for optical sectioning using the ApoTome is thus the acquisition of the three images. When compared to capturing images with widefield illumination followed by deconvolution image processing, the composite image resulting from the ApoTome is available immediately instead of after 15-20 minutes, as required for processing by the deconvolution computation. The total exposure of the specimen is slightly greater using the ApoTome because the grid projection is typically not completely opaque. However, the axial resolution is comparable to that achieved by either confocal or deconvolution techniques.
Contributing Authors
Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.