Imaging of thick specimens in fluorescence microscopy is compromised by signal originating from regions that are removed from the focal plane. The result is that sharp image information originating from the focal plane is overlaid with blurred image information arising from distant areas, thus reducing contrast and resolution in the axial (Z) dimension. Furthermore, three-dimensional reconstruction of the specimen is not possible under these conditions. Optical sections of the specimen, which only extract information from the region that corresponds to the objective depth of field, can alleviate blurring and enable volume rendering of stacks to generate three-dimensional images. Aside from using confocal techniques, optical sections can also be obtained in widefield fluorescence microscopy using structured illumination, as has been implemented in the ApoTome attachment manufactured by ZEISS.
The tutorial initializes with an animation of the ZEISS ApoTome in action featured in the upper left-hand side of the window. Beneath the ApoTome model is a cutaway view of the light path showing the aperture grid and the rotating optical glass plate that directs a projection of the grid across the specimen. Presented in the Preview Window is a projection of the grid on the specimen, as would be observed in the software. In order to operate the tutorial, use the ApoTome Position buttons to toggle the ApoTome grid in (Position 2) and out (Position 1) of the light path. When the ApoTome is inserted into the light path, the Grid Projection buttons can be used to manually control the projection position through the use of the Grid Projection Control slider. After the grid is removed using the position control buttons, an aperture diaphragm is slid into position whose size can be varied with the Iris Diaphragm Control slider.
The principle behind the ApoTome implementation of structured illumination is presented in the tutorial. A grid created by evaporating metal on the surface of an optical-grade glass window is inserted into the field diaphragm plane in the illumination pathway of a fluorescence microscope and projected onto the specimen. The projected grid image is translated over the specimen using a plane-parallel glass plate that is tilted back and forth in the light path. At least three raw images of the specimen are acquired with the grid structure superimposed in different positions. These images are subsequently processed in real time using the microscope software to create an optical section. The underlying principle is that the projected grid becomes visible in the focal plane as a result of specimen features being excited by the structured light. In regions where no light reaches the specimen (in effect, the dark grid lines), no fluorescence is generated. The software determines grid contrast as a function of location and removes the out-of-focus image information before collating the three images into a final optical section.
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.