The Principle
	Start animation (111 KB)		The imaging principle used with the ApoTome is referred to in the literature as "grid projection" or "structured illumination". The fundamental theory was described in publications on interferometry as early as in the nineteen-sixties. A grid of stripes of defined width is projected onto the focal plane of the objective and shifted laterally in three defined steps relative to the sample (see illustration). A CCD camera takes a picture in each grid position. The three "raw" images are combined into a resultant image by on-line computation. The resultant image is an optical section through the sample, from which all image slices originate. Free of artifacts, with out-of-focus information removed. This image has an improved signal-to-noise ratio, and approximately doubles resolution in axial (Z) direction. Making an optical section is a prerequisite for computing 3D visualizations of specimens.		ApoTome Principle Technology System Inside4D Gallery System Requirements
	In-focus range Enlarge		Why is the resulting image obtained with structured illumination an optical section? When we observe a specimen with a microscope, we can distinguish between two ranges in the axial (Z) dimension. All specimen details located within the depth-of-field range of the objective used are seen in sharp focus (the yellow area in the schematic illustration). Specimen details outside that range appear out of focus, i.e. blurred. In fluorescence microscopy, the out-of-focus portions of the image strongly interfere with the image information of the depth-of-field range.
	In-focus range of the grid Start animation (116 KB)		The grid of stripes is imaged in the depth-of-field range of the objective. However, same as the specimen image, the grid image is in focus within a narrow depth range only. Below and above this in-focus range, the grid image gets out of focus and finally disappears (see illustration). If the grid is shifted to three different positions (see animation), the image data (gray levels) originating from out-of-focus ranges of the three raw images do not differ. As the data (gray levels) from outside the depth-of-field range are homogeneous, the algorithm used can remove the out-of-focus shares of the image.