Structured illumination is a widefield technique in which a grid pattern is generated through interference of diffraction orders and superimposed on the specimen while capturing images. The grid pattern is shifted or rotated in steps between the capture of each image set. In the example illustrated below, the image set is composed of 5 individual subsets, each captured after rotating the grid by 60 degrees. Following processing with a specialized algorithm, high-frequency information can be extracted from the raw data to produce a reconstructed image having a lateral resolution approximately twice that of diffraction-limited instruments and an axial resolution ranging between 150 and 300 nanometers.
The tutorial initializes with a green pseudocolored widefield image of the specimen appearing in the Widefield Image window and a grayscale version in the Raw Image window. In order to operate the tutorial, click on the AutoPlay button or use the mouse cursor to click on one of the Modulation Angle buttons. The different grid orientations will be displayed superimposed over the raw image and the associated power spectrum will be shown in the Fourier Spectrum window. Once all 5 of the image sets are captured, the high resolution reconstructed image will appear in the Superresolution-SIM Image window.
In regards to the example presented in this tutorial, a total of five images are captured for each orientation angle of the grid to generate one image subset. After image subsets are obtained for each of the five grid orientations, the collection can be analyzed to produce a final high-resolution image. In general, a somewhat lower lateral resolution of 130 nanometers and an axial resolution of 350 nanometers can be obtained based on the reconstruction of only 10 to 15 images of this type. The relationship between the number of grid rotations and the expected image resolution is complex and heavily depends on the grid frequency and number of rotation angles, as well as the geometrical relationship between the grid lines and prominent specimen features. More simply stated, the final resolution can be enhanced by increasing the number of grid rotations at the expense of sampling speed and increased photobleaching. Among the benefits of high resolution structured illumination are the widespread availability of dyes and fluorescent proteins for labeling specimens and the ease of conducting multicolor imaging. The primary drawback is the length of processing time (1 to 30 seconds) necessary to generate high resolution images.
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.