Three Methods for Shading Correction on the ... - UT Southwestern

Method 2: • Acquire your images as usual @ 1x zoom and 512 x 512. • Using fluorescent plastic slides as homogeneous samples, take “flat field” images in each of the color channels at exactly the same settings as your data. We have ordered a set of fluorescent slides and will make them available in the 780 room. o Be sure to choose a field of view without scratches or other marks o If the image is saturated, adjust the laser intensity downward. DO NOT change the gain, offset or pinhole. • Go to the Processing tab and use the flat field images to apply shading correction of your data images (see below). Method 2 can also be carried out in ImageJ: • Acquire data as <strong>for</strong> Method 2 • Open the uncorrected LSM510 image and the flat field image in ImageJ • Process > Image Calculator (see right) • Then convert to 8-­‐bit: Image > Type > 8-­‐bit

Method 3: Low pass filtering • If you don’t have a flat field image <strong>for</strong> your images that need to be corrected you can use the FFT Bandpass function in ImageJ to correct <strong>for</strong> shading. This is less ideal but still acceptable in most cases. • Process > FFT > Bandpass • Filter large objects down to 100 pixels:

Figure 1. Uncorected Image Figure 2. Zen <strong>Shading</strong> Corrected Figure 3. <strong>Shading</strong> corrected in ImageJ Figure 4. FFT Method