Boulder Laboratory for 3-Dimensional Electron Microscopy of Cells FINDHOTPIXELS(1) FINDHOTPIXELS(1) NAME findhotpixels - find pixels to exclude from X-ray analysis in SerialEM SYNOPSIS findhotpixels [options] input_image output_model DESCRIPTION Findhotpixels will analyze a set of CCD camera dark reference images to identify relatively hot pixels in the CCD chip and will output a list of the pixels in a format suitable for placing in the property file for SerialEM. Automatic removal of X-rays from dark references can then be activated in SerialEM, and it will then be able to avoid identifying a hot pixel as an X-ray. It is recommended that three long-exposure (e.g., 3 sec) dark reference images be used for this analysis. The program works by finding pixels that deviation from the image mean by a specified threshold. The lower the threshold, the more pixels it will find. You may need to run the program several times to find a threshold that gives a reasonable number of hot pixels (under 100). A threshold on the order of 0.2% of the dynamic range of the camera should be appropriate. Hot columns are excluded from the analysis. With 4-port readout cameras from Gatan, it is important to exclude a fairly large number of bright columns near the edges; otherwise you will need to set the threshold higher than the optimal level for finding the hot pixels in the rest of the camera. The program could be smarter about this and use a deviation from a local mean, but there is no point in doing so under SerialEM uses a local criterion as well. The program outputs a list of the hot pixels coordinates and their values above the image mean. At the end is a set of HotPixels and HotColumns entries suitable for pasting into the SerialEM properties file. The program can be run either interactively or with command line arguments processed by the PIP interface (see the man page for pip). With command line arguments, the options are: -input OR -InputFile File name Name of input image file with dark reference images. If this option is not entered, the first non-option argument is used -output OR -OutputFile File name Name of output model file with a point at every hot pixel. If this option is not entered, the second non-option argument is used. -columns OR -HotColumns List of integer ranges List of hot columns to exclude (numbered from 0). Ranges may be entered. -threshold OR -ThresholdDifference Floating point Threshold difference from mean value for seeking hot pixels -help OR -usage Print help output -StandardInput Read parameter entries from standard input. If no command line arguments are entered, input is requested interactively as follows: Name of input image file with dark reference images Name of output model file with a point at every hot pixel List of hot columns to exclude (numbered from 0) Threshold difference from mean value for seeking hot pixels H^HHI^HIS^HST^HTO^HOR^HRY^HY Written by David Mastronarde in 2003. Added to IMOD, 10/17/06.