Image Processing

Various kinds of simple filters can be applied with these controls. The filter will always be applied to the current section. A sequence of filters can be applied. The program keeps a list of the applied filters so that it is easy to perform the same operation on a different section or on the data as a whole.

Note that many of these filters can be applied to a whole image stack through the Clip and Mtffilter programs. The List button can be used to get a list of the current command(s) in the Information window, and Process File... in the File menu can be used to apply the command(s) (if possible) to the whole image file or to the subvolume delineated by the rubber band in the Zap window. This menu item is available when data are loaded unbinned and when this dialog is open.

Image data are always analyzed as 8-bit bytes, even when they have been loaded into the program as 16-bit integers. In the latter case, the current setting of the Low and High sliders in the Information window determines how data are scaled to bytes. For best results, be sure that the Low and High sliders are set so that images have a good contrast with a fairly wide range between the Black and White sliders. Using the autocontrast function generally leaves the sliders in positions that will allow good scaling when processing.

General Controls
Fourier Filtering
Fourier Transforms
Smoothing
Median Filtering
Anisotropic Diffusion
Edge and Sharpen
Threshold

General Controls

Single-click in the list of filters to select the current filter to be applied to the data; in most cases there will be further parameters to select.
Apply: This button or the A hot key will apply the current filter to the ORIGINAL image data. Double-clicking in the filter list is the same as pressing the Apply button. The program initializes the list of applied filters with the selected operation.
More: This button or the B hot key will apply the filter to the CURRENT image data, as modified by previous filter operations. The program adds this new operation to the list of applied filters. If you change sections away from modified data, this button acts like Apply instead.
Less: removes the last operation from the list of applied filters and reprocesses the current section without that operation.
Do Same: applies the current list of filters to the current section. If the parameters of the current filter are modified, those new parameters are applied. Thus, this button can be used in two ways: to apply the same sequence of filters to a new section, or to reapply the filters to the current section with a change of parameters in the last operation.
Toggle: lets you toggle between the original and modified image by pressing and releasing the button.
Reset: Pressing this button, applying a filter to a different section, closing the window with Done, or flipping the data volume will all restore the original image data for a section, unless you press the Save button first.
Save: will permanently replace the image data in memory with the processed data. If necessary, the data can be restored with Edit-Image-Reload. Also, if image data are loaded into a cache of limited size, processed images will disappear if those sections get reloaded from the file.
List: will output to the Information Window the list of commands that could be applied to the image file to achieve the current result. If an operation cannot be done at the command line for whatever reason, the list includes "Cannot...". The commands will include file data mode changes as necessary to prevent truncation of the data for the particular sequence of operations.
Autoapply: If this is checked, the program will automatically apply the current filter(s) to a new section when the section is changed. This will happen only if the section being left has been filtered.
Autosave: If this is checked, the program will automatically save the modified data into program memory when the section is changed. Use this feature with caution. There is no protection against filtering the same section twice if you reverse directions. Moreover, you need to wait until you see a processed slice before moving to the next slice.

Fourier Transforms

To take a Fourier transform (FFT), the program will pad the image into a square array slightly larger than the original image, taper the image at its edges to minimize edge artifacts, take the FFT, apply log scaling, and clip out the portion that fits into the original image size. For a non-square image, the FFT will thus be isotropic (X and Y scales the same) but truncated in one dimension. The panel will show the range of frequencies that appear in the X and Y dimensions. Binning can be used to see the whole transform for a non-square image, and also to reduce noise and execution time. With binning, the smaller FFT will be embedded into a black background.

To do an FFT of a subregion, turn on Use Zap window subarea. If the rubber band is on in the active Zap window, the FFT will be taken of the area inside the rubber band. Otherwise, the area used will be the portion of the image showing in the window.

The Compute frequency button can be used to determine the frequency at a particular location in the FFT. First, click on that location with the first mouse button, or deposit a model point there, then press the button. If you are in model mode and there is a current model point, its position will be used; otherwise the current image position is used. The current image position is rounded to the nearest pixel while a model point can provide subpixel accuracy if needed. The program will compute the frequency in reciprocal pixels then divide by the pixel size in the model header to get the frequency that is show (e.g., reciprocal nanometers). The inverse of this value is also shown to provide a resolution value in real space units.

The panel also shows the scale that is used to convert from pixels in the FFT to frequency units.

Fourier Filtering

The Fourier filter is done by taking Fourier transforms and its parameters are the radius and sigma parameters used in many other IMOD programs. Namely, the Low frequency sigma is the sigma of an inverted Gaussian starting at the origin, used to attenuate low frequencies. Low pass filtering is done with a Gaussian starting at the High-frequency cutoff and with a sigma given by the High-frequency falloff. The units are cycles per pixel, ranging from 0 to 0.5. The slider for the low frequency sigma only goes up to 0.2, and when you click the slider to move it by one step, the step increases from 0.0001 at 0/pixel to 0.001 at 0.005/pixel. The same filtering can be done at the command line with "mtffilter -high sigma -low cutoff,falloff", where "sigma", "cutoff", and "falloff" are the values used in this panel.

If Filter Slicer image instead is checked, this filtering will no longer be done on the image data for the current section, but instead on the image being displayed in any Slicer windows that are open. The filtering is the last operation applied to the Slicer image before display, so it can be used to filter a Fourier transform for a Slicer where "FT" is on. While it is possible to combine this Slicer filter with one or more other filters on the image data for the current section, there are several limitations. The Slicer filter will always be applied at the end regardless of where it is added in the sequence of filters. It will only be applied once, using the last parameters set for it. Fourier filtering of the current section cannot be combined with Slicer filtering in a sequence of filters; the current setting of this checkbox determines what operations occur.

Smoothing

Smoothing replaces each pixel by a weighted sum of neighboring pixels. The image is multiplied by a small square matrix of weighting values, called a kernel. The standard 2D smoothing filter uses a 3x3 kernel with weights:

    1  2  1
    2  4  2
    1  2  1

A Gaussian function can be used for the weights if a standard deviation for the Gaussian is set in the Kernel sigma spin box. Initially this box shows None, which means that the standard kernel will be used. The latter gives the same result as a kernel sigma of 0.85 pixel. The lowest sigma value available is 0.5 pixel, since values below this give insignificant filtering. Note that this sigma specifies pixels in real space, unlike the sigmas in Fourier filtering. For 2D filtering, the program uses a 3x3 kernel for sigma up to 1.0, a 5x5 kernel for sigma up to 2.0, and a 7x7 kernel for higher sigma values. The computation will take longer with the larger kernels.

Select Filter in 3D cube to filter isotropically, in Z as well as in X and Y. The kernel will be 3x3x3, 5x5x, or 7x7x7 depending on sigma. In Clip, 3D filtering is specified by entering a negative value for the "-n" option, the iteration number.

The Rescale to match min/max makes the rescaling that happens after many kinds of filtering optional for smoothing. This rescaling may make the intensities vary from section to section, since it is based on the minimum and maximum rather than a more robust measure like standard deviation. Such variation can be a problem when using the Edit - Contour - Auto window because it would make the threshold vary unpredictably, so it is recommended that this option be turned off if smoothing is used with autocontouring.

Median Filtering

Median filtering replaces each pixel by the median value of neighboring pixels, where the Size parameter determines the size of the block of pixels. The default is to do the filtering in 3D, and take the median in a cube of voxels. Filtering in 2D, considering only the pixels on the current section, can be done if you turn off Compute median in 3D cube. With 2D filtering, you can iterate by pressing More. However, with 3D filtering, pressing More will not have much effect and will not be the same as iterative filtering in 3D, because only the central slice has been filtered.

Anisotropic Diffusion

This panel provides parameters for running a simple anisotropic diffusion algorithm using the Perona and Malik filtering method, based on a program by Alejandro Canterero. The gradients in this method are simply pixel-to-pixel differences. The ratio between these pixel-to-pixel differences and the threshold K determines how much diffusion is allowed between pixels. Note that much better results will generally be achieved with Nad_eed_3d, There is an interface for using that method in Etomo, available through the File-Parallel Processing menu entry.

The Edge Stopping Function radio buttons allow you to choose between the Rational edge stopping function and the Tukey biweight stopping function, which correspond to options -cc 2 and -cc 3, respectively, in Clip. The Tukey biweight will preserve more local structure than the rational edge stopping function.

The Iterations value controls how many iterations are run when Apply or More is pressed. The total number of iterations done is also reported. The routine will give the identical result if a certain number of iterations are done with multiple steps using More rather than all at once.

The K entry sets a threshold for the edge stopping function; when the number is too low virtually nothing will happen. The rational edge function may require smaller values than the Tukey biweight. The unscaled value is the number that should be specified with the -k option in Clip to achieve the same filtering on the raw data from the image file as is seen on the scaled byte data in 3dmod.

The Lambda value controls the so-called time-step; if images become noisier this probably needs to be reduced.

Edge and Sharpen

The 5 different edge-enhancing filters and the Sharpen filter all work the same as the operations by the same name in Clip.

Threshold

This filter will convert an image to black and white for intensities below and above the threshold selected by the slider. Below the slider, a text line will show the corresponding threshold value that could be applied to the image file, for example with "clip threshold". Grow thresholded area will increase the white area by about a pixel on all sides, and Shrink thresholded area will decrease it by about a pixel. These two options can be used together to achieve some smoothing of the thresholded area. Apply changes automatically will make the image be reprocessed after every change in the threshold value, which is more convenient than pressing Do Same repeatedly. If your image data are loaded as integers (there are 4 sliders in the Information window), turn off the Float button there to prevent the Low and High sliders from being changed, which will result in a gray image.

Although thresholding can done in Clip, the grow and shrink operations are not available there. Also, because other filter operations make it difficult to relate the threshold to a value in a file, it is possible to include thresholding when processing the file with the File-Process menu item only if thresholding is the first operation.

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