Process > Combine > EDF

The Extended Depth of Field, or EDF, feature allows you to derive a single image from a series of images that are focused on different depths along the Z-plane.  This tool uses the multi-plane focus feature along with a contrast detection algorithm to analyze the series of images to either extract the one that represents the best-focused image, or to create a composite image that takes the best-focus data from the various images of the series and combines them into one extended depth of field image.

To perform an EDF operation and obtain an EDF image, you can provide either a single image sequence file, or you can provide a set of individual image files, or a combination of both.

Click the EDF tool's down arrow to show the EDF drop-down menu. The three options of the EDF pull-down menu are described below.

EDF: This tool opens the EDF dialog box and adds the currently active image or image sequence, and any images/sequences selected in the Quick Start Guide: Using the Images Panel, to the Selected Images panel of the EDF dialog box.

Tip: If you are providing a single sequence file, simply open it into the application workspace before clicking on this tool. Optionally, you can also open multiple individual images and select them all in the Images Panel by holding the <Ctrl> key down as you click on their thumbnail image. If you do this prior to clicking on the EDF option, they will all be added to the EDF dialog box's "Selected Images" panel.

At this point, you can review the options available on the EDF dialog box to 'tune' the EDF function, make any modifications as needed, and click the Apply button on the EDF dialog box to perform the EDF operation. Click here for more information about the controls available on the EDF dialog box.

EDF Selected: Click this tool to instantly perform an EDF operation on the currently active sequence file or on the currently selected set of images in the Images Panel. When you click this option, Image-Pro performs the EDF operation according to the EDF options (i.e., on the EDF dialog box) that were last used. Note that when you click on the EDF Selected tool, whatever settings are currently set for the EDF tool will be applied, whether or not the EDF dialog box is displayed.  If, by clicking the EDF Image tool you do not obtain the best-focus results you require, then adjust the controls on the EDF dialog box, and try again.

NOTE: This tool is only enabled if the current image is a sequence file, or if multiple images are selected in the Images Panel. You can select multiple images in the Images Panel by holding the <Ctrl> key down as you click on their thumbnail image.

Set Selected to EDF: This tool functions exactly the same as the EDF tool described above.

The EDF Dialog Box

When you click on the EDF tool or its Add selected to EDF menu option, the EDF dialog box is displayed. To use this dialog box to obtain an extended depth of field image from a series of images:

  1. Open the images you want to combine in the Image-Pro workspace.
  2. Click the All button on the EDF dialog to add the images to the Selected Images list box.
  3. Choose your output parameters.

    1. Indicate your output options:

      • Generate composite best-focus image: Clicking this button creates a single image composed of the best pixels from the set of input images, as determined by the Focus Analysis Options.

      • Return best-focus frame: Clicking this button creates a new workspace containing a copy of the selected frame with the most in-focus material. If an ROI is drawn in the image, Image-Pro finds the most in-focus material in that ROI.

      • Activate best-focus frame: When you select this option, Image-Pro advances the sequence to the frame that has the most in-focus material. If an ROI is drawn in the image, Image-Pro finds the most in-focus material in that ROI.

      • Normalize Illumination: Use this option when the illumination or intensity of emitted light will change in the time that it takes to acquire all of the planes. Basically, this option corrects for uneven illumination as the Z plane changes.

      • Bottom Up: If you click this button, the order of the images is reversed. The image at the top of the list will be the last image in the stack, and the image at the bottom of the list will be the first image in the stack. Note that the perceived “position” of the images will affect the Topographic Map, and may affect which pixel is selected for some of the focus analysis operations.  

    2. Choose your focusing method:  

      • Local Contrast: This method considers areas with high/maximum contrast to be in focus. Pixels are analyzed in the area around the current pixel location. The pixel from the plane with the largest variance or local contrast is selected.

      • Stack contrast: The mean value of the pixels is calculated. The pixel from the plane with the largest variance from the mean is selected.

      • Maximum Intensity: This method is used for samples that are more intense than the background such as light emitting fluorescent dyes. The pixel from the plane having the highest intensity at the current pixel location is selected.

      • Minimum Intensity: This method is used for regions that are darker than the surrounding area to be in focus. The pixel from the plane having the lowest  intensity at the current pixel location is selected.

      • Small Edges: Edges are extracted from the average of the stack assuming a moderate blurring.

      • Medium Edges: Edges are extracted from the average of the stack assuming a mid-scale blurring.

      • Large Edges: Edges are extracted from the average of the stack assuming a large-scale blurring. 

    3. Select the Edge filter that extracts edges defining in-focus pixels. 

      • Dilated Variance 3x3 - Variance 3x3 filter followed by Dilation 5x5

      • Variance 3x3 - Variance 3x3 filter

      • Dilated Variance 5x5 - Variance 5x5 filter followed by Dilation 5x5

      • Variance 5x5 - Variance 5x5 filter

      • Variance 7x7 - Variance 3x3 filter followed by Dilation 5x5 

  4. Indicate the Significance Threshold.

    The Significance Threshold defines threshold for significant edge pixels.  The pixels (after edge filter) above the threshold are considered in-focus pixels; others are considered out of focus pixels. If, for a given pixel position, none of the frames contain edge intensities above the threshold, the height of the pixel (in 3D) is assigned to the frame with the closest in-focus pixel. The threshold percent is relative to the total accumulated value of edge image. The range of the value is from 0 to 100.  The value = 50 (default) will set the threshold to a level of Median intensity, cutting 50% of pixels. The value 80 will take only the 20% of pixels with highest contrast. Use a higher threshold on noisy images.

  5. Indicate if you want to generate a topographic map and, if so, your topographic map options:

    • Generate Topographic Map: Clicking this button creates a topographic map from the focused image.
    • Create Intensity Calibration: Check this box if you want to generate an intensity calibration for the combined EDF image.
    • Create Surface Plot: Check this box if you want to generate a surface plot image of the combined EDF image.
    • Smoothing: Choose a smoothing factor for the topographic map image. 

  6. When all the parameters have been set, click the Apply button.