Image Processing

On the Processing tab you can apply image processing functions (IP functions) to acquired or loaded images. The basic workflow is quite simple:

• Acquire or open an image that you want to process.
• Open the Processing tab.
• When you open the tab, the last active image (e.g. from Acquisition tab) will be used as the input image for the processing function.
• If you want to select another input, load the new image under Image Parameters > Input. Note that the image must be opened in ZEN before.
• Select the desired processing mode (Single/Batch). Per default, single processing mode is selected. Batch processing allows to apply a specific processing function automatically to a batch of images. Images processed with Batch mode are by default saved with a lossless compression. The compression setting can be changed in Tools > Options > Processing.
• Select the desired processing function under Method, e.g. Color Balance.
  You can search for processing functions in the Search field. Therefore, just enter the initials of the functions you want to search.
• Set the parameters of the function under Parameters.
  If you need help for a specific function and its parameters press F1 on your keyboard. You will find detailed descriptions for each functions in the online help.
• To see how the functions works you can click on the Preview button under Image Parameters > Output.
• Click on the Apply button to apply the processing functions to the image.
  This will create a new image in a new image container. The original image will not be changed.

In this topic we will show you how to extract individual fluorescence channel images of a multichannel image.

1. You have acquired or opened a multichannel image.
   

1. Select the Processing tab. Open the Method tool and select under Utilities the entry Create Image Subset.
2. In the section Image Parameters, open the Input tool and select the multichannel image as Input image.
3. In the section Method Parameters, open the Parameter tool and the select the entry Channels. Deactivate the channels you do not want in the extracted image (e.g. the red and the blue fluorescence channel).
   
4. Click on the Apply button.

1. In the example image, you have extracted the green fluorescence channel image of the multi-channel image. Activate the corresponding channel and deactivate the other channels to extract further individual fluorescence images. You can now save the extracted sub images as separate files.
   

In this topic we will show you how to crop a region of interest (ROI) of an image.

1. You have acquired or opened a multichannel image.

1. On the Processing tab, open the Method tool and under Utilities select the entry Create Image Subset.
2. In the Image Parameters section, open the Input tool and select the image.
3. In the Method Parameters section, open the Parameters tool.
4. Open the Z-position dimension setting and select Extract Range of the drop-down list. Set the start position and the end position using the slider or the input field.
5. Open the Region setting and select Rectangular Region of the drop-down list.
6. Click in the image on the start position of the ROI and draw a rectangular region.
   
7. You have marked the ROI which should be cropped.
7. Click on the Apply button.

1. The marked ROI with the defined dimensions is extracted of the image. You can now save or process this image.
   

In this topic we will show you how to create an extended depth of focus (EDF) image of a Z-Stack image. The focus planes of all the z-positions will be calculated to one EDF image.

1. You have acquired or opened a z-stack image.

1. On the Processing tab, open the Method tool and select under Sharpen the entry Extended Depth of Focus.
2. In the Image Parameters section, open the Input tool and select the Z-Stack image as input image.
   
3. In the Method Parameters section, open the Parameters tool.
4. Select Wavelets of the Method drop-down list.
5. Select High of the Z-Stack Alignment drop-down list.
6. Click on the Apply button.

1. All focus planes of the Z-Stack image are now calculated to one EDF image.
   

Depending on your loaded image, you can split certain dimensions, e.g. channels or timepoints, and save them to separate images. First, you select the method, and in the Parameters tools, you specify the dimension. Each of the dimensions is only visible if the corresponding dimension is present in the input image.

Create Image Subset and Split opens the resulting images in the ZEN document area. Therefore, for this image processing function, Split Dimension is limited to a maximum of 20, e.g. timepoints, tiles, or slices. To split images along a dimension with more than 20 elements, use Create Image Subset and Split (Write files), which creates image files in the specified folder.

1. You have loaded an image with more than one channel and not more than 20 timepoints.

1. Select Processing tab > Method tool, and select under Utilities the entry Create Image Subset and Split or Create Image Subset and Split (Write files). When using the first option, you can specify the output in the Output tool.
2. To create a subset from the available dimensions, make your settings in the sections Channels and Time.
3. In the Parameters tool, in the Split Dimension section, select the dimension along which you want to split the image, e.g. Channel or Time.
   In the Channels list, all channels of the loaded image are listed. Unselect channels you want to exclude.
   In the Time section, select the subset of timepoints which you want to include. If you have an image with more than 20 timepoints, and you limit the range to below 20 elements, Time will appear in Split Dimension allowing you to split the data set along this dimension (for Create Image Subset and Split).
   In the Region section, select the region which you want to extract from the image, e.g. Full or Rectangle Region.
   Select Rectangle Region and draw a rectangle in the 2D view to specify the region you want to extract.
   When you use Create Image subset and Split (Write files), you also need to specify the output folder for the images.
4. In the Input tool, specify whether you want to set the input automatically and which image you want to display after processing. Check the according options.
5. In the Output tool, specify how to proceed with the new image. Select if you want to overwrite an existing image with the same name or if you want to create a new output. The output tool is only available, if you selected the Create Image Subset and Split method.
6. Click the Apply button.

1. The new images are created according to your specifications, see Create Image Subset and Split or Create Image Subset and Split (Write files).

The image processing functions Analyze to Label Image and Analyze to Label Image Batch label images based on an existing image analysis setting and the parameters which you select from the function. For general information about the image processing, see Image Processing Workflow. For a description of all the parameters of Analyze to Label Image, see Analyze to Label Image and for Analyze to Label Image Batch, see Analyze to Label Image Batch.

The image analysis with these functions offers a wide variety of parameter options and the outcome completely depends on the combination of them. Therefore, this chapter can only give an example to illustrate the use and outcome of the function. In general, the labeling is possible as regions, contours, and double contours. For all three the same options are given in Label Mode.

As an example, the following two channel image and image analysis setting with three classes are used to illustrate the functions:

Example: Applying One channel and One channel per class

With these options, you can initialize your output image either as an image with one labeled channel or as an image where the labels for each class have a separate channel.

Labeling regions with the maximum pixel type as an 8 bit black and white image generates the following output for the first class:

Labelling the other two classes with the option One channel per class additionally creates the output for the other two classes:

If you want the regions to have the same color as defined in the image analysis setting, you can, for example, choose the option Region - Region Class Color and 24 Bit RGB as Pixel Type:

As stated before, you can choose the same options (labeling with pixel type maximum and region class color) for contours or double contours as well.