zen lite manual 6e 06-image analysis - zmb uzh · image analysis 6 zen lite 2012 manual after...

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ZEN lite 2012 Manual 1

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In the module Image Analysis you will use a Wizard, to create with ZEN lite (blue edition) a measurement program to perform automatic measurements. In this wizard you will define in 7 steps all pre-adjustments for the pre-processing of the images, the object detection via thresholds, the further processing of the resulting binary images, the definition of the parameters to be measured and finally the measurement of all objects.

At last you will save this created measurement program for a later usage. You may change and adapt all measurement programs to new conditions at any time. All intermediate steps you may set as interactive in order to adapt the variables (i.e. thresholds or measurement frames) to the specific needs during the automatic process.

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As an introduction and to explain the different adjustment possibilities of each intermediate step of the Wizard we will use an image of Beads. The acquired image has been loaded and is displayed as active image in the Center Screen Area.

Image Analysis

2 ZEN lite 2012 Manual

In order to use the module Image Analysis the module has to be licensed and activated. Start under Tools the function Modules Manager.

Please check under ZEN lite if the module Image Analysis has been selected.

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If in addition to ZEN lite you have activated the module Interactive Measurement and/or the module Image Analysis, you will additionally get the Analysis tab in the Left Toolbar. Open the field Image Analysis and start Setup Image Analysis to get access to the wizard.

Under Program you load an existing image analysis setup, or you enter the name for a new program under the options via New.

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For our example we entered the name Beads, that we stored via the Save-icon for a future use.

Next, via the button Setup Image Analysis, you start to define the sequence of the automatic measurement program.

With this button you open the Image Analysis Wizard.

The Image Analysis Wizard is organized in 7 separate steps. In each step you will define the settings for the automatic image processing and object measurement. Later in the run phase you may skip single steps. In some steps you may define a stop command, in order to interactively adapt some setting variables.

Image Analysis


Step 1: Classes

For the definition of a class select Class 1 in the hierarchic tree and click to the button Add class.

This will add the channel name from the open image to the class (in the example C1).

If the channel is already defined by a name different to the channel name of the image, you first have to delete the class via the button Delete class (see also example 2 Epithelial cells, chapter 6.6.8).

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Finally assign a color for the binary image display in the later image overlay to the class.

Open the color selection and select one of the pre-defined colors, or define an alternative color via the button Custom.

Proceed to the next step via one of the buttons Next.

Step 2: Frame

In step 2 you will define a measurement frame. You may select a rectangle, a circle or a polygon. You may select already existing frames using the arrow function and delete them individually.

Image Analysis


After selecting the frame type you first have to draw the frame shape in the image. Then you may change the position (Left, Top) and size (Width, Height) via the input fields. Under Mode you have the choice if you want to exclude objects from the measurement that are cut by the frame, or if you want to cut the objects at the frame border.

Select the checkbox Interactive, if you want to define the measurement frame interactively in the running program. The running program will pause at this position.

With the button Next you will proceed to the next step.

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Step 3: Automatic Segmentation

Via Automatic Segmentation you create a binary image for the measurement of single objects. You may select several image pre-processing steps for a better recognition of the objects.

Under Smooth you have the choice from different grey-image operations. Under Sharpen you use edge filters. Additionally you may exclude all small objects via the preset under Minimum Area.

In the section Threshold you segment the objects by their grey values. Under Threshold definition you will set the start values at its best via the button Automatic. You will see the upper and lower threshold range shown in the histogram. You may interactively change these limits in the Histogram. As an alternative select under Pick Behavior +, to add grey regions or – to delete grey regions.

In the resulting binary regions you may fill holes by selecting the Fill function, or you cut touching objects by selecting one of the Separate functions.

Image Analysis


After a click at the cursor position as a result you will see the binary image overlay via the selected grey values in the surrounding of the cursor. You will find the threshold limits in the histogram. You add unrecognized object areas by further clicks, or by changing the limit lines in the histogram.

You may select the Lowpass-Filter to smooth the grey values prior to the threshold settings. As an alternative you may select a Gauss or a Median filter.

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Result after increasing the steepness of the object borders via an Edge Enhancement. In our experiment we used the Unsharp Masking filter.

Finally exclude under Minimum Area all objects smaller than10 from the measurement. This will avoid resting noise points or small objects close to the objects borders to falsify the measurement.

Result of the binary image overlay after selecting grey values by a click into one object at the cursor position. The threshold limits are indicated in the histogram.

Image Analysis


By selecting the function Fill you will close all holes in binary regions.

By selecting the Separate function Watersheds you automatically will separate all touching objects.

Finally you may optimize the object recognition by adapting the thresholds in the histogram.

Please select Interactive if you eventually want to control and re-adjust the thresholds with each new image.

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Step 4: Condition

You may define conditions via measurement parameters to include or exclude objects from the measurement. For this option you accept the function Edit.

For the beads we used a circularity with values from 0.966 to 1.000. This will exclude edge-cutting and touching objects from the measurement.

Step 5: Interactive Segmentation

Here you process the binary image prior to the measurement interactively. Selecting Draw you create objects manually. For this function you may use different graphic tools.

Via Erase you will delete objects from the image, Cut will separate touching objects, Merge will join separated objects, and Fill will close holes in the selected objects.

Using the automatic segmentation you will usually skip this step. For this simply de-select the checkbox Interactive.

Image Analysis


Step 6: Features

Via Region Features you select those parameters that shall be displayed with each object in the resulting image. For this click to the button Edit.

Under Annotations you select the graphic elements that shall appear in the resulting image. For the selection click to the button Edit.

In our example the Draw Center was selected to be displayed for each object in the resulting image.

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Note: You have to check the selected Features under Display, in order to use it as a label in the resulting image.

For labeling in the image you may select several measurement parameters. Those parameters that should be shown in the resulting image you have to check under Display ( in the example: Region ID).

After you have defined all Features and Annotations proceed via Next to the last definition step.

Image Analysis


Step 7: Measure

In the last step the measurement will be performed and the annotations will be shown in the resulting image. Please check again the correct performance before you click the button Finish.

With the Finish button you will close the Wizard and save your definitions as a measurement program under the pre-defined name.

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Now check your created measurement program via the button Analyze Interactive.

The program will pause at all steps defined with Interactive in order to perform changes.

In our example program only for the threshold setting a pause was defined. You may adapt the thresholds in the Histogram for a correct object recognition. Click the button Finish to perform the remaining program steps.

Image Analysis


With the end of the program run the resulting image including binary overlay and parameters (centers of object and object IDs) will be displayed. Aside the image the results table with all measurement values is shown.

Whenever you did any changes during the measurement procedure that you also want to use in the future, please save the program again under Program (this will overwrite the old values), or save under a new name.

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If you do not need any changes during the running program start via the button Analyze. All steps with defined Interactive function will now be skipped, and the pre-defined values will be used.

As a result also in this case the image will be displayed with all binary overlays and parameters. In addition you will also get the table with the measurement results.

For a further processing of the measurement results click to the button Create Tables on the Analysis tab under the display-options.

Image Analysis


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You will receive two data documents by default. On the Regions tab you will find all summarized values (field data) from the image as a table. In our example this is just the Count (number of objects).

On the tab Region you will receive a table with all measurement values for each single object and for each defined parameter in the image.

You have access to graphic presentations of the values. To do so select one column in the table, click to New Data Source under Data source, and finally under View select the table graphic and the diagram type. You will find the description for examples with the interactive measurements (chapter 6.5).

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With large number of objects mostly size distributions by number are required. For these distributions first select the Processing tab.

Under Method select Calculate Histogram in the region Extras.

From the setup possibilities select the type of Class Boundaries, the Class Count (in our example 16), and finally the Display Mode (count, percent, sum, cumulative; in our example Count). You may select single Columns via the input of the column number (1, 2, 3, or 4 instead of “All”), or via the selection

button .

Image Analysis


For the execution click the button Apply.

As a result for each measurement parameter you will receive a table with the calculated class limits and the count per class.

For a single parameter select the corresponding column in the table, and click under options on the Data Source tab the button New Data Source.

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In some applications logarithmic class limits are preferable for an optimum distribution.

For these distributions select the function Logarithmic under Parameters.

Now you will receive a count distribution with logarithmically calculated class limits.

For our example we selected under Display as a Diagram Type the Line-diagram.

Image Analysis


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For this example a two-channel fluorescence image of epithelial cells was used. As channel names we used DAPI and FITC corresponding to the staining.

We started the evaluation using the program Beads. In this program the channel name C1 was defined. Therefore we receive a message, as the channel C1 is not existing in the active image.

In this case proceed to step 1 Classes to define the correct channel name.

First select Class 1 and click to the button Remove Class. This will erase channel C1.

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Now click to the button Add Class.

This will change the channel name to the name of the first channel of the active image (DAPI).

Next adjust the thresholds to the recognition of the nuclei according to the description above.

To separate touching nuclei we selected in this example the Watersheds algorithm.

The Navigator will be of help checking the accuracy of the thresholds in the enlarged part of the image.

Image Analysis


In step 6 Features define the desired parameters via the button Edit under Region Features.

Now perform the measurement (in our example using Analyze).

As a result you will receive the table with all your defined measurement parameters for each object. For a further processing proceed as described in the beginning (chapter 6.6.6).

zen lite manual 6e 06-image analysis - zmb uzh · image analysis 6 zen lite 2012 manual after...

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