GSA_Image_Analyser_eng

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GSA Image Analyser .................................................................................................................3

1. General information ...............................................................................................................3

2. Description of functions ......................................................................................................... 3

2.1 Creation of graphic data ................................................................................................... 3

2.2 Selection of devices.......................................................................................................... 3

2.3 Scan ..................................................................................................................................42.4 Twain input mask .............................................................................................................5

2.5 Filemanagement ...............................................................................................................5

2.5.1 Open files ..................................................................................................................5

2.5.2 Close files..................................................................................................................5

2.6 Create image objects ........................................................................................................ 5

2.6.1 Create images ............................................................................................................6

2.6.2 Calibrate input devices .............................................................................................. 6

2.6.3 Draw objects..............................................................................................................7

2.7 Video interface .................................................................................................................8

2.8 Stack processing ...............................................................................................................9

2.8.1 Data file list ...............................................................................................................92.8.2 Create a stack ............................................................................................................9

2.8.3 Monitor windows .................................................................................................... 10

2.8.4 Changing alpha values in the stack ......................................................................... 10

2.8.5 To apply an image for the purpose of analysis........................................................ 11

3. Work space (canvas) ............................................................................................................ 12

3.1 Colour indication and defining of limit values for object identification........................ 13

3.2 Apply limit values to images.......................................................................................... 15

4. Analysis functions ................................................................................................................16

4.1 Area ................................................................................................................................ 16

4.1.1 Output of determined object areas according size groups....................................... 17

4.2 Density ........................................................................................................................... 18

4.3 Length............................................................................................................................. 18

4.4 Object counting ..............................................................................................................19

4.4.1 Settings for object counting of overlapping objects................................................ 20

4.4.1.2 Separation of overlapping objects by object area comparison............................. 21

4.4.1.3 Separation of overlapping objects by intensity comparison................................. 21

4.4.1.4 Separation of overlapping objects by object recognition (circle or ellipse)........ 22

4.5 Number of intersection points with a raster ................................................................... 22

4.6 Manual counter............................................................................................................... 23

5. Colour distribution ...............................................................................................................25

6. Settings ................................................................................................................................. 256.1 Limit values for object identification............................................................................. 25

6.2 Units ............................................................................................................................... 26

6.3 Cursor and raster ............................................................................................................ 26

6.4 White balance................................................................................................................. 26

6.5 Language........................................................................................................................ 27

6.6 Dpi and colour depth ...................................................................................................... 27

6.7 Magnification coefficient ............................................................................................... 27

6.8 Scan method ................................................................................................................... 28

6.9 Save image data..............................................................................................................28

7. Extra .....................................................................................................................................28

7.1 Cut and count ................................................................................................................. 287.2 Calibrate ......................................................................................................................... 29

7.3 Loupe.............................................................................................................................. 30


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7.4 Measurement Scale ........................................................................................................ 30

7.4.1. Hide and unhide ..................................................................................................... 30

7.4.2 Position.................................................................................................................... 30

8 ? ............................................................................................................................................. 31

8.1 Registration .................................................................................................................... 31

8.2 Help ................................................................................................................................ 328.3 Update ............................................................................................................................ 32


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GSA Image Analyser

1. General information

The GSA Image Analyser is a tool specially designed for the creation and analysis of 2D

image objects. The objects can be identified on the basis of their specific colouration and bedistinguished in this way from their environment. For an analysis the following automatic

functions are provided :

number of identified objects

2D object area computation

object area ratio to total area (density)

determination of object length

counting of intersection points in a raster

visualization of colour distribution

Additional functions : manual counting by marking areas

manual counting by image splitting

creation of images

image processing functions

2. Description of functions

2.1 Creation of graphic data

To create the required graphic data a Twain interface is provided. By means of this interface

all optical devices such as scanner, digital cameras, video cameras or microscopes can be

controlled by the program.

2.2 Selection of devices

To select the needed Twain device you have to use the menu item File > Select Source. A

window appears listing all existing devices.

Figure 1. Device selection window


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2.3 Scan

Use the menu item File > Scan in order to open the desired device program for the creation

and export of image files.

Figure 2. Scan mask

In figure 2 a control program of a Canon colour picture scanner is shown.

Detailed operating instructions have to be extracted from the program documentation of theproducer.

In general you have to consider which colour mode or resolution of the target file is selected.

The colour mode selection depends on how individual image objects differ from each other. If

a greyscale analysis allows a clear differentiation so the mode Greyscale can be reasonable.

Otherwise the mode Colourhas to be selected.

Concerning the image resolution you have to take into account that a higher resolution leads

to a more precise analysis on the one hand. On the other hand it requires a longer processing

time and more memory capacity.

Devices used for enlarging images (e.g. microscope) require the input of a magnification

factor. A corresponding window appears automatically or can be opened by the menu item

Options > Magnification factor(see chapter 6.7).

Figure 3. Input mask magnification factor Figure 4. Control mask - Mikokular (microscope)

If the magnification factor is unknown, such as at photographs made by a digital camera, it is

necessary to take a test photo with a scale (e.g. ruler). The magnification factor then can be


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determined on the basis of the difference between the scale and the length measuring of the

scale done by the program after scanning (see chapter 4.3).

2.4 Twain input mask

To use the Twain input mask of the selected Twain device you have to activate the option File

> Show twain input mask. Otherwise it is tried to import the image on the basis of standard

settings :

Scanner (default):

resolution 100 dpi

colour depth 24 Bit

width 210 mm

length 297 mm

Mikrokular (microscope)

resolution 320 x 240 dpi

colour depth 24 Bit

2.5 Filemanagement

2.5.1 Open files

By default the image file formatMYP is offered.MYP is a program internal format, including

a packed image without loss and additional needful information such as the magnificationfactor and more.

Image files of the formats BMP and JPEG can be loaded too. In this case parameters

concerning resolution, colour depth (menu item Options > dpi and resolution) or

magnification factor have to be defined manually (see chapter 6.6 and 6.7).

2.5.2 Close files

By default image files are stored in the format MYP (see chapter 2.5.1).

It is also possible to store it in a BMP format, but then all additional parameters(magnification factor ) get lost.

Please note : The image of the work space is stored in WYSIWYG mode. If the image should

have been changed, these changes are stored too, e.g. raster display or display mode

black/yellow (see chapter 6.3 and 3.2).

2.6 Create image objects

This function provides the area computation of objects whose contours are captured by a

graphic input device.


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2.6.1 Create images

To create the image please select the menu item File > Create.

Figure 4.: Creation of an image

The creation of a drawing area (A2 A5) requires a calibrating of the input device (seeCalibrate input device ).

2.6.2 Calibrate input devices

With this function the resolution capability of the mouse is determined (DPI) .

Figure 5.: Calibrate the input device

1. Select calibration distance and unit2. Press the left mouse button and go along the section of measurements

Press the button System parameters mouse in order to configure the mouse, if the resolution of

the mouse should be too large. A DPI value bigger then 500 leads to a slowdown of the

program execution, because the image size has to be matched with these settings.

Select the button Finish Calibration for applying the determined settings.


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2.6.3 Draw objects

It is possible to draw objects either on an empty work space (format DIN A2 A5) or on a

loaded template.

Objects can be generated by two different methods :1) Freehand2) Connected lines

1) Freehand

Set the mouse on the object (arrow or the like) first, press the left mouse button then and go

around the object with the mouse button keeping pressed.

After releasing the button the starting point will be connected automatically with the end point

and the object is filled.

Please note : The starting point is located in the upper left region of the drawing area. The

visible changes automatically when drawing (within the format definition A4, A5). If the

defined working space is left while drawing a system tone raises.

Press the buttonApply to make the created image available in the main program (only

available in a full version) for an automatic computation of the object area.

2) Connected lines

Set the mouse on the object and press the left mouse button. The starting point is marked on

the drawing area as a small black point. Move the mouse to the next object point and press the

left button. The points are connected automatically. Additional points have to be marked in

the same way.

Press the right mouse button at last to connect the starting and end point. The object is filled

automatically.Please note : Keep the left button pressed to activate the auto scroll function. In this case press

the right button to mark single points. Both buttons have to be released to finish the process.

Press the right button once again and the object will be shown as described above. If the

defined work space is left a system tone raises.

Press the buttonApply to transfer the created image to the main program (only available in a

full version) for an automatic computation of the object area.

The NUM buttons 4,8,6 and 2 can also be used for scrolling.


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2.7 Video interface

As shown in figure 8 it is possible to import images directly

by diverse video equipment such as frame grabber, video grabber etc.

Figure 6. Video

Select the desired video input and please enter the camera resolution

and magnification factor into the provided fields (seel also chapter

7.2 Calibration). Press then the button 'Picture' to import

the image as a snapshot in your programme.


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2.8 Stack processing

In stack processing mode multiple images (e.g. image sequences) can be opened concurrently

in a stack. The target is to generate a new image resulting from multiple source images.

Figure 7. Opening in batch mode

2.8.1 Data file list

An unlimited number of images can be listed to generate the desired image stack.

The list entries can be sorted either automatically (click on column header) or manually (use

right mouse button or button on the left side of the table or function keys F5 to F8). It is

furthermore possible to rotate an image within the defined stack. To display an image on the

monitor on the left side simply click on the corresponding list entry. All created lists can be

stored for later processing.

2.8.2 Create a stack

With a click on the button Create stack all images of the list will be merged into a stack in

the given order. The result is to be seen on the monitor on the right.


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2.8.3 Monitor windows

The monitor windows are intended to control images respectively to select images from the

list. Several options for image scaling are offered. The mode Scale allows to zoom in and

out by the function keys F9 or F10.

Figure 8: Monitor windows

2.8.4 Changing alpha values in the stack

It is possible to change the visibility (alpha values) of single stack images in order to createindividually a new image.

2.8.4.1 To change alpha values harmonically

Figure 9: To change alpha values harmonically

With the left slide control it is possible to define the master image of the stack. For example,

there are 10 images in the stack and the controller is at five, that means the 5th

image is fully

visible. All other images, lying before or behind, are damped visible corresponding to the

slider on the right. Concerning our example we have the following alpha-values:

Image 1 : 0; Image 2 : 21; Image 3 : 99; Image 4 : 177; Image 5 : 255

Image 6 : 177; Image 7 : 99; Image 8 : 21; Image 9 : 0; Image 10 : 0

That means, the images 1, 9 and 10 will have no influence on the resulting image.


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By moving the left slider you can go through the stack. The image blending is, corresponding

to the setting of the right slider, more or less fluently (on 100 % the master image is equal to

the output image).

2.8.4.2 To change alpha values individually

Figure 10: To change alpha values individually

With this setting you are able to set alpha values for every single image of the stack

individually. The result (a blended image) is displayed in the right monitor window. An alpha

value of 255 means a high visibility, 0 means invisibility.

2.8.5 To apply an image for the purpose of analysis

After the completion of the image the result can be transferred to the main program. Press the

button Apply to do so. You then will be asked to enter the image resolution, the file type

and the used image magnification manually. These values are the basis for the following area

or length calculation .


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3. Work space (canvas)

All images loaded or manually created are displayed on the canvas. Use the menu itemEdit >

Rotate 90 degrees to rotate the canvas by 90 degrees in a clockwise direction. This process

can be repeated as often as the user need to.

Additional menu optionsEdit (canvas)

Centre centering the image on the canvas

Filter selection of filters for image representation Nearest

Linear

Spline

Lanczos

Mitchell

Zoom for zooming in and out

Cut and paste to cut and paste a region marked by the mouse (keep right buttonpressed and shift mouse)

Toolbar image editing

Figure 7. Canvas Figure 8. Toolbar


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3.1 Colour indication and defining of limit values for object identification

Colour representation :

When moving the mouse pointer over an image the colour ratio of the touched pixels will be

shown in the fieldsRed, Green andBlue. To define a colour average of the relevant pixels

keep the key STRG pressed simultaneously. The automatically calculated average value willbe buffered and also displayed in the fields Red, Green and Blue. Please press the key S to

load the calculated average colour value into the windowLimit values for object

identification.

Figure 9. Average colour values and positive/negative maximum values

To apply the values shown in the fieldsRed, Green andBlue to the object identification you

have to press the buttonAdd( see figure 9 ). The colour values and the reached minimum

and/or maximum values are inserted into the table and are considered for the object

identification. Generally it is possible to add more values here.

When moving across the object it is advisable to enlarge the image in order to avoid to crossthe object edge.

Please note : Press the keyR to delete all entries of the colour value buffer. So a new average

colour value can be defined.

The procedure just described can be carried out also directly in the maskLimit values for

object identification. Look for the desired image detail ( if necessary zoom in ), keep the key

STRG pressed and move with your mouse pointer across the part of the image whose limit

value have to be determined. The result will be displayed in the right window by pressing the

key S ( the option Preview has to be activated before). To see the result already when moving

the mouse across the image area you have to activate the optionApply limit values

immediately. If the values shown in the fieldsRed, Green andBlue are to be used for the

object identification, then please press the buttonAdd.


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Figure 10. Analysis of three image regions and determination of the corresponding tolerance values for object identification

Background identification :

The determination of the entire object area requires a differentiation from background.

Proceed analogously to the method described in section Colour representation and save all

determined background colour values in the listLimit values for background identification.

Automatic extension of limit values:

Please note, for this function an active background recognition is essential.

This function analyses all pixels (yellow), which could not be classified

and tries to assign it automatically to one of the two groups (object or

background). Can an assignment be made the limit values of the corresponding

group will be extended in a way that the pixel matches the group.

By activating the option "With distance consideration" also pixels can

be classified where an assignement of two colours in both groups would

be possible. In that case the choice of the group will

be made on the base of the minor distance of the third colour.


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Greyscale representation:

Principally the procedure is identically to those described before. Instead of the three basic

colour values a grey tone is shown. Here not the colour average of the relevant pixels is

determined, but the maximum value.

Figure 11. Limit value for object identification in greyscale representation

In figure 11 you can see some defined limit values for the identification of very dark (0-50)

and bright (200-255) objects.

Black-and-white representations:

Only objects with a greytone of 0 (black) are identified. All other values are ignored.

3.2 Apply limit values to images

In order to apply the determined limit values (see chapter 3.1) to the image you have to

activate the checkboxApply limit values to the image. A computation process starts and the

result is shown in form of a black/yellow representation. Black means, that the object could be

identified and used for the analysis. To check the identified objects you can press the button

Original Image or the key [O]. So you can switch between both representations (original andblack/yellow) very quickly.


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Figure 12. Original image and object identification

Areas represented in grey had been identified as the background. This area will not be

considered for the analysis (object area computation). The total object area (yellow and black)

will be schown in the lower status bar.

4. Analysis functions

4.1 Area

The area of all identified objects of the image is computed and displayed considering the

selected unit of measurement (see chapter 6.2). The computation can be restarted by pressing

the buttonReset.

An image detail can be picked for a more precise analysis. Mark the desired image detail with

the right mouse button keep pressed or use the menu itemEdit > Cut and paste.

Within the representation of the applied limit values it is possible to click directly on the

objects of interest. The corresponding area is computed and the selected object is displayed in

another colour (Red). Furthermore the number of pixels is shown in the lower status bar. This

value will be used in the process of an automatic object counting.

Additionally you have the ability to research a custom area with a free-hand-drawing tool.


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Figure 13. Area computation of an object

4.1.1 Output of determined object areas according size groups

This function checks all determined object areas of the image and

arranges them according to size. To call this function please

press the key 'Calculate' in the toolbar for object counting (seechapter 4.4). Object areas which do not meet the specified

criterias for minimum and maximum number of pixels are not included

in the output.

Figure : Option 'Output of object areas according size groups'

The dimensions of the used size groups are freely selectable.

For that the desired number of pixels is to be specified(the resulting area value is displayed in brackets). The total

number of size groups then results from the largest object of the image.

For example:

Number of pixels of the largest object: 8680 pixel

dimension of classes in pixel: 500 (14,33 mm)

18 size groups will be generated.


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Figure: Output of the area analysis

4.2 Density

This function computes the proportion from object area (all identified object) to total image

area. Press the button Computate (available only in mode yellow/black representation) to start

this process. In general it provides the proportion from the object area ( yellow and black ) to

the determined area (black). If you did mark single objects (by mouse click) then the

proportion from the marked objects to the total object area (yellow and black) is provided.

4.3 Length

A manual measurement of the object length can be made by the left mouse button keepingpressed. The length of the covered line will be shown in the fieldLength depending on the

defined unit of measurement.

All measured values will be added simultaneously in the field Total. Press the button Reset in

order to reset the calculated value.

To start an automatic calculation of the length of all identified objects please press the button

Calculate (only available in the yellow / black representation ). As a result you will see the

contours of the corresponding objects on the image (see chapterLength determination of plant

roots ). Again you can use the free-hand-drawing tool for custom areas in this view.


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Figure 14. Automatic object length calculation

4.4 Object counting

With this function all identified objects can be counted automatically. The number of objects

can be reduced by using the optionsMinimum number of pixels per objectand Maximum

number of pixels per object. Click on the single object to determine its number of pixels.

Figure 15. Object counting

This function is only available in the yellow / black representation. All counted objects can be

displayed in red colour by pressing the button Show objects.

Additionally you have the ability to research a custom area with a free-hand-drawing tool.


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Figure 16. Presentation of the counted objects

4.4.1 Settings for object counting of overlapping objects

The separation of overlapping object can be made by comparison of either object surfaces or

intensities. In addition, there is the possibility to separate circular or ellipsoidal objects by

means of object recognition.

Figure 17. Settings for object separation


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4.4.1.2 Separation of overlapping objects by object area comparison

The reference area can be specified by hand. The specification is made in pixel. Its best to

click on the reference object within the yellow/black presentation to determine the number of

object pixels and to fill it into the corresponding field automatically.

The second possibility for specifying a reference area is to determine either the average value

or the median of the object areas. All objects fulfilling the conditions described in chapter 4.4

are now compared with the reference area. Whole divisors are increasing the object counter,

the remainder will be treated depending on similarity defaults (sliding control).

Figure 18. Object separation by using the average value of object areas

4.4.1.3 Separation of overlapping objects by intensity comparison

Another way for counting overlapping objects is by using the object intensity. This option is

available individually or combined with area separation (chapter 4.4.1.2) or object recognition(chapter 4.4.1.3). Completely overlapping objects so can be counted separately providing that

the resulting intensity differs from the intensity of the single objects.

If both options (intensity and object area) are used the separation by object area comparison

takes priority. This means that only objects are treated by intensity comparison which could

not be separated by object area comparison. The sensitivity of the intensity comparison (total

intensity of all objects, AVG and/or median) to the average object intensity can be controlled

by means of a sliding control.


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Figure 20. Image with counted intersection points

4.6 Manual counter

Here the objects can be counted manually (see also chapter 6.9).

Figure 21.: Scan method manual counter

Please select Options > Scan method> Manual counter.


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Afterwards you can specify the size of marking with Options > Marker. This is a circle,

which is drawn around the clicked area. The colour of marking can be set over Options >

Marker > Colour for marking.

With every left mouse button click the corresponding position will be marked and the counter

counted up. The last counting can be reset if you press the Button Undo.Please press the buttonResetif you want to delete the image and counter.

Figure 22.: Manually counted cell nucleus


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5. Colour distribution

This function provides an analysis of the colour distribution of the working space. This can

be helpful to determine the limit values for object identification.

Figure 23. Colour distribution 3D view

The Maneuvering within the representation can be done over a bar of buttons or by mouse.

Figure 24. Button bar

For zooming in and out please use the corresponding loupe buttons. For marking a specific

part you have to mark it by mouse with the left button keep pressed.

For scrolling please use either the corresponding arrow buttons or the mouse function when

keeping the right button pressed.

A changing between work space and colour distribution view can be made by the menu

optionsExtras > Determine colour distribution orExtras > Show work space.

6. Settings

6.1 Limit values for object identification

All limit values for object identification determined manually or automatically for the colour

and/or grey tone analysis can be managed here(see 2.3.1). The values are stored at the end of

the program and loaded automatically with the next start.


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6.2 Units

The following units of measurement for the output of the result can be selected : meter,

millimetre, centimetre and/or inch.

6.3 Cursor and raster

When marking an image region or measuring the object length by mouse, the marked region

is drawn. These markings get lost when releasing the mouse button. To show it permanently

use this menu option.

When counting intersection points the raster used is placed on the image. The drawing can be

switched off too.

6.4 White balance

With this function an automatic image import is made by the selected device. No objects

should be located on the image. The maximum values of the found pixels (grey tone) are

indicated and can be used for a better definition of limit values in the process of the object

identification. Ideally the value should be 255 (white) for a scanner, that means no objects

(e.g. dust) was found. If the determined value should be smaller than 255, then an object

identification is only possible below the measured value.

For example :

Figure 25. Recommended threshold value for object identification

A detailed evaluation can be made by the chart of the colour distribution (see also chapter 5).


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Figure 26. Colour distribution - total and a range around the grey tone 74

6.5 Language

The offered program languages are German and English.

6.6 Dpi and colour depth

To set the required resolution and colour depth you can use this menu item.

When creating images by a twain interface or loading MYP images this will be done

automatically. The loading of formats like BMP and JPEG requires a manual input of thesevalues. The results of the analysis depends directly on the parameter settings for image

resolution

Figure 27. Resolution and colour depth

6.7 Magnification coefficient

The magnification coefficient as well has a big influence on the analysis results. The manual

input is required if images are created by a microscope or a digital camera (see chapter 2.3).


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6.8 Scan method

Use this menu item to select one o the following analysis method :

* 2D area calculation or

* counting raster intersection points (see chapters 4.1 and 4.5).

6.9 Save image data

If this option is selected, the user will be requested for saving the image date after scanning or

creating. So the original image data can be stored automatically.

7. Extra

7.1 Cut and count

This menu item provides a tool for the manual counting of image objects. The current image

file will be divided into subframes according to the setting given in field Cut after.

Figure 28. Setting for image cutting

Press the button Cutin order to cut the image and to reset the counter readings.

Figure 29. Cut and count

All subframes are listed in the left scrollbox. The active frame marked with a red border will

be displayed on the work space.


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In the example shown in figure 29 the image was cut into 15 subframes. By means of the keys

5 (forward) and 4 (back) a change of the active subframe is possible. The counter will be

updated by the keys + or -.

All functions can be controlled over a popup menu.

Figure 30. Popup menu right mouse button

Press the buttonReset counterin order to reset the counter of the active subframe.

The total counter reading will be shown after reaching the last subframe.

7.2 Calibrate

With the help of this function the DPI parameters can be matched with the actual proportions.

This will be necessary for instance if the magnification factor of a digital camera is unknown.

An image containing a scale is needed here (see figure 31).

Figure 31. Image calibration by scale


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1. Select calibration distance and unit

2. Press the left mouse button and go along the line3. Press the button Finish calibration at the end

7.3 Loupe

This function will open an additional loupe window.

The magnification factor is freely selectable.

Abb.: Loupe window

7.4 Measurement Scale

A measurement scale is displayed in the opened image. Position and dimension of the scale

are freely selectable. The unit and distance of the scale can be choosen by using the menu

item Options -> Measurement Scale.

7.4.1. Hide and unhide

With this function the measurement scale can be hided and unhided. The display of the

measurement scale has no effect on any analysis functions.

7.4.2 Position

The measurement scale can be freely positioned within the main image. With the position

window opened the user can define the start and end position by mouse click. To do this move

the mouse to the desired starting position in the image and press (and hold) the left mouse

button. Now drag the mouse to the end position and leave the hold of the left mouse button.

The measurement scale will be displayed then. After closing the position window all analysisfunctions will be available again.


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Abb.: Measuremnet scale

8 ?

8.1 Registration

Here the user is requested to enter his registration data. Please enter the received licence

number and registration name. After a successful validation the demo program will be

converted to a full version.

Figure 32.: Registration


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