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Calibration of the image Z Value

Calibration of the image Z Value 2


Z Value Calibration
The Define Z Calibration dialog box ..........................................4
Compared to the Calibrate Image... command .........................7

Step-by-Step Instructions 9

Defining a Z Calibration using a Calibration Standard ..............9


Conducting a simplified re-calibration .....................................11
Z Calibration of an Image ........................................................13
Z Calibration of an Input Channel ............................................14

Integrated Z Value 14
ZKal0300E
Calibration of the image Z Value

Calibration of the image Z Value


The Calibration of the image Z Value module is used when the gray values of your
images have a definite physical significance and you wish to define the relationship
between the gray values and the physical quantity.

Define Z Cali-
bration

After installing the Calibration of the


image Z Value module, the Define Z
Calibration... command will appear in
the Image menu beneath the Calibrate
Image... command.

Available This command is only available if an 8- or 16-bit image is in the active


image buffer.
Application Z calibration is necessary to be able to conduct a quantitative analysis
of brightness information in the same way that an X and Y calibration
is necessary for measurements of length or area within an image.
The relationship between gray value and Z value (a physical quantity
such as, eg, height, temperature, optical density) is generally not
simply linear, but rather is dependent in a complex manner on the
acquisition conditions (eg, illumination conditions within a micro-
scope) and on camera characteristics. This is why Z calibration - in-
volving use of a calibration standard - is necessary. Using the Define
Z Calibration... command in the Image menu you can conduct this
calibration with ease. You are given a lot of freedom to declare arbi-
trary and even unusual relationships between gray value and Z value.
What will Following the Z calibration of an image, the measurement of pixel va-
happen... lues using the Pixel Value or Intensity Profile commands (Measure
menu) provides a result that is not the gray value, but rather the value
of the declared physical quantity.

2
Calibration of the image Z Value

An image’s calibration is permanently linked to that image and is thus


saved along with it. This applies to both saving the image in a file
directory (using the Save As... command in the File menu) and when
you archive it in a database.
Example Using an infrared camera you have acquired the temperature distri-
bution of house facades. You have assigned the physical quantity of
temperature and a unit of °C to the gray values of your images. You
have determined which gray values correspond to which tem-
peratures by using a calibration acquisition. You have thus conducted
the Z calibration. When you now select the Pixel Value command in
the Measure menu and click on a number of pixels in the image
selected, analySIS® will enter the temperature values into the
measurement sheet directly. During the measurement procedure the
current gray value as well as the corresponding temperature value
appear at the lower left in the status bar.
How to... How to install the Calibration of the image Z Value module:
If your current analySIS® configuration is already making the Define
Z Calibration... command in the Image menu available to you, this
means that the module has already been installed.
1) Open the Special menu.
2) Select the Add-In Manager... command.
→ The Add-In Manager dialog box will be opened.
3) Select the Calibration of the image Z Value check box from the list
of Available add-ins.
4) Click on the Close button.
5) Now you’ll have to end analySIS® and then re-start it.

3
Calibration of the image Z Value

The Define Z Calibration dialog box


After initiating the Define Z Calibration... command in the Image menu, the Define Z
Calibration dialog box will be opened. This is where you conduct the calibration of the
Z value by assigning selected gray values to the numerical value of the physical quantity.

This is what the


dialog box looks like
when an 8-bit gray-
value image (gray
values 0 - 255) is in
the active image
buffer and it’s your
first time opening
the dialog box. The
default setting has 6
pairs of values en-
tered. These pairs
are comprised of
the gray value and
the assigned
Z value.

Diagram The diagram gives you a visual overview of the relationship between
the gray values and the physical quantity. The gray values are en-
tered on the horizontal axis of the diagram and the numeric values of
the physical quantity on the vertical axis of the diagram. Red X’s de-
note the position of the declared pairs of values. A small red square
indicates the value pair currently selected.
List of value pairs The list on the lower right includes all defined value pairs of gray and
Z values. If you have not yet conducted a Z calibration on an image,
the Z values appearing will simply be the gray values. You must al-
ways have a minimum of two value pairs defined.
Change gray- Select the Change grayvalue offset check box to make sure that
value offset when you alter the gray value of one value pair, the gray values of all
other value pairs will be altered by the same amount.
Application This check box makes re-calibration a lot easier in many cases. Let’s See also
just say, for example, that you’re working with a light microscope. How to...: Simplified re-
You have acquired a whole series of images whose gray values you calibration 11
have calibrated as optical density with the aid of a calibration standard
(a stepped gray wedge). If the acquisition conditions have changed
because you have increased illumination of the specimen, the pre-
vious calibration will no longer be valid. In this case you do not how-
ever have to redefine all value pairs for the now-necessary re-
calibration. You need to redefine just one value pair using the original
stepped gray wedge. You thus have to enter the new gray value for

4
Calibration of the image Z Value

only one value pair into the Grayvalue field, or, using the Hotspot
button you can read it out interactively from the image of the stepped
gray wedge. If the check box has been selected the gray values of
the other value pairs will be altered by the same amount. The re-cali-
bration is now completed. The shape of the curve displayed in the
diagram remains the same - only the coordinate system is displaced.
Error message You may receive the following error message when you have this kind
of global displacement of the gray values of all value pairs:

Check and see which value pair is the source of this error message:
which value pair has a gray value that would go beyond the valid value
range upon a further displacement? Gray values ranging from 0 to
65535 are permitted for 8- and 16-bit gray-value images for the cali-
bration. You have to delete this value pair before you can continue
the global gray-value displacement. You do this by selecting the
relevant entry by clicking on it with the mouse in the value-pair list.
You then click on the Delete button. The value pair concerned is ge-
nerally the first or last entry on the list.
Position You determine the position of the individual value pairs in the Posi-
tion group by entering the respective gray and Z values. The value
pair shown will be the one that you had previously selected via
mouseclick in the value-pair list.
Grayvalue The gray value of the value pair currently selected is shown in the
Grayvalue field. Enter a new value or use the little arrow keys if you
wish to alter the gray value.
An 8-bit gray-value image usually has a gray-value range of 0 - 255;
a 16-bit image of 0 - 65535. Entries greater than 255 are however
permitted in the Grayvalue field to make re-calibration easier. The
greatest value available is 65535, as is the case with 16-bit images.
Z Value The Z Value field contains the numeric value of the physical quantity
of the value pair currently selected. Enter a new value or use the little
arrow keys if you wish to alter this numeric value.
The Z Value field is not available if the Change grayvalue offset
check box has been selected. This is because in this case only al-
teration of the gray values is to take place.
Fitting function Select the functional relationship - from the Fitting function list - that
best describes the relationship between the pixel gray values and the
corresponding numeric values of the physical quantity. You have four
different polynomial fits available to you: of order 1, order 2, order 3
and order 4. The order corresponds to the number of the function pa-
rameters that are calculated by an optimization algorithm such that
the function best fits the whole of the coordinate pairs (the value pairs
defined by you).

5
Calibration of the image Z Value

Fitting function for If you have calibrated the gray values of your images, eg, to optical
optical density density, for example, then a polynomial of order 2 should be the fitting
function that is most suitable.
New Click on the New button to add another gray-value/Z-value value pair.
The currently-selected entry in the value-pair list will be duplicated
and can then be edited in the Position group fields or using the
Hotspot button.
Delete Click on the Delete button to remove the currently-selected entry from
the value-pair list. This button is only available if the list contains more
than two entries. This is because a calibration always requires at
least two value pairs.
Hotspot Click on the Hotspot button to interactively read out the gray value of
a value pair directly from the image when defining that value pair. For
a step-by-step instruction on the steps typically taken for Z calibration, See also
see below. How to...: Z Calibration
using a Calibration
Before clicking on First select - via mouseclick - the value pair from the list whose new Standard 9
this button gray value you wish to read out from the image. In addition, the active
image buffer should contain an image that is suitable for execution of
Z calibration. If you wish to calibrate the gray values to optical density,
then this image ought to be, eg, the acquisition of a stepped gray
wedge. This is comprised of a striped pattern of varying optical densi-
ties whose numeric values are known.
This kind of step-
ped gray wedge (or
"stepped density
filter") serves as
calibration standard
for a Z calibration in
which the gray va-
lues are assigned to
optical densities.
You read out the
gray values suc-
cessively using the
mouse-controlled
Hotspot. You have
to transfer the re-
spective optical
densities below the
stripes yourself.
What will After clicking on the Hotspot button the mouse cursor appears within
happen... the image located in the active image buffer. The "Hotspot" appears
next to the mouse cursor. The "Hotspot" is an elliptically-shaped area
with a plus sign and an ellipse indicating its center and size. You can
position the Hotspot by moving the mouse. You can alter size and
shape of the Hotspot by moving the mouse while keeping the left
mouse button depressed. Once you have positioned the Hotspot at
the desired image area, you rightclick to conclude the interactive gray-
value reading out. The mean gray value of the Hotspot area will auto-
matically be entered into the list of value pairs.
Use the middle mouse button or the [Esc] key to cancel this procedure
without having a gray value read out.

6
Calibration of the image Z Value

Quantity Enter the name of the physical quantity into the Quantity field. If you
conduct a pixel-value measurement in the calibrated image, this
name will appear as the header of the corresponding column in the
evaluation sheet.
Unit Click on the Unit... button to determine the unit of the physical quan-
tity. The Set Unit dialog box will be opened. This is where you can
select common physical units. If the unit you’re looking for isn’t there,
then you can enter any other unit you like (up to 8 letters) into the
Basic unit field. After clicking on OK you return to the Define Z Cali-
bration dialog box. The unit so determined will appear in the list of
value pairs as well as in the Z Value field.
If you conduct a pixel-value measurement in the calibrated image, this
unit will appear directly beneath the header of the corresponding
column in the evaluation sheet.
File Click on the File... button to save or load a Z calibration along with all
the value pairs and any other settings you’ve defined. The File In/
Output dialog box will be opened. This dialog box offers you the
option of saving Z calibrations independent of images to the hard disk
and of loading them.
Application Use the File... button to transfer the Z calibration of one image to
another. You’ll find the usage of this button described in greater detail
in the step-by-step instructions.

Compared to the Calibrate Image... command


Two Z-calibration There is another command in the Image menu which is also for con-
methods ducting Z calibrations but should not be used together with the Define
Z Calibration... command.
Calibrate Image The command referred to is the Calibrate Image... command which
opens the Calibrate Image dialog box. The Z Calibration tab then
offers you the option of defining a functional relationship between gray
values and Z values. You have a choice of 6 different functions whose
course you determine by entering two value pairs (referred to as
"Control points" in this context) and up to three parameters.
Define Z Cali- The additional Define Z Calibration... command provides you grea-
bration ter latitude for defining arbitrary and also unusual relationships bet-
ween gray value and Z value. This is why this command is advan-
tageous. Instead of just having two value pairs, you have as many
value pairs available as you like and whose number and position you
can define as you please. Instead of these pairs being combined with
a pre-defined function, they are combined with a polynomial fit that
adapts to the coordinates of the entered value pairs as optimally as
possible.
Application You can use both the Calibrate Image... command (Z Calibration
tab) and the Define Z Calibration... command to conduct a Z cali-
bration.
Calibration data is In both cases, the calibration data is managed as a part of the image
part of image and saved along with the image when you place the image in a file
directory or in a database.

7
Calibration of the image Z Value

Methods not to be If you wish to view or edit the Z-calibration data of an image which was
combined calibrated using the Calibrate Image... command then select the
Calibrate Image... command once again.
If you wish to view or edit the Z-calibration data of an image which was
calibrated using the Define Z Calibration... command then select the
Define Z Calibration... command once again.
Warning Calibration data can be lost if you use the ‘other’ command (ie, the
one you didn’t use for calibration) to view or edit the data. If you use
the ‘wrong’ command by accident and the ‘wrong’ dialog box is thus
opened, simply exit the dialog box by clicking on Cancel. This is how
you avoid losing any data. If you at this point click on OK, your data
will be partially lost.
Not compatible The reason for this possible loss of data is that when you click on OK
a re-calibration takes place using precisely that data that is located in
the fields of the newly-opened dialog box. If you’ve opened the
‘wrong’ dialog box that data cannot be complete because the two
calibration methods are fundamentally different and the calibration
data sets are not compatible for conversion.
For example, if you have an arbitrary number of freely-defined value
pairs, it is not possible to construct a simple mathematical function
(from these value pairs) which is defined by only two value pairs and
a maximum of 3 parameters. This is why only the declared physical
quantity and the unit appear in the Calibrate Image dialog box when
the active image was calibrated using the Define Z Calibration dialog
box. The remaining fields contain default entries.
Note The best thing for you to do is to decide for one of the two Z-calibration
methods. You thus avoid the described compatibility problems that exist bet-
ween the two methods.

8
Calibration of the image Z Value

Step-by-Step Instructions
Defining a Z Calibration using a Calibration Standard
How to... How to generate a Z-calibration curve for gray-value images via a
calibration standard and save it in a file:
You wish to acquire 8-bit gray-value images of backlighted specimens
using your light microscope in order to be able to examine their optical
density. You conduct the Z calibration necessary here (declaring
which gray value corresponds to what optical density) using a cali-
bration standard. This calibration standard is usually a stepped gray
wedge that, in our example, is to have eight different optical densities.
1) Place the calibration standard in the specimen mount of your micro-
scope.
2) Make sure that acquisition conditions are good while keeping an eye
on the live-image quality on the PC monitor.
→ To do this, select the input channel for your microscope using the
Set Input... command. The online histogram can be very helpful
for controlling illumination. You activate the online histogram by
using the Configure Input... command and then by selecting the
corresponding check box in the Display tab. The live-image ac-
quisition is started by using the Acquire command. You terminate
live-image acquisition via the Snapshot command. All com-
mands mentioned here are located in the Image menu.
3) Select the Snapshot command in the Image menu.
→ An image of the calibration standard will be acquired and written
into the active image buffer.
4) Check the image quality once again: is the brightness distribution of
the backlighted calibration standard clearly visible in the gray-value
image? There should be no saturation.
→ As needed, you can apply the Intensity Profile and/or the
Histogram... commands in the Measure menu to quantitatively
check the gray values.
→ If you’re not satisfied with image quality then repeat step 3 at
improved acquisition conditions.
5) Make no further adjustments to acquisition conditions once you’re
satisfied with image quality.
6) Select the Define Z Calibration... command in the Image menu.
→ The Define Z Calibration dialog box will be opened.
→ The fields contain the default entries. The default Z values appea-
ring are simply the gray values.
7) Enter the name of the physical quantity into the Quantity field. In our
example this name is "Optical Density" or the abbreviated "OD".
8) Click on the Unit... button.
→ The Set Unit dialog box will be opened.
→ This is where you set the unit for the physical quantity desired.
The "No Unit" default entry is the correct entry in the Basic unit
field because optical density does not have a unit.
9) Click on OK.
→ You will return to the Define Z Calibration dialog box.

9
Calibration of the image Z Value

10) Click on, eg, the lowest entry (255 / 255,000000) in the list of value
pairs to select that entry.
11) Click on the New button two times.
→ This is how you obtain 8 value pairs from the standard 6 value
pairs you begin with. You need precisely this number of value
pairs if you wish to use all 8 steps of the stepped gray wedge for
the Z calibration.
12) Click on the first entry (0 / 0.000000) in the list of value pairs to select
that entry.
13) Click on the Hotspot button.
14) Position the Hotspot onto the brightest gray-wedge step - ie, the step
with the lowest optical density.

→ How you position the Hotspot and alter its size and shape is See also
described above in the section on the Hotspot button. You can What will happen, Hot-
cancel the measurement by middleclicking (with the middle spot, Define Z Cali-
mouse button) or pressing the [Esc] key. bration dialog box 6
15) Now rightclick once.
→ You will return to the Define Z Calibration dialog box.
→ The gray value that has been read out (the mean of the Hotspot
area) has been entered into the list of value pairs.
→ The read-out gray value will only then be shown in the Grayvalue
field of the Position group once you have clicked in that field.
16) Enter the corresponding numeric value of the optical density into the
Z Value field of the Position group.
→ As soon as you press the [Enter] key or move the mouse cursor to
another field this Z value will be included in the list of value pairs.
17) Repeat the steps beginning at step 12 for all further value pairs. This
is how you have one gray value after another of all the 8 gray-wedge
steps of the calibration standard read out via the Hotspot (always
entering the corresponding Z value yourself).
18) Then select the most suitable polynomial from the Fitting function
list.
→ The "Polynomial, order 2" entry should fit this example well.
→ Generation of the Z-calibration curve is now concluded. This
Z-calibration curve is applicable to images that have been ac-
quired using the same acquisition conditions as with the calibra-
tion standard.
19) Click on the File... button.
→ The File In/Output dialog box will be opened.
20) Enter a suitable name for the Z calibration into the File field.

10
Calibration of the image Z Value

21) Click on the Save button.


→ The data of the Z calibration curve will be saved and the File In/
Output will be closed.
→ You return to the Define Z Calibration dialog box.
22) Click on the OK button.
→ The Define Z Calibration dialog box will be closed.
You conduct the Z calibration of an image by transferring the cali- See also
bration data you’ve just saved to this image. To find out how to do How to...: Z Calibration of
this, have a look under the step-by-step instructions called "Z Cali- an Image 13
bration of an Image".
You can also have the calibration data you’ve saved automatically See also
transferred to each newly-acquired image. To do this, have a look at How to...: Z Calibration of
the step-by-step instructions on "Z Calibration of an Input Channel". an Input Channel 14

Warning This Z calibration only remains applicable to all subsequently- See also
acquired images as long as acquisition conditions are not altered. If How to...: Simplified re-
you, for example, adjust the microscope illumination to be brighter or calibration 11
less bright then you’ll have to conduct a re-calibration.

Note Please make sure that when selecting value pairs that you - as much as is
possible - cover the entire gray-value range. If you, eg, have not defined a
value pair close to the lower end of the gray-value scale, the curve will simply
be extrapolated into this range. This can result in inaccuracies when mea-
suring the Z value of dark specimen regions.

Conducting a simplified re-calibration


How to... How to conduct a simplified re-calibration using your calibration stan-
dard when illumination brightness of your microscope has changed:
You have readjusted the illumination brightness of your light micro- See also
scope. This is why the Z calibration that you had conducted using the How to...: Z Calibration
calibration standard is now no longer valid. You now need a re-cali- using a Calibration
bration for any images acquired afterwards. Standard 9
In our example the alteration of the acquisition conditions can be de-
scribed via an alteration to the gray-value offset. This is why a
simplified re-calibration is feasible.
You have saved the original Z calibration via the File... button in the
Define Z Calibration dialog box.
1) Place the 8-step calibration standard (the one you used to conduct
the original calibration) into the specimen mount of your microscope.
2) Select the Snapshot command in the Image menu.
→ An image of the calibration standard will be acquired and written
into the active image buffer.
→ If you from now on change the illumination conditions once again,
you will have to re-acquire the calibration standard using the
Snapshot command.
3) Select the Define Z Calibration... command in the Image command.
→ The Define Z Calibration dialog box will be opened.
4) Click on the File... button.
→ The File In/Output dialog box will be opened.

11
Calibration of the image Z Value

5) Select the name of the file containing the original calibration from the
list located beneath the File field.
6) Click on the Load button.
→ The File In/Output dialog box will be closed and you return to the
Define Z Calibration dialog box.
→ The settings of the saved original Z calibration have now been
loaded into the Define Z Calibration dialog box.
7) To select an entry, click on one of the 8 entries in the list of value
pairs.
8) Select the Change grayvalue offset check box.
9) Click on the Hotspot button.
10) Position the Hotspot onto the gray-wedge step corresponding to the
value pair selected.
→ How you position the Hotspot and adjust its size and shape is See also
described above in the section on the Hotspot button. You can What will happen, Hot-
cancel the measurement by middleclicking or pressing the [Esc] spot, Define Z Cali-
key. bration dialog box 6
11) Now rightclick once.
→ You will return to the Define Z Calibration dialog box.
→ The new read-out gray value (averaged from the area of the
Hotspot) has now replaced the previous gray value of the selected
value pair in the list of value pairs.
→ At the same time, all other gray values of the other value pairs
have been altered by the same amount.
→ The Z values are not altered.
→ The shape of the curve in the diagram has not changed; the
system of coordinates has simply been displaced.
→ In some cases you may receive an error message telling you that See also
this gray value is not available. Please have a look above to know Error message, Change
what to do in this case. grayvalue offset,
→ The generation of a new Z calibration curve is thus concluded. Define Z Calibration
dialog box 5
You can apply this Z calibration to images acquired under the
same acquisition conditions you’ve just used for the acquisition of
the calibration standard.
12) Click on the File... button.
→ The File In/Output dialog box will be opened.
13) Enter a suitable name for the new Z calibration into the File field.
14) Click on the Save button.
→ The data of the new Z calibration curve will be saved and the File
In/Output dialog box will be closed.
→ You will return to the Define Z Calibration dialog box.
15) Click on the OK button.
→ The Define Z Calibration dialog box will be closed.
You conduct the Z calibration of an image by transferring the cali- See also
bration data you’ve just saved to that image. To find out how you do How to...: Z Calibration of
that, have a look at the step-by-step instructions called "Z Calibration an Image 13
of an Image".

12
Calibration of the image Z Value

You can also have the calibration data you’ve saved automatically
transferred to each newly-acquired image. To do this, have a look at See also
the step-by-step instructions on "Z Calibration of an Input Channel". How to...: Z Calibration of
an Input Channel 14
Warning This Z calibration as well only remains applicable to all subsequently-
acquired images as long as acquisition conditions are not altered. If
you, for example, adjust the microscope illumination to be brighter or
less bright then you’ll have to conduct a re-calibration.

Warning This sort of simplified re-calibration only resets the gray-value offset,
thus displacing the calibration curve in relation to the coordinate
system without altering the shape of the curve.
If you’re not sure whether the alteration of the acquisition conditions
can be adequately described in this manner, then you’d be well
advised to conduct a completely new Z calibration.

Z Calibration of an Image
How to... How to conduct a Z calibration of a gray-value image:
You have defined a Z calibration using a calibration standard. The ca- See also
libration data has been saved in a file. How to...: Z Calibration
You now proceed to conduct the Z calibration of an image by trans- using a Calibration
ferring the saved calibration data to this image. Standard 9
How to...: Simplified re-
This image has to have been acquired under the same acquisition calibration 11
conditions using the same bit depth as the image of the calibration
standard.
1) Make sure that the image that you wish to Z calibrate is located in the
active image buffer.
→ You can do this by clicking, eg, on the image or on the image
buffer containing it in the image buffer box.
2) Select the Define Z Calibration... command in the Image menu.
→ The Define Z Calibration dialog box will be opened.
3) Click on the File... button.
→ The File In/Output dialog box will be opened.
4) Select the correct file name from the list beneath the File field.
5) Click on the Load button.
→ The File In/Output dialog box will be closed and you return to the
Define Z Calibration dialog box.
→ The settings of the Z calibration saved have now been loaded into
the Define Z Calibration dialog box.
→ If necessary, you can now edit the settings.
6) Click on OK to have these settings transferred to the image.
→ The image has now been Z calibrated.
→ If you click on Cancel instead of OK, the settings will not be
transferred to the image and the image will not be Z calibrated.
Note You can use the File... (Save/Load) button in the Define Z Calibration dialog
box at any time to transfer a Z calibration from one image to another. If you
have, eg, a series of images that were all acquired under the same acquisition
conditions, but only one has been Z calibrated, you can use the above button
to have all the other images Z calibrated subsequently.

13
Calibration of the image Z Value

Z Calibration of an Input Channel


How to... How to conduct a Z calibration of an input channel:
You have defined a Z calibration using a calibration standard. You’ve See also
saved the calibration data in a file. How to...: Z Calibration
You now wish to have images that are newly acquired to be auto- using a Calibration
matically Z calibrated at acquisition. This is done by determining for Standard 9
How to...: Simplified re-
the input channel that the calibration data will automatically be trans- calibration 11
ferred to each newly-acquired image.
The input channel must be the same one used to acquire the cali-
bration standard; acquisition condition must remain constant.
1) Select the Configure Input... command in the Image menu.
→ The Configure Input dialog box will be opened.
2) Select the Macro tab.
3) Type the following Imaging-C command line into the Postproces-
sing steps field:
DefineImageZCalibration(Name:="Filename");
→ Don’t type in Filename; type in the name of the file containing the
desired Z-calibration data.
4) Select the Enabled check box to the right of this field if it has not yet
been selected.
5) Click on the OK button.
If you now go ahead and acquire an image using the Snapshot com-
mand in the Image menu, it will be Z calibrated automatically.
Warning The automatic transferral of calibration data will also only result in a
correct Z calibration of the images if the acquisition conditions have
not changed since definition of the Z calibration.

Integrated Z Value
Use the iZpar module to make an additional particle parameter - called Integrated Z
Value - available to you for image analysis. This particle parameter is the product of the
area of a particle and its mean Z value.

Available The Integrated Z Value additional particle parameter is available in


the Particle Measurements dialog box once you’ve installed the
iZpar module. This dialog box is opened by selecting the Define
Measurements, Particle Measurements... command in the Analy-
sis menu.
Application Z calibration involves your defining a quantitative relationship bet-
ween the gray values of an image and their physical significance. For
example, you can assign the gray values as Z value to an optical
density (OD). In many cases it’s not only this Z value that is of in-
terest, rather its integrated value within a certain area: mean{Z value}
∗ area. You can use the Integrated Z Value particle parameter for
these sorts of measurements of integrated quantities.

14
Calibration of the image Z Value

Example For example, the integrated optical density (IOD) is the decisive quan-
tity with regard to gel analysis. In order to evaluate the characteristic
band structures you can define the bands you’re interested in as par-
ticles. The integral Z value of a particle is thus the integrated optical
density of the band: IOD = mean{OD} ∗ particle area.
How to... How to install the iZpar module:
If your current analySIS® configuration is already making the Inte-
grated Z Value particle parameter available to you in the Particle
Measurements dialog box, this means the module has already been
installed.
1) Open the Analysis menu.
2) Select the Define Measurements, Particle Measurements...
command.
→ The Particle Measurements dialog box will be opened.
→ The Integrated Z Value particle parameter is not in the Available
list.
3) Click on the Install... button.
→ The Install Modules with Measurement Parameters dialog box
will be opened.
4) Click on the Add Module... button.
→ The Select Module with Measurement Parameters dialog box
will be opened.
5) Search for the iZpar.sxu file in the ...\analySIS\Module\iZPar folder.
Select the file by clicking on it once.
→ The file name will appear in the Filename field.
6) Click on the Open button.
→ You’ll return to the Install Modules with Measurement Para-
meters dialog box.
→ The module has now been loaded and will appear in the Modules
list.
7) Click on the OK button.
→ You’ll return to the Particle Measurements dialog box.
→ The Integrated Z Value particle parameter is now listed in the
Available list. It’s position is alphabetical. You may have to use
the scroll bar to scroll through the entries listed there before you
see it.
8) Click on the OK button.
If you now, after installing the module, select the Define Measure-
ments, Particle Measurements... command (in the Analysis menu)
you will see the Integrated Z Value entry in the Available field of the
Particle Measurements dialog box. To use this parameter during
measurement, select its entry in the list via single leftclick and click on
the Add>> button. The entry will thus be moved over to the Current
list. Exit the dialog box via the OK button.
For further information on how you can use particle parameters for
image analysis, have a look at our User’s Guide.

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