Manual Image Pro Plus 4

Image-Proâ Plus
Version 4.5 for Windowsâ
Updates and Changes
Media Cybernetics, Inc.

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(301) 495-3305, FAX (301) 495-5964
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Introducing Image-Pro Plus Version 4.5 .................................................................1-1
System Requirements.................................................................................1-1
What's New in Version 4.5 ........................................................................1-2
File Menu .................................................................................................................1-4
File Signature .............................................................................................1-4
Edit Menu.................................................................................................................1-5
Process Menu ...........................................................................................................1-6
Large Spectral Filters .................................................................................1-6
Color Composite ......................................................................................1-11
Image Signature .......................................................................................1-17
Extended Depth of Field ..........................................................................1-18
Sequence Gallery .....................................................................................1-23
Measure Menu........................................................................................................1-25
Co-Localization........................................................................................1-25
Help Menu..............................................................................................................1-28
Solutions Zone .........................................................................................1-28
New File Format Information.................................................................................1-30
Supported File Formats............................................................................1-30
New Auto-Pro Functions..........................................................................................2-1
Image-Pro Plus v. 4.5
Introducing Image-Pro Plus Version 4.5
Welcome to Image-Proâ Plus version 4.5, the most complete and powerful image analysis
program available today. This version of Image-Pro Plus is designed to run under the
Microsoftâ Windows 32-bit systems: Windowsâ ‘98, Windows â 2000, and Windows NTâ
4.0. These advanced image processing features are provided through the Microsoft
Windows graphical user interface (GUI), delivering powerful imaging capabilities in an
easy-to-use tool. In corresponding package materials, Image-Pro Plus is also referred to as
Image-Pro and IPP.
System Requirements
To install and work with Image-Pro Plus 4.5, you will need the following equipment
and software:
n Pentium CPU, running at 120 MHz or higher, configured with Windowsâ

‘98, Windows â 2000, Windows NTâ 4.0, or Windows â ME
n A minimum of 128 MB of RAM
n 82 MB of disk space to hold the program files. (The installation process
provides options for a scaled-down version of the application that requies less
space.)
n A Windows-compatible mouse, keyboard, and CD-ROM drive
n A VGA (or better) resolution 256-color display device.
The following hardware is optional:
n Image Capture Board (frame-grabber)
n Scanner
n Printer.
Page 1-1
Image-Pro Plus v 4.5
What's New in Version 4.5
The following improvements have been made to Image-Pro Plus v. 4.5:
n Extended Depth of Field (EDF) /Multi-plane focus has been added to the
Process menu. This feature allows you to take a stack of images and chose the
in-focus parts to create a new composite image that is completely in focus.
n Color Composite Module, similar to the Color Composite Module found in
Fluoro-Pro, has been added to the Process menu. This feature creates and
configures color composite images from black and white source images. Any
number of different colors can be used to create the final image. In addition,
standard dye sets can be imported to use with this feature (e.g. DAPI, FITC,
etc.).
n Co-Localization has been added to the Measure menu. This feature allows
you to determine the existence of two or more molecule types in precisely the
same spatial position. The material under examination may be treated with
different types of dyes to indicate the presence or absences of areas of co-
localization or overlap. The Co-Localization command in can be used to
measure the amount of co-localization in a color image, in a pair of gray
images, in an image sequence, or in an AOI on a color image.
n The Sequence Gallery feature has been added to the Process menu. This
feature creates thumbnail gallery of the images in a sequence.
n Additional File Format Support has been added so that Image-Pro Plus,
version 4.5 will be able to read files in the Molecular Dynamics’ * .gel,
MetaMorph’s *.stk, and AutoQuant *.DEB and *.AVZ formats as well
as other commonly-used confocal file formats.
n Large Spectral Filters have been added to the Process menu. This feature
allows the user to pick the kernal size for some of the most common filters
from the filters dialog, up to a maximum size of 4001 by 4001.
n A Digital Image Signature feature has been added to the Process menu.
When activated, this feature generates a unique string for a given image.
Comparing the signatures enables you to tell if the image has changed from a
previous point.
n A Digital File Signature has been added to the File menu. This feature
complements the Audit Trail and Image Signature features to provide
further auditing and control for our FDA regulated customers. The feature will
create a “fingerprint” string for a file so that we can tell if any part of it (image
or text tag, etc.) has been modified.
Page 1- 2
n An Audit Trail feature has been added to the Application page of the
Preferences dialog .When selected, IPP will log all recordable events to a
daily audit log. The log contains the timestamp and macro call for each IPP
event. This enables you to track the history of your image processing session.
n Apply to Sequence options have been added so that the Extract, Merge, and
Convert functions of the Color Channel can be used with sequence
operations.
n Standard Position Information has been added to the General page of the
Image Information tab dialog on the Edit menu. This feature displays the X,
Y, and Z positions of each frame of the image, if this information is known.
n Link to Solutions Zone: The Help menu now contain a link to the Media
Cybernetics’ Solutions Zone . The Solutions Zone is an information and
resources web page which can be accessed freely by Media Cybernetics’
customers. This page contains downloadable solutions that range from device
drivers, plug-ins, macros, hardware, and services. Solutions are created by
Media Cybernetics’employees, dealers, and users. Solutions created by Media
Cybernetics’ employees can be downloaded at no cost. Some solutions created
by dealers or other users may have a cost listed in their description. Media
Cybernetics does not set any of these prices.
The Solutions Zone is searchable by keyword, product (i.e. Image-Pro Plus,
Scope-Pro, etc.), by solution type (i.e. device driver, plug-in, etc.), or
application (biological sciences, industrial, or forensics). Click this link to visit
the Media Cybernetics' Solutions Zone web page:
http://www.solutions-zone.com/
n New Auto-Pro functions have been added to support the Digital Signature,
Spectral Filters, Color Composite, Extended Depth of Field,
Co-Localization, and Sequence Gallery features.
Information about all of these new features can be found in the following pages.
Page 1-3
File Menu
The following new features appear on the File menu of Image-Pro Plus version 4.5:
File Signature
The File Signature feature calculates and displays the signature of a selected file. The
signature enables you to determine if a file has changed from a previously known point.
When activated, you will first see the standard File Open dialog. Use the File Open
dialog to select the desired file. When you click the Open button, you will see the File
Signature dialog:
You may copy this value to a string on the Clipboard, but you cannot edit it.
The File Signature is different from the Image Signature function in that headers and
other file information, such as image type, are included with the File Signature. The
Image Signature appears on the Process menu.
Page 1- 4
Edit Menu
The following item appears on the Edit:Preferences menu on the Application tab.
Generate Audit Trail log: Check this box to create an audit trail of all the
recordable events during your image processing session. This enables you to
reconstruct your actions should your session terminate unexpectedly. Audit trail
logging will be turned off during macro execution, except that the act of running the
macro will be recorded. The daily audit trail log file can be found in the Logs folder of
the Image-Pro Plus folder on your hard drive.
Page 1-5
Process Menu
The following new features appear on the Process menu in IPP 4.5:
Large Spectral Filters

The large spectral filters are filters of large aperture that can be used for various
purposes in image processing. These filters can be applied to an AOI, active image, or
active sequence. The size of the large spectral filters is not limited. All the large
spectral filters are very fast regardless of the size.
When you select the Large Spectral Filters command, you will see the following
dialog:
The main problem with large kernel filters is the slower filtering speed. Usually, the
filtering process is based on the convolution of a kernel and an image. Convolution
filters process image neighborhoods by multiplying the values within a neighborhood
by a matrix of filtering coefficients called a kernel. The result replaces the center pixel
in the image neighborhood (see Convolution filters in the previous section). The time
for this filtering method increases proportionally to the size of the kernel. For kernels
with sizes larger than 20-30, the process is unacceptably slow.
All filters in the Large Spectral Filters group use an algorithm that eliminates most of
the multiplication and sum operations, which increases the filtering speed
significantly. In theory, the size of the filters is not limited. However, Image-Pro Plus
limits the size of the Large Spectral Filters to 4Kx 4K.
Page 1- 6
Filter Types and Parameters

There are 5 types of large spectral filters: LoPass, HiPass, BandPass and two Edge
filters. Each filter type has its own set of related parameters.
LoPass: A LoPass filter removes image noise or extracts the background .
Width: Indicates the width of the filter being applied.

Height: Indicates the height of the filter being applied.
Passes: Indicates the number of times the filter should be applied to the image. For
example, an image that has been softened by one pass of the LoPass filter, will be
softened further by a second pass.
HiPass: The HiPass filter increases image sharpness and contrast settings. When used
with large aperture and high strength setttings, this filter can perform automatic image
binarization.
Page 1-7
Strength: Enter a value from 1 - 100 that reflects how much of the filtering effect
you want applied to the image. A value of 100 specifies that you want the full strength
(100%) of the filtered result applied to each pixel. Values less than 100 cut the full
weight of the filter - a value of 10 indicates that only 10% of the difference between the
filtered pixel value and the original pixel value should be applied, a value of 20
indicates that 20% of the difference should be aplied, and so forth.
Passes: Enter the number of times you want the filter applied to your image. When
a filter is applied multiple times, its effect is amplified by each pass.
Square: When this box is checked, the filter has a square matrix.
BandPass: The BandPass filter can simultaneously reduce noise and increase image
contrast, which can be very useful on low-contrast, noisy images.
The BandPass filter is a combination of the LoPass and HiPass filters. First an image
is processed by a LoPass filter with a given number of passes, and then by one pass of
the HiPass filter. For example, the dialog shown above defines the BandPass filter
with 3 passes of a 5x5 LoPass filter, and one pass of a 27x27 HiPass filter. The shape
of a BandPass filter is always a square.
Filter combinations such as these allow you to suppress hi-frequency components of
the image spectrum, which generally represent image noise. You may also amplify
middle components of the spectrum, corresponding to the edges and fine details of
various objects, and increase the contrast in these areas.
Hi Size: Indicates the size of the HiPass filter to be used for a BandPass filter.
Lo Size: Indicates the size of the LoPass filter to be used for a BandPass filter.
Page 1- 8
indicates that 20% of the difference should be applied, and so forth.
a filter is applied multiple times, its effect is amplified by each pass. An image that has
been softened by one pass of the BandPass filter, will be softened further by a second
pass.
Note: BandPass filters provides clearer images with smaller LoSize and more passes.
Edge: The Edge filters extract and enhance positive or negative edges. The Edge +
filter extracts positive edges (bright features on a dark background) from an image; the
Edge – filter enhances negative edges (dark features on a bright background).
Using non-square Edge filters, you can create filters that will enhance either vertical or
horizontal edges on the image.
indicates that 20% of the difference should be applied, and so forth.
a filter is applied multiple times, its effect is amplified by each pass. An image that has
been modified by one pass of the selected filter, will be modified further by a second
pass.
Page 1-9
Preview and Spectrum

On-image Preview. Check this box to see results of the filtering process before the
filter is actually applied to the image. The active image or an AOI is used as the
preview area.
Spectrum: Clicking this button displays a window that contains a spectrum of the
current filter. This graph helps to set the correct filter parameters to achieve the desired
results. The background of the Spectrum window shows a range of spatial harmonics
for corresponding frequencies. The spatial harmonics are vertically oriented and their
scale is equal to image scale with 100% zoom. The background is used to evaluate the
size of objects, which will be enhanced or filtered out.
Apply: Click this button to apply the specified filter. Before clicking this button, be
sure you have selected the filter you want to use (e.g., LoPass, Edge), and set the
pareamtersthat you need. Be sure to select the filter type first, because your choices are
determined by the type of filter selected.
Undo: Click this button to reverse the effects of the filter you just applied.
Close: Click this button when you have finished working with the filters.
Page 1- 10
Color Composite
Use the Color Composite dialog box to create and configure color composites using
black and white source images. You can access the Color Composite dialog box
through the Color Composite menu item from the Process menu.
You can combine gray-scale images into a color composite. Any group of gray-scale
images that are of the same size can be mixed in a color composite. Images of 8-, 12-,
16-bit integer or floating point format are combined into a 24-bit color composite. Each
input channel will have individual LUT adjustments, as well as a registration offset to
line it up with the rest of the images.
Page 1-11
Source Images: The Source Images combo box displays the images available for
color mixing. You need to click the New button first before you can fill the Source
Images list box with image names.
Add: Click the Add button to add an image to the mix (you will be prompted for the
color to be assigned to that image).
The size of the color composite is determined by the first input image selected. Once
this is entered, the list is further reduced to images of the same size as the initial
selection.
Delete: Use this button to delete the selected image from the list.
Colors: The Colors list box displays the images selected for color mixing.
To change the hue within the list box, double-click to select the image name (the Select
Hue dialog box appears) or highlight the name and click the Set Hue button.
To delete colors/images from the Colors list box, first click on the color/image to
highlight it then press the <Delete> button within the Color Composite dialog box.
Color Bar: The picture item box on the left side of the Colors list indicates the hue
associated with each image in the composite. The Color Bar will scroll with the
contents of the Colors list box.
Set Hue: This option determines the hue (Color) value in the final image display. Set
Hue is used as an initial value for color compositing. Clicking on the Set Hue button
brings up the Select Hue dialog box.
Page 1- 12
Directly select a hue by:

Dragging the Hue Value Slider to the corresponding value on the Hue Bar (range of 0
to 359°); or
Clicking the appropriate hue button on the Color Palette. The Hue Bar represents hue
degrees in the standard color wheel representation, and the Color Palette contains a
subset of possible hues (pure red, green, blue, cyan, magenta, and white).
Note: You can navigate through the Select Hue dialog box by using the <Tab>
and <Shift + Tab> keys to cycle through the items in the dialog box.
Intensity is determined by the image the hue is applied to, and saturation will always
be 100%.
The only exception to this is the “white” hue, which has by definition a saturation of
0%. Selecting a value greater than or equal to 360 (dragging the Hue Value slider over
to the far right) is a choice of white, which results in a non-tinted (gray) image.
Page 1-13
The elements of the Select Hue dialog box are:
Hue Bar: The Hue Bar shows a rainbow displaying the hues underneath the
appropriate point of the Hue Value slider.
Note: If Hue Value Slider is the selected (highlighted) selected control, then you can
also control the slider with keyboard strokes:
Use the left/right arrow < ß à>keys to move the slider incrementally.
Press the <Page Up> and <Page Down> to move the slider in “chunk” sections along
the Hue Bar.
Use the <Home> and <End> keys to move the slider to the beginning and end of the
Hue Bar.
Selected: This color block displays the new hue selected by the Hue Value slider bar
or the Color Palette.
Selected Hue: The Selected Hue text field displays the corresponding numeric hue
value of the Selected color block described above. The Selected Hue value is
expressed as a degree between 0 and 359, corresponding to the hue degree in HSI (Hue,
Saturation, and Intensity) color space.
Current: The Current color block displays the starting hue of the channel.
Color Palette: The Color Palette proves an alternate method of selecting a hue.
Clicking on an item in the Color Palette will set the Hue Value slider to the correct
value and update the displays in both the Selected color block and Selected Hue text
field.
For example, clicking on the white button on the Color Palette will set the Hue Value
slider to the right, change the Selected color block to white, and cause the Selected
Hue text box to display “White” with an associated value of >360.
Adjust: The controls in this section are usd to adjust the color composite contribution
from each image, or for the color composite preview itself. You can adjust the input
image that is currently selected in the Colors list by clicking the slected button.When
you want to adjust the color composite preview, or you want no image is selected in the
Colors list, use the All button.
Page 1- 14
Registration: Registration allows you to correct for mis-registrations of images
caused by filter-induced optical shifts. Use the directional keys to shift the selected
image in the x- and/or y- direction, with respect to the rest of the input images. One
click moves that channel one pixel in the direction indicated. Blank areas are filled with
black. The key labeled “0,0” will re-center the selected image.
The registration shift for the selected channel or the overall image is displayed in the
static text box below the directional keys.
Contrast: Use the BCG sliders along with the Best Fit and Reset buttons to adjust
the contrast of the selected image or the composite.
BCG sliders: The Brightness, Contrast, and Gamma sliders are the same as the BCG
sliders in Image-Pro Plus. LUT adjustments are clipped to output 0-255 in each
channel. You can also adjust each channel individually or collectively by using the
Selected or All radio buttons (to the left of the BCG sliders in the Adjust group box).
Best Fit: Click this button to perform a Best-Fit contrast stretch on the selected input,
automatically setting the Brightness and Contrast settings accordingly (Gamma will be
reset to 1.0.). This method is identical to the Best Fit contrast adjustment under the
standard BCG tool for Image-Pro Plus. Best Fit is useful for first pass adjustments.
Reset: Pressing the Reset button automatically resets the BCG settings. If the Adjust
radio button is set on All, the BCG settings for the overall color image will be reset.
Frame: Use the frame slider to adjust the frame of the selected image that is used for
the preview, or the active frame of the composite. (Please see the discussion of Color
Composite and Image Sequences that follows this section.)
Background: Select one of the available images to be the background color in this
combo box. The rest of the image planes are subtracted from the background image —
effectively punching “holes” into it and allowing the foreground colors to show without
mixing with the background. This is most effective where the background covers large
areas and the other images have smaller objects within the background objects.
Background allows the other inputs to be displayed “in front of” the background
inputs, minimizing color mixing. For example, Image-Pro can display a red dot on a
blue background without turning the dot to magenta.
Selecting None causes all images to be equally mixed.
New: Click on the New button to start a new color composite image.
The Source Images combo box will reset to include all gray scale images (including
floating point images). Selecting an image from the list will create a new composite the
same size as the initial selection. The remaining source images available for selection
will match the image size of the newly formed composite image.
Page 1-15
Color Composite and Image Sequences

When working with single-frame images, the Frame slider and the Make Sequence
button will both be disabled and have no effect. When working with image sequences,
the Frame slider affects both the single-frame preview and the way that the sequence
will be built when Make Sequence is invoked.
Frame: The single-frame color composite workspace that is created once you've added
at least one image, is built from the frame of each image that is indicated by the Frame
slider. Note that when the image is added, the frame setting for that image defaults to
the currently active frame of the sequence. Changing the Frame slider for an image
does NOT change the image's active frame.
As you activate different images (click them in the Colors list), the Adjust radio button
switches to Selected to indicate the settings for that image's contribution to the color
composite. The Frame slider will update to indicate the currently selected frame that is
contributing to the color composite preview, and you can adjust the slider to select a
different frame to contribute. If you change the Adjust radio button to All, then you
can use the Frame slider to change the frames of ALL images. This has the same effect
as the sequence tool bar for sequences but the color composite preview is still a single-
frame workspace.
The frame settings for each image determine how those sequences will be aligned when
Make Sequence is used to create a color composite sequence. To illustrate, consider
two workspaces of 5 and 7 frames, where the frame setting is frame 4 for the first
sequence and frame 3 for the second (numbering from 1, with the selected frames being
represented by Frame # (F1, F2, etc.) in the following diagram):
S1 S2 Result
F1
F2 F1 F2/F1
F3 F2 F3/F2
F5 F4 F4/F5
F5
F6
F7
Page 1- 16
Note that the first frame of sequence S1 is ignored because of the way the two
sequences are aligned. Note also that the “All” frame setting for this color composite
will start at 3 - this is the point of alignment in the resulting sequence. If the sequences
have been captured using the same Zstack settings, you will seldom if ever want to mis-
align the sequences this way, but it also makes for some interesting effects. Note also
that the length of the sequence built by Make Sequences is the amount of overlap
between the sequences with the specified alignment, or 4 frames in this case.
Image Signature
The Image Signature feature calculates and displays the signature of the active image.
The signature enables you to determine if an image has changed from a previously
known point. When activated, you will see this dialog:
You may copy this value to a string on the Clipboard, but you cannot edit it. If you
activate a different image, the Update button is also activated. Click the Update button
to see the signature of the new image.
This feature differs from the File Signature in that it only considers visible image
parameters to generate the string. File Signature is located on the File menu.
Page 1-17
Extended Depth of Field

The Extended Depth of Field feature comprises the multi-plane focus feature, along
with a new contrast detection algorithm and an option to return a topographic map of
the image. This feature allows you to combine individual images or sequences into a
single, in-focus, composite image.
When you choose this option from the Process menu, you will see the dialog below:
Image List Tab

The Available Images contains a list of all the images or sequences that could be used
to create a best-focus image. Any number of single-frame images or image sequences
can be moved from the Available Images list to the Selected Images list. Use your
cursor to highlight the name of the image or sequence that you want to move into the
Selected Frames area.
Available images that are sequences will indicate how many of the frames are selected
for the operation. If Apply to Sequence (on the Sequence toolbar) is not selected, only
the active frame will be used. This indicates that Apply to Sequence is not active.
When Apply to Sequence is active, the length of the active portion of the sequence
will be indicated.
Page 1- 18
After the first image is selected, the Available Images list will be filtered to list only
images that are the same type and size as the selected image (no conversion or resizing
is supported).
Add: Click this button to move the highlighted image or sequence from Available
Images into Selected Frames. The Selected Frames are used to create the image
stack. Add will add frames from any images selected in the Available Images list to
the Selected Frames list, and is enabled whenever there are one or more images
selected. When an image with multiple frames is added, the Selected Frames list will
indicate the range of frames that will be used, e.g. Heart.seq (5-25).
Add All: Click this button to move all the images or sequences from Available
Images into Selected Frames. Add All is always enabled, and will add all images
from the Available Images list to the Selected Frames list. This feature does not
check to see if there are duplicate images, so if there are already images in the Selected
Frames list, the whole list from Available Images will be added to them.
Remove: Click this button to move the highlighted image or sequence from Selected
Frames back into the Available Images list. Remove will remove any images selected
in the Selected Frames list, and is enabled whenever there are one or more images
selected.
Clear: Click this button to erase the images or sequences from the Selected Frames
list. Clear is always enabled and will remove all images from the Selected Frames list.
Stack Order: The order of the images in the Selected Images, in combination with
the Stack Order controls, list will determine the order of the stack.
Top Down: If you click this button, the first image at the top of the list will be the first
image in the stack and the bottom image the last.
Bottom Up: If you click this button, the order of the images is reversed. The image at
the top of the list will be the last image in the stack, and the image at the bottom of the
list will be the first image in the stack. Note that the perceived “position” of the images
will affect the Topographic Map, and may affect which pixel is selected for some of
the focus analysis operations.
Move Up/Move Down: The Move Up and Move Down buttons ere enabled when
only one image is selected in the Selected Images list, and only if the selected image
does not have Z position information. These buttons will move the selected item up or
down in the list.
Page 1-19
Select Default Plane: This feature is enabled whenever there are images in the
Selected Frames list. Clicking this button displays the following dialog:
Select as Default Plane: Click this button to set the selected image frame as the
default plane to use when the multi-plane focus algorithm does not find “in-focus”
material on any of the supplied planes. Since one or more of the source images may be
multi-frame image sequences, any sequence images are expanded to list each included
frame, with the frame number indicated in parenthesis.
Cancel: Click this button to cancel the default plane select and return to the Extended
Depth of Field dialog.
At the bottom of the Extended Depth of Field dialog box are 3 controls that are shared
by all tab pages, as shown here:
The message box at the far left contains a text field indicating the current number of
images and frames in the stack. This field is updated whenever any of the pertinent
settings change. The first portion of the field indicates the number of images; the
second portion of the field indicates the number of frames currently selected for
inclusion in the stack.
Create: Click this button to start the process of creating the image stack. This button
will only be enabled when there are at least two selected image/frames. The dialog will
not close when the stack is created.
Cancel: Click this button to close the dialog. If images have been added but an image
stack has not been created, you will see a message asking you if you want to continue.
Page 1- 20
Focus Settings Tab

The Focus Settings tab page allows you to select the various focus analysis and output
options.
The Output options provide the same functionality as the multi-plane focus and
software auto-focus options on the Acquire feature of Scope-Pro.
Generate composite best-focus image: Clicking this button creates a single
image composed of the best pixels from the set of input images, as determined by the
Focus Analysis Options.
Return best-focus frame: Clicking this button creates a new workspace containing
a copy of the selected frame with the most in-focus material.
Normalize Illum ination: Use this option when the illumination or intensity of
emitted light will change in the time that it takes to acquire all of the planes. Basically,
this option corrects for uneven illumination as the Z plane changes.
An example would be a sample that uses a fluorescent dye that degrades with exposure
to light. The first captured plane will have a higher intensity then the last plane
captured. In this case all captured planes will be normalized before being processed
into the final image.
Select Focus Regions for: There are four options to select what area is “in-focus”:
Page 1-21
Maximum Local Contrast: This method considers areas with high/maximum
contrast to be in focus. Pixels are analyzed in the area around the current pixel location.
The pixel from the plane with the largest variance or local contrast is selected.
Maximum through-depth contrast: The mean value of the pixels is calculated.
The pixel from the plane with the largest variance from the mean is selected.
Max. Intensity: This method is used for samples that are more intense than the
background such as light emitting fluorescent dyes. The pixel from the plane having the
highest intensity at the current pixel location is selected.
Min. Intensity: This method is used for regions that are darker than the surrounding
area to be in focus. The pixel from the plane having the lowest intensity at the current
pixel location is selected
The Topographic Map options allow you to select whether or not to generate a
topographic map from the focus, and if so, whether to add an intensity calibration.
Generate Topographic Map: Clicking this button creates a topographic map from
the focused image.
Display calibrated height: Clicking this button adds an intensity calibration that
translates the plane number values in the image to the height. This option will be
disabled if Generate Topographic map is not selected, and also if the Z position of
the image frames is not known.
Activate Selected Image: When this box is checked, selecting an image in the in
the selected frames list will also cause the associated workspace to be activated.
Page 1- 22
Sequence Gallery
The Sequence Gallery feature creates a workspace from an active image sequence.
The individual frames in the sequence are displayed in a gallery format; hence it creates
a “sequence gallery.” Clicking the Sequence Gallery menu item displays the
following dialog:
Create Gallery: Clicking this button creates a sequence gallery workspace from the
active workspace. This feature is disabled if there are no active images, or if the active
image is a single-frame workspace. Here is a sample sequence gallery:
Page 1-23
Bring Sequence to Top: Clicking this button activates the sequence workspace on
which the gallery is based, and is only enabled when the active workspace is a
sequence gallery.
Update: Clicking this button will update the sequence gallery with any changes to the
image sequence in the active workspace.Update will be disabled until a sequence
gallery is the active workspace, and a change is made to the sequence that the gallery
cannot track, or when other changes are made and Update in Background is not
selected.
Update in Background: Checking this box provides a live link between the
sequence workspace and its gallery so that any changes made to the sequence are
updated in the gallery as they are made.
OK: This button closes the Sequence Gallery dialog.
The red square in the Sequence Gallery workspace indicates the active frame in the
larger Image-Pro workspace. Use your cursor to click on different frames in the
Sequence Gallery, and the red square will move to the selected frame, and the active
image in the Image-Pro workspace will change to the selected frame as well.
Note: When a change is made to the length of the original sequence, e.g. frames are
added or removed, the sequence gallery workspace will no longer be associated with
the sequence, and the Update and Update in Background links will no longer be
active. The only valid option will be Create Gallery.
Page 1- 24
Measure Menu
The following features appear on the Measure menu in IPP 4.5:
Co-Localization
Co-localization describes the existence of two or more molecule types in precisely the
same space. The material under examination may be treated with different types of
dyes to indicate the presence or absence of areas of co-localization or overlap. The Co-
Localization command can be used to measure the amount of co-localization in a color
image, in a pair of gray images, in an image sequence, or in an AOI on a color image.
Selecting Co-Localization from the Measure menu displays the following dialog box:
Operations with original color or gray images: Co-localization operates on a pair of

grayscale images or two planes of one color image. An AOI on a grayscale image will
be ignored; however if there is an AOI on a color image, the calculations will be
limited to the area of the AOI. Otherwise, calculations will be performed on the whole
image.
Active Image: Indicates which image is currently in the active workspace. This image
will be used as the baseline for the co-localization calculations.
nd
2 Image: Indicates the second image in the pair of grayscale images for co-
localization. This control will be disabled (grayed out) if the active image is color.
Page 1-25
If the original images are grayscale, the next four options will create a color image
showing the two source images merged according to the Color Pair that has been
selected, in addition to any Co-Localization plots that are specified.
Create gray co-localization with frequencies: Choosing this option will create a
new, untitled grayscale image that displays the co-localization areas of the two
channels selected (these can either be two grayscale images, or two channels of a color
image) when the Forward button is clicked.
Create color co-localization: Choosing this option will create a new, untitled color
image that displays the co-localization areas of the two channels selected (these can
either be two grayscale images, or two channels of a color image) when the Forward
button is clicked.
Create 3D view of color co-localization: Choosing this option will create both a
gray scale and a color Co-Localization plot of the selected area of the active image and
uses Image-Pro’s Surface Plot feature to create a three-dimensional image of the color
co-localization when the Forward button is clicked.
Calculate correlation between images: Choosing this option will use the
Pearson Correlation Coefficient to measure the amount of co-localization in the
selected image or AOI when the Forward button is clicked. The results are displayed
in the Output Window, which can be accessed from the Macro menu.
Color pair: Use this drop-down list box to select the two colors that Image-Pro will
use to define the co-localization area. When using gray scale images as the data
source, this drop-down list determines the colors that will be assigned to each source
image in making the color composite image and the color co-localization image. When
a color image is the data source, this drop down determines which two of the three
RGB planes will be used for the co-localization calculations.
Forward: Click this button to begin the co-localization analysis process.
Operations on the co-localization plot: These selections affect the way in which co-
localization is displayed on the image that Image-Pro created.
Use an AOI on co-localization plot to select area for analysis: Create an AOI
on the co-localization image to define the range of intensities for each color on which
the co-localization calculations should be performed. Also, notice that as the AOI is
drawn, or moved on the co-localization plot, a three-color mask, based on the bounding
box of the AOI, is applied to the source color image. The two colors from the imaging
show non-co-localized pixels within the area selected by the AOI range of intensities.
The third color, a summary of the channel colors, represents the co-localized pixels.
For example, if the channels are red and green, the co-localized pixels are shown in
yellow.
Page 1- 26
Create mask of co-localizing objects: Click this radio button to create an image
mask using Image-Pro's Grid/Mask feature. The image mask will show the areas of co-
localization, as defined by the AOI on the co-localization plot.
Calculate co-localization coefficients: Clicking this radio button uses the
Pearson Correlation Coefficient to measure the amount of co-localization in the
selected image or AOI. The mathematical results are displayed in the Output Window.
Analyze: Click this button to execute the operation selected above.
Close: Click this button to close the Co-localization dialog.
Co-Localization Plot: The Co-Localization plot is a 256 x 256 pixel image
displaying a scatter plot, with the origin in the bottom left corner, in which the
intensity of each pixel represents the frequency with which that color pair exists in each
image. For example, if pixel 10, 12 has an intensity of 2013, that means that there are
2013 pixels with an intensity of 10 in the first channel that also have an intensity of 12
in the second channel.
Page 1-27
Help Menu
The following information appears on the Help menu of Image-Pro Plus version 4.5:
Solutions Zone
The Solutions Zone is an information and resources web page which can be accessed
freely by Media Cybernetics’ customers. This page contains downloadable solutions
that range from device drivers, plug-ins, macros, hardware, and services. Solutions are
created by Media Cybernetics’ employees, dealers, and users. Solutions created by
Media Cybernetics’ employees can be downloaded at no cost. Some solutions created
by dealers or other users may have a cost listed in their description. Media Cybernetics
does not set any of these prices.
The Solutions Zone is searchable by keyword, product (i.e. Image-Pro Plus, Scope-Pro,
etc.), by solution type (i.e. device driver, plug-in, etc.), or application (biological
sciences, industrial, or forensics).
Click this link to visit the Media Cybernetics' Solutions Zone web page:
http://www.solutions-zone.com/
Device Drivers
Device drivers are software plug-ins that help a user control a specific piece of
hardware that is connected to their computer. From time to time, Media Cybernetics
will post new and updated drivers on the Solutions Zone. If your device is not working
correctly, please check first to see if there is an updated driver available before
contacting Technical Support.
To search for a device driver, type the name of the device for which you are seeking a
driver (e.g. ORCA II). After the search is complete, click on a driver so that you can
read the description and updated information (if applicable). The next screen will allow
you to download the driver directly to your computer.
Plug-ins
Plug-ins are software programs that work within Image-Pro to provide a new function
or feature. Many times, users have asked how rather complicated functions can be
accomplished using Image-Pro Plus. In some cases, our engineers and other Image-Pro
Plus users have taken it upon themselves to write new plug-ins so that these complex or
novel tasks can be accomplished. We feel that if one user asks a question, there are
probably many more that will have to have the same question answered. That is why
these plug-ins are placed on the Solutions Zone.
Page 1- 28
A plug-in search is best accomplished by selecting Application Programs/Plug-Ins
from the Solutions Type drop-down menu on the front page of the Solutions Zone. You
should then limit your search with a keyword based on what you would want the plug-
in to do (e.g. trace object). Once you have some options to choose from, click on the
plug-in title for more information. The page with information on the plug-in will have a
link to allow you to download the plug-in directly to your computer.
Macros
One of the major advantages of using Image-Pro Plus is the ability to record and use
macros. Macros are programming scripts that call the programming language of Image-
Pro Plus to automatically execute tasks. Our engineers and other users have written
macros to solve problems and add to the capabilities of Image-Pro Plus.
A macro search is accomplished in a similar manner as a plug-in search. Select Macros
from the drop-down menu on the front page of the Solutions Zone. You should then
limit your search with a keyword based on what you would want the macro to do (e.g.
sort objects). Once you have some options to choose from, click on the macro title for
more information.
The page with information on the macro will have a link to allow you to download the
plug-in directly to your computer.
Hardware and Services

Dealers and other users have specialized hardware and macro/plug-in authoring
services that are listed on the Solutions Zone. Hardware devices can best be found by
selecting Other Hardware Driver/Component from the Solutions Type drop-down
menu on the front page of the Solutions Zone.
Macro/plug-in authoring services can best be found by selecting
Consultants/Programming Services from the Solutions Type drop-down menu on the
front page of the Solutions Zone. After selecting a specific item for more information, a
description of the hardware or service will appear as well as contact information.
Do you have a solution that you think others could benefit from? Submit it to the
Solutions Zone today! Just click
http://www.solutions-zone.com/addedit.htm
and fill out the form on the webpage.
Page 1-29
New File Format Information
Supported File Formats

Image-Pro Plus supports the following file formats. A brief description of each format
will list the name used by Image-Pro in the File:Open and File:Save As dialogs, the
typical file extensions that identify the file format, and any limitations such as formats
that are supported for File:Open only.
AutoQuant ( *.DEB, *.AVZ)

Image-Pro supports many varieties of the AutoQuant file formats, which will have a
*.DEB or .AVZ file extension. AutoQuant files are supported in File:Open only.
AVI (*.AVI).
Image-Pro supports many of the video varieties of the Microsoftä AVI file format,
which will usually have the *.AVI file extension. The varieties that will be available
in your copy of Image-Pro depend on the AVI file compression support that has been
installed by the operating system and other multi-media components.
BioRad (*.PIC)
Image-Pro supports many varieties of the BioRad file format, which will have a
*.PIC file extension. BioRad files are supported in File:Open only.
BMP (*.BMP, *.DIB, *.RLE)

Image-Pro supports all known varieties of the Microsoftä BMP file format,which will
usually have the *.BMP file extension but may also have a *.DIB or *.RLE xtension.
Dr. HALO (*.CUT)

Image-Pro supports all varieties of the Dr. HALO CUT format that many previous
Media Cybernetics applications supported. CUT files are supported in File:Open only.
Page 1- 30
Flat
Image-Pro supports loading a wide variety of proprietary file formats using a
configurable file description. This capability can also be used to define, load and save
simple proprietary file formats of your own. Please refer to your Image-Pro Plus
Reference Guide for more information about flat file formats.
Gatan DigitalMicrograph
Image-Pro supports many varieties of the Gatan DigitalMicrograph file format, which
will have a *.DM3 file extension. DM3 files are supported in File: Open only..
Image-Pro Workspace (*.IPW)

Image-Pro supports a proprietary file format with the *.IPW file extension.
JPEG (*.JPG)
JPEG files will have a *.JPG or JPEG file extension. Image-Pro supports all standard
varieties of the JPEG file format..
Kodak PhotoCD (*.PCD)

Image-Pro supports all known varieties of the Kodak PhotoCD file format, which will
have a *.PCD file extension. PCD files are supported in File:Open only.
Molecular Dynamics (*.GEL)

Image-Pro supports many varieties of the Molecular Dynamics file format, which will
have a *.GEL file extension. GEL files are supported in File:Open only.
MetaMorph (*STK)
Image-Pro supports many varieties of the MetaMorph file format, which will have a
*.STK file extension. STK files are supported in File:Open only.
Park (*.HDF)
Image-Pro supports many varieties of the Park Scientific file format, which will have
an *.HDF extension. HDF files are supported in File:Open only.
PICT (*.PCT, *.PICT)

Image-Pro supports many varieties of versions 1 and 2 of the Macintosh PICT file
ormat, which will usually have a *.PCT or *.PICT file extension. PICT files may
contain vector graphs (e.g. lines and text) as well as raster image or bitmap data; only
the bitmap data will be read.
Page 1-31
PostScript (*.EPS)
Image-Pro supports Encapsulated PostScript files, which will have an *.EPS file
extension. EPS files are supported in File:Save As only.
Sequence (*.SEQ)
Image-Pro supports multiple-image TIFF files with the .SEQ file extension. For
additional information on TIFF files saved by Image-Pro Plus, please refer to your
Image-Pro Plus Reference Guide.
TIFF (*.TIF)
Image-Pro supports all known varieties of the TIFF (the Tag Image File Format) file
format, including most multiple-image varieties. For additional information on TIFF
files saved by Image-Pro Plus, please refer to your Image-Pro Plus Reference Guide.
TrueVision (*.TGA)
Image-Pro supports all known varieties of the Targa and TrueVision file format with
the *.TGA file extension.
Zsoft (*.PCX)
Image-Pro supports all known varieties of the ZSoft PCX file format, which will have a
*.PCX file extension. PCX files are supported in File:Open only.
Zeiss (*.LSM)
Image-Pro supports many varieties of the Zeiss file format, which will have a *.LSM
file extension. LSM files are supported in File:Open only.
Page 1- 32
Image-Pro Plus v. 4.5
New Auto-Pro Functions

The following new Auto-Pro functions have been created to support Image-Pro Plus
version 4.5.
IpCmpAdd
Syntax IpCmpAdd (DocId, Hue)
Description This function adds a document to the active composite image.

Indicates the image to add to the color
Parameters DocId Integer composite.
Indicates a color for the source image. Hue may

Hue short
be any number from 0 to 360, or be one of the
following predefined values:
HUE_RED = 0
HUE_GREEN = 120
HUE_BLUE = 240
HUE_YELLOW = 60
HUE_CYAN = 180
HUE_MAGENTA = 300
HUE_WHITE = 361
HUE_DEFAULT - use color specified in Hue
property, if any (if no default is specified,
HUE_WHITE will be used)
HUE_QUERY - displays Hue dialog to set
color interactively
IpCmpAdd (0, HUE_QUERY) ‘Adds document 0, queries the user for

Example the hue.’
IpCmAdd(DOCSEL_ACTIVE,HUE_RED) Adds the active image
Return 0 if successful, an error code otherwise.

Value
Page 2-1
IpCmpDel
Syntax IpCmpDelete (DocId)
Description This function removes a document from the active composite image
Indicates the document ID of the image to
Parameters DocId short remove from the color composite.
IpCmpDelete(0) ‘Remove the image with a document ID of 0’
Example
0 if successful, or IPCERR_INVARG (bad docID) or IPCERR_FUNC (not a composite active)

Return if failed
Value
IpCmpGet
Syntax IpCmpGet (Command, DocId, Value)
Description This function gets the values for color compositing.

Must be one of the following:
Parameters Comman short LUT_BRIGHTNESS
d LUT_CONTRAST
LUT_GAMMA
SHIFT_X
SHIFT_Y
COMP_HUE
COMP_BACKGROUND
COMP_FRAME
COMP_NUMFRAMES
GETNUMDOC
GETDOCLST
For the LUT , FRAME, NUMFRAMES, and

DocId short
SHIFT commands, Doc Id is the the DocID of the
source image to inquire or DOCSEL_ACTIVE for
the color composite itself.
Value indicates the variable that will receive the

Value long
selected setting’s current value.
Page 2- 2
Comments See the following:

COMMAND DocID VALUE
LUT_BRIGHTNESS DocID of the source Current LUT value.

LUT_CONTRAST image to inquire Gamma is scaled by a
LUT_GAMMA about, or factor of 100 so that it
DOCSEL_ACTIVE to can be integrated.
inquire about the color
composite preview
image’s setting.
SHIFT_X DocID of the source Current pixel shift

SHIFT_Y image to inquire
about, or
DOCSEL_ACTIVE to
composite preview
image’s setting.
COMP_FRAME DocID of the source Current frame

image to inquire
about, or
DOCSEL_ACTIVE to
composite preview
image’s setting.
COMP_HUE DocID of the source Hue

image to inquire
about.
COMP_BACKGROUND none DocID of the

background,
-1 if none
Page 2-3
COMMAND DocID VALUE
COMP_NUMFRAMES DocID of the source Number of frames in

image to inquire the source image or
about, or composite.
DOCSEL_ACTIVE to
composite preview
image’s setting
GETNUMDOC none Number of documents

(images) in the color
composite.
GETDOCLIST none An integer array of the

document Ids of all the
documents in the color
composite. Use the
GETNUMDOC
command to get the
number of values that
will be returned.
dim parm1 as integer
ret = IpCmpGet (LUT_BRIGHTNESS, 0, parm1)
Example Print parm1
Return 0 if successful, an error code otherwise

Value
Page 2- 4
IpCmpNew
Syntax IpCmpNew (DocId, Hue)
Description This function creates a new color composite, based on the size of the supplied
image.
Indicates the image to use for color compositing
Parameters DocId short
Indicates a color for the source image. Hue may

Hue short
be any number from 0 to 360, or be one of the
following predefined values:
HUE_RED = 0
HUE_GREEN = 120
HUE_BLUE = 240
HUE_YELLOW = 60
HUE_CYAN = 180
HUE_MAGENTA = 300
HUE_WHITE = 361
HUE_DEFAULT - use color specified in
Hue property, if any (if no default is specified,
HUE_WHITE will be used)
HUE_QUERY - displays Hue dialog to set
color interactively
IpCmpNew (0, HUE_RED) ‘Adds Document 1’
Example

Value
Page 2-5
IpCmpSet
Syntax IpCmpSet (Command, DocId, Value )
Description This function sets the values for color composites.

Parameters Command short LUT_BRIGHTNESS
LUT_CONTRAST
LUT_GAMMA
SHIFT_X
SHIFT_Y
COMP_HUE
COMP_BACKGROUND
COMP_RESET
COMP_BESTFIT
COMP_FRAME
For the LUT, SHIFT, RESET, BESTFIT, and

DocId short
FRAME commands, parameter is the DocID of
the souce to adjust, or DOCSEL_ACTIVE for the
color composite itself.
Value long New value for the specified setting.

Not used with COMP_RESET or
COMP_BESTFIT.
Comments For COMP_RESET or COMP_BESTFIT the value argument is ignored, and a

LUT bestfit or reset is performed on the specified image. COMP_HUE cannot
be adjusted on the color composite preview image. For COMP_BACKGROUND
the document ID argument is ignored, and the value argument is used to
specify the background document, or –1 to reset.
Page 2- 6
COMMAND DocID VALUE
LUT_BRIGHTNESS DocID of the source New LUT value.

LUT_CONTRAST image to adjust, or Gamma is set to
LUT_GAMMA DOCSEL_ACTIVE to gamma*100 to allow
adjust the color integration.
composite preview
image’s setting.
SHIFT_X DocID of the source New pixel shift

SHIFT_Y image to adjust, or
DOCSEL_ACTIVE to
adjust the color
composite preview
image’s setting.
COMP_RESET DocID of the source none

COMP_BESTFIT image to adjust, or
DOCSEL_ACTIVE to
adjust the color
composite preview
image’s setting.
COMP_FRAME DocID of the source New frame

image to adjust, or
DOCSEL_ACTIVE to
adjust the color
composite preview
images’s setting
COMP_HUE Document ID of the Hue

source image to adjust
COMP_BACKGROUND none DocID to use for

background,
-1 to reset
IpCmpSet (LUT_BRIGHTNESS,0,87)
Example
Page 2-7

Value
IpCmpShow
Syntax IpCmpShow (flag)
Description This function shows or hides the color composite dialog.

Parameters flag short COMP_SHOW = 1, show dialog
COMP_HIDE = 0, hide dialog
IpCmpShow (COMP_SHOW)
Example

Value
IpCoLocForward
Syntax IpCoLocForward (SecondImage, ColorPair, Type)
Description This fu4nction calculates the co-localization scatterplot and parameters
Parameters SecondIma Integer Document ID of the image to use as the second

ge grayscale channel (the active image is used as
the first channel). The second image ID should
be –1 if the first image is a color image.
Page 2- 8
ColorPair Interger Indicates the color that should be used:

CP_RED_GREEN Red for first channel,
green for second
CP_BLUE_RED Blue for first, red for
second
CP_GREEN_BLUE Green for first,
blue for second
CP_GREEN_RED Green for first,
red for second
CP_RED_BLUE Red for first,
blue for second
CP_BLUE_GREEN Blue for first,
green for second
Type Integer Indicates the type of output desired:

CLOC_FWDMASK: Creates a color composite
workspace only if the input images are
grayscale. It will create and return the grayscale
scatterplot.
CLOC_FWDCOLOR : Creates a color

composite workspace only if the input images
are grayscale. It will create and return the color
scatterplot.
CLOC_FWD3D: Creates a color composite

workspace only if the input images are
grayscale. It will also create a grayscale
scatterplot that should be the active image for
the surface plot. It will create and return the
color scatterplot.
CLOC_FWDPARAMS: Calculates the first set of

co-localization parameters and sends them to
the output window.
Comments This function, IpCoLocForward, can end up creating up to 3 new workspaces

(or none at all) depending on the type of image being operated on, and the co-
localization option selected.
Return Returns the Doc ID of the co-localization scatterplot for all output types
Value except CLOC_FWDPARAMS. A negative return value indicates an error.
Page 2-9
IpCoLocGetDocument
Syntax IpCoLocGetDocument (DocType, DocID)
Description This function gets the document IDs of the documents created by
IpCoLocForward.
Parameters DocType Integer Identifies the document type to return. Must be

one of the following:
CLDOC_COLORCOMPOSITE – Return the
document ID of the color composite or color
input image.
CLDOC_SCATTERPLOT - Return the
document ID of the co-localization scatterplot.
CLDOC_3DMASK – Return the document ID of
the grayscale scatterplot used for surface
plotting.
Parameters DocID Integer An integer variable to recieve the document ID.
Return 0 if successful. A negative return value indicates an error .

Value
Comments The document ID returned by CLDOC_COLORCOMPOSITE may be the

color iput image or a new document containing a color composite created
from the grayscale imput images. The document ID returned by
CLDOC_SCATTERPLOT will be the same ID returned by IpCoLocForward.
The document ID returned by CLDOC_3DMASK should be the active image
for proper display of the co-localization surface plot.
Example ret = IpCoLocGetDocument(CLDOC_COLORCOMPOSITE, ColorImg)
See Also IpCoLocForward
Page 2- 10
IpCoLocGetForward
Syntax IpCoLocGetForward (SecondImage, ColorPair, Data)
Description This function gets the co-localization overlap parameters of the original
image.
Parameters SecondImage Integer Document ID of the image to use as the second

grayscale channel (the active image is used as
the first channel). The second image ID should
be –1 if the first image is a color image.
ColorPair Interger Indicates the color that should be used:

CP_RED_GREEN Red for first channel,
green for second
CP_BLUE_RED Blue for first, red for
second
CP_GREEN_BLUE Green for first,
blue for second
CP_GREEN_RED Green for first,
red for second
CP_RED_BLUE Red for first,
blue for second
CP_BLUE_GREEN Blue for first,
green for second
Data Single Data should be an array of 10 singles, which will

receive the forward parameters:
Data(0) - Pearson's correlation Rr
Data(1) - Overlap coefficient R
Data(2) - Overlap coefficient k1
Data(3) - Overlap coefficient k2
Data(4..9) - reserved

Value
Page 2-11
IpCoLocGetInverse
Syntax IpCoLocGetInverse (Data)
Description This function gets the co-localization parameters.
Parameters Data Single Data should be an array of 10 singles, which will

receive the forward parameters:
Data(0) - Co-localization coefficient M1
Data(1) - Co-localization coefficient M2
Data(2 - 9) - reserved

Value
IpCoLocInverse
Syntax IpCoLocInverse (Type)
Description This function calculates co-localization parameters on the basis of the active
AOI on the co-localization plot.
Parameters Type Integer CLOC_INVMASK: Creates a mask of co-

localizing pixels on the base of an AOI on the
image of co-localization plot
CLOC_INVPARAMS: Calculates the second set

of co-localization parameters and sends them to
the output window.
Return If the input is CLOC_INVMASK, the return value is the document ID of the
Value mask image.
If the input is CLOC_INVPARAMS, the return value is 0.
Page 2- 12
IpCoLocShow
Syntax IpCoLocShow (show)
Description This function shows or hides the co-localization dialog.

Parameters Show Integer COMP_SHOW = 1, show dialog
COMP_HIDE = 0, hide dialog

Value
IpDocGetPosition
IpDocGetPosition(DocID, PositionID, Frame, DocPosition)
Syntax
Description This function gets the position of the specified frame in the specified image.
Parameters DocId Integer Indicates the image of interest.

DOCSEL_ACTIVE can be used to inquire
about the active image.
PositionI Integer Indicates the axis of interest. Must be one

d of the following:
INF_XPOSITION = Absolute position
along X axis in calibrated units
INF_YPOSITION = Absolute position
along Y axis in calibrated units
INF_ZPOSITION = Absolute position
along Z axis in microns
Frame Long Indicates the frame of interest, which must

be between 0 and the number of frames in
the image or composite – 1, or the value –1
which specifies the active frame.
Page 2-13
DocPositi Any DocPosition is the IPDOCPOS variable

on that will receive the position information.
The IsKnown element will indicate whether the desired position information
Comments is known for the image. The Position element will indicate the position
value.
Note that the Z-axis position is always expresed in microns, regardless of the
current spatial calibration.
The IPDOCPOS type is defined as follows
IPDOCPOS Gets the image position Is known as Interger If non-zerio, IsKnown indicats the positon is
information. known. If zero, position is unknown
Position as Single Position along the specified axis.
Return 0 if successful. Non-0, otherwise.

Value
Dim posX As IPDOCPOS, posY As IPDOCPOS
ret = IpDocGetPosition( DOCSEL_ACTIVE, INF_XPOSITION, 0, posX)
Example ret = IpDocGetPosition( DOCSEL_ACTIVE, INF_YPOSITION, 0, posY)
If (posX.IsKnown = 0 Or posY.IsKnown = 0) Then
MsgBox("X/Y position is not known.")
Else
MsgBox("Position X , Y: " & CStr(posX.Position) & ", "
& CStr(posY.Position))
End If
See Also IpDocSetPosition, IPDOCPOS
IpDocSetPosition
IpDocSetPosition(DocID, PositionID, Frame, Position)
Syntax
Description This function sets the position of the specified frame in the specified image.
Parameters DocId Integer Indicates the image of interest.

about the active image.
Page 2- 14
PositionId Integer Indicates the axis of interest. Must be one

of the following:
INF_XPOSITION = Absolute position
along X axis in calibrated units
INF_YPOSITION = Absolute position
along Y axis in calibrated units
INF_ZPOSITION = Absolute position
along Z axis in microns
Frame Long Indicates the frame of interest, which must

be between 0 and the number of frames in
the image or composite – 1, or the value –1
which specifies the active frame.
Position Double This is the new position value.
Comments Note that the Z-axis position is always expresed in microns, regardless of the
current spatial calibration.
Return 0 if successful, a negative error code if failed.

Value
See Also IpDocGetPosition, IPDOCPOS
IpEDFAdd
Syntax IpEDFAdd(DocId)
Description
This function adds all the frames of the specified image to the current
multi-plane focus stack.
Page 2-15
Parameters DocId Integer Specifies the image to add to the existing EDF
stack. Note that the images must be of the
same image type and size. A document ID of
DOCSEL_ACTIVE can be used to add the
active image. A document ID of
DOCSEL_ALL can be used to add all images.

Value
Comments This function can be used to add the first image as well as subsequent
images, however using IpEDFNew for the first image will assure that the
stack does not contain any unwanted images from previous stacks.
See Also IpEDFNew
IpEDFCreate
Syntax IpEDFCreate(Type)
Description
This function creates the extended depth of field image
Parameters Type Integer Determines the type of Extended Depth of

Field image that will be created from the
current stack. Must be one of the following:
EDF_COMPOSITE Creates a
composite image from the best-focus areas
selected from multiple input frames.
EDF_BEST_FOCUS Returns a new image
using the single frame having the largest
amount of in-focus area.
Return Document ID of the new image if successful, a negative error code if

Value failed.
Page 2- 16
Comments Use IpEDFNew and IpEDFAdd to build the image list that will be used as
the stack of pre-focus images. If the topographic option is selected, use the
IpEDFTopoMap function to create the topographic map.The in-focus
material is determined by the current analysis criteria.
See Also IpEDFNew, IpEDFGet
IpEDFGet
Syntax IpEDFGet(Attribute, Value, Frame)
Description
This function gets an attribute of an Extended Depth of Field image.
Parameters Attribute Integer Attribute indicates the sequence gallery

attribute to get, from the following:
EDF_NORMALIZE Indicates whether
the frame intensities should be normalized
prior to focus analysis.
EDF_CRITERIA Indicates the criteria
used to analyze the frames for in-focus
material. (See below for constants.)
EDF_TOPO_MAP Indicates whether a
topographic map image should be created.
EDF_ TOPO_CALIBRATED Indicates
whether the topographic map image should
be given an intensity calibration that gives
each pixel a value corresponding to the Z
position of the plane that it was extracted
from.
EDF_ORDER Indicates whether the image
list will be used from top to bottom
(EDF_TOPDOWN) or bottom to top
(EDF_BOTTOMUP).
EDF_DEFAULT_FRAME Returns the

default plane document ID (in Value) and
frame number (in Frame).
Value Integer This is an integer variable in your script that

will receive the attribute’s value.
Page 2-17
Frame Long This is a long variable in your script that will

receive the attribute’s value.

Value
Comments The Frame parameter is not used and can be IPNULL for all commands
except EDF_DEFAULT_FRAME
The following constants will be used with EDF_CRITERIA attribute to

indicate the type of focus analysis:
EDF_MAX_LOCALCONTRAST: Pixels will be examined in
a local neighborhood around the target pixel and the degree
of local contrast determined for each plane. The pixel from
the plane with the greatest local contrast will be selected.
EDF_MAX_INTENSITY: Pixels in the same location on
each plane will be examined and the pixel with the highest
intensity will be selected.
EDF_MIN_INTENSITY: Pixels in the same location on each
plane will be examined and the pixel with the lowest
intensity will be selected.
EDF_MAX_DEPTHCONTRAST: Pixels in the same
location on each plane will be compared to the mean
intensity of all pixels at that position, and the pixel with the
greatest contrast from that mean intensity will be selected
See Also IpEDFSet
IpEDFNew
Syntax IpEDFNew(DocId)
Description
This function starts a new Extended Depth of Field stack with frames from
the specified image.
Page 2- 18
Parameters DOcId Integer Specifies the first image to add to a new EDF
stack. A document ID of DOCSEL_ACTIVE
can be used to add the active image. A
document ID of DOCSEL_ALL can be used
to add all images.

Value
Comments This function guarantees that a new stack is started (any existing stack is
discarded). Note that the first image added to the stack determines the
image type and size required of all subsequent images added. Note also that
all frames of the image will be added to the stack .
See Also IpEDFAdd
IpEDFRemove
Syntax IpEDFRemove(DocId)
Description
This function removes the specified image from the current Extended
Depth of Field stack.
Parameters DocId Integer Specifies the image to remove from the

existing EDF stack. A document ID of
DOCSEL_ACTIVE can be used to remove the
active image. A document ID of
DOCSEL_ALL can be used to remove all
images.

Value
See Also IpEDFAdd
Page 2-19
IpEDFSet
Syntax IpEDFSet(Attribute, Value,Frame)
Description
This function sets the Extended Depth of Field attributes

attribute to set, from the following:
EDF_NORMALIZE Indicates whether
the frame intensities should be normalized
prior to focus analysis.
EDF_CRITERIA Indicates the criteria
used to analyze the frames for in-focus
material. (See below for constants.)
EDF_TOPO_MAP Indicates whether a
topographic map image should be created.
EDF_ TOPO_CALIBRATED Indicates
whether the topographic map image should
be given an intensity calibration that gives
each pixel a value corresponding to the Z
position of the plane that it was extracted
from.
EDF_ORDER Indicates whether the image
list will be used from top to bottom
(EDF_TOPDOWN) or bottom to top
(EDF_BOTTOMUP).
EDF_DEFAULT_FRAME Returns the
default plane document ID (in Value) and
frame number (in Frame).
Value Integer The value that the attribute will be set to.
Frame Long This is a long variable in your script that will


Value
Page 2- 20
Comments The EDFTopoMap attribute will affect only the way the EDF operates from
the dialog. To create a topographic map from a macro, use
IpEDFTopoMap.
The Frame parameter is not used and can be IPNULL for all commands
except EDF_DEFAULT_FRAME
The following constants will be used with EDF_CRITERIA attribute to

indicate the type of focus analysis:
EDF_MAX_LOCALCONTRAST: Pixels will be examined in a
local neighborhood around the target pixel and the degree of local
contrast determined for each plane. The pixel from the plane with
the greatest local contrast will be selected.
EDF_MAX_INTENSITY: Pixels in the same location on each
plane will be examined and the pixel with the highest intensity
will be selected.
EDF_MIN_INTENSITY: Pixels in the same location on each
plane will be examined and the pixel with the lowest intensity will
be selected.
EDF_MAX_DEPTHCONTRAST: Pixels in the same location on
each plane will be compared to the mean intensity of all pixels at
that position, and the pixel with the greatest contrast from that
mean intensity will be selected
See Also IpEDFGet, IpEDFTopoMap
Page 2-21
IpEDFShow
Syntax IpEDFShow(Show)
Description
This function shows or hides the Extended Depth of Field dialog box.
Parameters Show Integer An integer value of 0 or 1 indicating whether

to show or hide the Extended Depth of Field
dialog
0 - Hide the Extended Depth of Field dialog.
1 - Show the Extended Depth of Field dialog

Value
See Also IpEDFNew,IpEDFAdd, IpEDFCreate, IpEDFTopoMap, IpEDFGet,

IPEDFSet
IpEDFTopoMap
Syntax IpEDFTopoMap()
Description
This function creates the Extended Depth of Field topographic map image.
Return DocId of the new image if successful, a negative error code if failed.
Value
Comments If the topographic map option is selected, use the IpEDFTopoMap function
to create the topographic map image.
See Also IpEDFAdd, IpEDFCreate, IPEDFNew
Page 2- 22
IpFsGet
Syntax IpFsGet (File,Attribute,Data)
Description Indicates the file signature attribute that should be returned.
Parameters File String Indicates the full path of the file name to
analyze.
Attribute Integer Indicates the file signature attribute that should

be returned.
Data Any Provides the user variable to receive the

attribute.
The Attribute parameter determines the type of data returned to the user’s
Comments variable, and can be one of the following:
FS_SIGNATURE The current file signature is returned as a 128-bit
number.
FS_COMPARE The 128-bit number provided is compared to the current
signature.
If they are identical, the return code will be 1, otherwise the
function returns 0.

Value
See Also IpFsGetStr
IpFsGetStr
Syntax IpFsGetStr (Attribute, Data)
Description Indicates the file signature attribute that should be returned.
Parameters File String Indicates the full path of the file name to
analyze.
Page 2-23
Attribute Integer Determines the type of data to be returned.

FS_SIGNATURE_STR The current file
signature is returned as a string
FS_COMPARE_STR The provided string is
compared to the current signature.
Parameters Data Any Provides the user variable to receive the

attribute.
When using FS_COMPARE_STR, returns 1 for identical signatures,

Return otherwise returns 0.
Value Will return an error code if failed.
See Also IpFsGet
IpIsGet
Syntax IpIsGet (sAttribute,Data)
Description Indicates the image signature attribute that should be returned.
Parameters Attribute Integer Identifies the type of data to be returned. Must

be one of the following:
IS_SIGNATURE The current image signature
is returned as a 128-bit number.
IS_COMPARE The 128-bit number provided
is compared to the current signarture.
Data Any Provides the user variable to receive the

attribute.
Return When using IS_COMPARE, returns 1 for identical signatures, otherwise

Value returns 0.Will return an error code if failed.
Page 2- 24
See Also IpIsShow, IpIsGetStr
IpIsGetStr
Syntax IpIsGetStr(sAttribute, Data)
Description Indicates the digital signature attribute that should be returned.
Parameters Attribute Integer Determines the type of data to be returned. Must be

one of the following:
IS_SIGNATURE_STR The current image
signature is returned as a string
IS_COMPARE_STR The provided string is
compared to the current signature.
Data String Provides the user variable to receive the attribute.

See comments below.
Return When using IS_COMPARE_STR, returns 1 for identical signatures, otherwise

Value returns 0.
Will return an error code if failed.
See Also IpIsShow, IpIsGet
IpIsShow
Syntax IpIsShow (Show)
Description Shows or hides the image signature dialog.

An integer value specifying whether to display
Parameters Show Integer or hide the image signature dialog.
0 = Hide Image Signature dialog
1 = Show Image Signature dialog.
Page 2-25
Return Returns 0 if successful, a negative error code if failed.

Value
See Also IpIsGet, IpIsGetStr
Page 2- 26
IpLSFltApply
Syntax IpLSFltApply(Type, Width, Height, Passes, Strength )
Description This function applies one of the Large Spectral Filters
Parameters Type Integer Type indicates the kind of filter that should be
applied. Must be one of the following:
LF_LOPASS a low-pass filter
LF_HIPASS a high-pass filter
LF_EDGEPL a bright-edge filter
LF_EDGEMN a dark edge filter
LF_BANDPASS a band-pass filter
Width Integer Width indicates the width of the filter to apply.
Height Integer Height indicates the height of the filter to apply.
Passes Integer Passes indicates the number of times the filter

should be applied.
Strength Integer Strength indicates the filter strength, where 100

is full strength and 0 is no effect.

Value
See Also IpLSFltShow
IpLSFltShow
Syntax IpLSFltShow(Show )
Description This function shows or hides the Large Spectral Filters dialog box.
Page 2-27
Parameters Show Integer An integer value of 0 or 1 indicating whether to

show or hide the Large Spectral Filters dialog
0 - Hide the Large Spectral Filters dialog.
1 - Show the Large Spectral Filters dialog

Value
See Also IpLSFltApply

IpSeqGCreate
Syntax IpSeqGCreate()
Description
This function creates a sequence gallery for the active image.
Return Document ID of the new sequence gallery if successful, a negative error code
Value if failed
Comments The sequence galleries themselves are Image-Pro workspaces, and can be
manipulated with the normal set of IpDoc and IpWs Auto-Pro functions.
IpSeqGGet
Syntax IpSeqGGet(Attribute, DocID, Value)
Description
This function gets a sequence gallery attribute.

attribute to get, from the following:
SEQG_TRACKENABLE Indicates whether
the gallery will track changes in the
sequence
SEQG_ISTRACKED Indicates whether
the specified workspace is a sequence and
is tracked by a sequence gallery
SEQG_ISGALLERY Indicates whether
the specified workspace is a sequence
gallery
Page 2- 28
DocId is ignored for
Integer SEQG_TRACKENABLE.
DocID For SEQG_ISTRACKED and
SEQG_ISGALLERY,
DocId indicates the document ID of the

workspace to inquire about.
about the active workspace.
Value Integer Value is an integer variable in your script to

Return 0 if successful, a negative error code if failed

Value
See Also IpSeqGUpdate
IpSeqGSet
Syntax IpSeqGSet(Attribute, Value)
Description
This function sets a sequence gallery attribute.

attribute to set, from the following:
SEQG_TRACKENABLE Indicates whether
the gallery will track changes in the
sequence.
Value Integer Value is the value to set the attribute to, where
1 indicates to track sequences changes, and 0
indicates that the function should wait for a
call to IpSeqGUpdate.

Value
See Also IpSeqGUpdate
Page 2-29
IpSeqGShow
Syntax IpSeqGShow(Show)
Description
This function displays or hides the sequence gallery dialog.
Parameters Show Integer A value of 0 or 1 specifying whether the

sequence gallery dialog is to be displayed
or hidden.
0 = hide the dialog
1 = show the dialog
ret = IpSeqGShow(1)
Example
This statement opens the Sequence Gallery dialog
ret = IpSeqGShow(0)
This statement closes the Sequence Gallery dialog.

Value
IpSeqGUpdate
Syntax IpSeqGUpdate(DocId)
Description
This function updates the sequence gallery to reflect any changes to the
sequence itself.
Parameters DocId Integer Indicates the document ID of the sequence

gallery to update.

Value
Page 2- 30

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Manual Image Pro Plus 4

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Image-Proâ Plus

Version 4.5 for Windowsâ

Updates and Changes

Media Cybernetics, Inc.

MAN SU 7842N40 20010530

All other trademarks in this document are trademarks or registered trademarks

U. S. GOVERNMENT RESTRICTED RIGHTS IN DATA CLAUSE

Introducing Image-Pro Plus Version 4.5

n Pentium CPU, running at 120 MHz or higher, configured with Windowsâ

The following hardware is optional:

n Image Capture Board (frame-grabber)

What's New in Version 4.5

The following improvements have been made to Image-Pro Plus v. 4.5:

Large Spectral Filters

Filter Types and Parameters

Width: Indicates the width of the filter being applied.

Width: Indicates the width of the filter being applied.

Preview and Spectrum

Directly select a hue by:

Color Composite and Image Sequences

Extended Depth of Field

Image List Tab

Focus Settings Tab

Operations with original color or gray images: Co-localization operates on a pair of

Hardware and Services

New File Format Information

Supported File Formats

AutoQuant ( *.DEB, *.AVZ)

BMP (*.BMP, *.DIB, *.RLE)

Dr. HALO (*.CUT)

Image-Pro Workspace (*.IPW)

Kodak PhotoCD (*.PCD)

Molecular Dynamics (*.GEL)

PICT (*.PCT, *.PICT)

New Auto-Pro Functions

Description This function adds a document to the active composite image.

Indicates a color for the source image. Hue may

IpCmpAdd (0, HUE_QUERY) ‘Adds document 0, queries the user for

Return 0 if successful, an error code otherwise.

0 if successful, or IPCERR_INVARG (bad docID) or IPCERR_FUNC (not a composite active)

Description This function gets the values for color compositing.

For the LUT , FRAME, NUMFRAMES, and

Value indicates the variable that will receive the

Comments See the following:

LUT_BRIGHTNESS DocID of the source Current LUT value.

SHIFT_X DocID of the source Current pixel shift

COMP_FRAME DocID of the source Current frame

COMP_HUE DocID of the source Hue

COMP_BACKGROUND none DocID of the

COMMAND DocID VALUE

COMP_NUMFRAMES DocID of the source Number of frames in

GETNUMDOC none Number of documents

GETDOCLIST none An integer array of the

Return 0 if successful, an error code otherwise

Indicates a color for the source image. Hue may

Return 0 if successful, an error code otherwise.

Description This function sets the values for color composites.

For the LUT, SHIFT, RESET, BESTFIT, and

Value long New value for the specified setting.

Comments For COMP_RESET or COMP_BESTFIT the value argument is ignored, and a

COMMAND DocID VALUE

LUT_BRIGHTNESS DocID of the source New LUT value.

SHIFT_X DocID of the source New pixel shift

AutoQuant ( .DEB, .AVZ)

BMP (.BMP, .DIB, *.RLE)

PICT (.PCT, .PICT)