Bio Image XD

BioImageXD User Manual 1 / 39
BioImageXD
USER MANUAL
Last updated 2007-08-21
by Pasi Kankaanpää
Important notes:
• This manual is still incomplete.

• This manual may contain errors.
• This manual does not yet contain any pictures.
• Only beta versions of BioImageXD have been released so far, and some
of the functionality of the software does not yet fully correspond to what
is described in this manual.
• A complete version of this manual will be released at the latest when the
first "production quality" version of the software is released.
• If you notice errors in this manual, please report them to
info@bioimagexd.org
• Only the design lead of the BioImageXD team is allowed to change this
document
Contents
1. Introduction
1.1. What is BioImageXD?
1.2. Who made BioImageXD and why?
1.3. Why was BioImageXD not built on existing open source applications?
1.4. How do I start using BioImageXD?
1.5. Some terminology
2. BioImageXD user interface

2.1. Main tool bar
2.2. View panel
2.2.1. Top and right side buttons and settings
Viewing angle
Zoom controls
Annotation controls
Original
ROI controls
Delete
Six arrow buttons
Channel display controls
2.2.2. Sliders
2.2.3. Eyedropper
2.2.4. Interpolation method
2.3. Task panel
2.4. File tree & visualization settings
3. BioImageXD features and functions

3.1. File input/output functions
3.1.1. Open dataset
3.1.2. Save dataset
3.1.3. Open settings
3.1.4. Save settings
3.1.5. Save snapshot image
3.1.6. File tree
3.2. Tasks
3.2.1. Merge
Alpha channel
Preview and help
Saving the results
3.2.2. Intensity Transfer Function (ITF)
“Brightness”, “contrast” and “gamma”
Other ITF adjustments
3.2.3. Colocalization
Threshold sliders
2D histogram (scattergram)
Automatic thresholding
P-value
Colocalization statistics
Colocalization color
3.2.4. Adjust
Transfer function
Interpolation
3.2.5. Process
Xxx
Xxx
3.3. Visualization modes
3.3.1. Slices view
3.3.2. Gallery view
3.3.3. Orthographical view
3.3.4. Maximum Intensity Projection view
3.3.5. 3D view
Lighting settings
Clipping plane
Frame
Orthogonal slices
Surface rendering
Volume rendering
Lights
Render window
3.4. Special or miscellaneous functions
3.4.1. Animator
Xxx
Xxx
3.4.2. Resampling switch
3.4.3. Quick help
3.5. Additional features and functions
3.5.1. File menu additional commands
Import
Export
Open settings for current
Save settings for current
Close task panel (?)
Quit
3.5.2. Edit menu additional commands
Undo
Redo
Command history
3.5.3. Settings menu additional features
Preferences
3.5.4. Tasks menu additional features
Resample dataset
Rescale dataset
3.5.5. Visualization menu additional features
Immediate updating
3.5.6. View menu additional features
3.5.7. Help menu additional features
About BioImageXD
Help
3.5.8. Other additional features
Histograms
Script editor
Channel palettes
1. Introduction
1.1. What is BioImageXD?
BioImageXD is a free, open source software package for processing, analyzing and visualizing
multidimensional image data. As the name implies, the software is primarily intended for biological data,
for instance confocal microscopy images of living cells, but it is fully suitable for working with any kind
of multidimensional image data.
BioImageXD has a simple, intuitive user interface based on a single large window, and its operations
have been designed to be consistent and functional. The software offers advanced file input/output
functions and multiple visualization modes (ranging from simple 2D slice view to advanced 3D
rendering), that can be freely combined with any of the numerous task modes (including channel
merging, colocalization analysis, intensity correction and various filters for removing noise,
segmentation, tracking, measurements etc.). Stunning movies can be created with the built-in animator
that allows full control of camera paths, viewing angles, time point changes etc.
BioImageXD is written in Python and C++, with wxPython being used for the graphical user interface
(GUI) and the Visualization Toolkit (VTK) and the Insight Segmentation and Registration Toolkit (ITK)
for image processing, analysis and rendering. Movies are created using ffmpeg.
1.2. Who made BioImageXD and why?

BioImageXD was made by a heterogeneous group of scientists and researchers (microscopists, cell
biologists, programmers etc.) from the universities of Jyväskylä and Turku in Finland, and the Max
Planck Institute for Cell Biology and Molecular Genetics, Dresden, Germany, with collaborators
worldwide.
Existing commercial software for processing and visualizing multidimensional microscopy images
seemed to us very expensive, inflexible and full of bugs. We had lots of great microscopy data, but no
good free software to process it reliably, and felt frustrated by the situation. We therefore developed
BioImageXD, and specifically made it open source so that anyone can use and modify it freely. Now,
even if the software doesn’t do exactly what you want or misbehaves, you can change or fix it yourself
(or ask us to do it) - without paying for expensive software licenses. In addition, you never have to
wonder what exactly the program does to your valuable data, because there are no undisclosed
algorithms, “black boxes” or other “trade secrets” that commercial programs often have. You can process
your data more reliably because you always know what you’re doing, and you can even freely change
algorithms or other details if you so choose. BioImageXD is free and operating system independent, so
basically any scientist in need of this type of software should be able to use it.
1.3. Why was BioImageXD not built on existing open source 3D rendering
and processing/analysis GUI applications?
Originally it was (MayaVi), but as our work progressed we realized that existing software structures and
interfaces were not always suitable for our purposes, and changing them would have meant more work
than doing completely new software. Not too long ago we were still using MayaVi for rendering (a few
years ago it seemed like the best available rendering tool for our purposes), but then it turned out that it
did some things differently from what we desired, and when our main programmer made a new renderer
in just one night, it was clear that BioImageXD was going to become a completely new piece of software.
This is not to say that other similar open source applications would be bad – quite the contrary! In fact,
we have gotten many good ideas and found useful solutions from them. In this particular case making a
new application just was a more practical solution. Please note, however, that we do leverage much
functionality from previously existing open source toolkits like the Visualization Toolkit (VTK) and ITK.
Plans also exist to interface BioImageXD with other software, such as ImageJ and PyMol.
1.4. How to start using BioImageXD?

Anyone can use BioImageXD, free of charge! To begin, go to the Download section of our website
(www.bioimagexd.net). Choose the software package that suits your needs and operating system, and
install it onto your computer according to the instructions specified. For Windows, just download the
appropriate file, then double-click it once download is complete and follow the instructions - within a few
moments BioImageXD will be ready for use. For Mac OS X, mount the disk image downloaded and drag
and drop the BioImageXD application into your Applications folder, and then drag that into your Dock, if
you like, for easy access.
1.5. Some terminology

Before reading further, you should make sure that you understand the meaning of the following terms.
(Note: the reader of this text is expected to have basic knowledge of computer programs and bioimaging.)
Dataset. In BioImageXD the word dataset is used to describe any 3-dimensional (3D) image data. In
practice a dataset is a collection of 2-dimensional (2D) images (lateral dimensions, X and Y) from
various depths (axial dimension, Z). A typical example is a Z-stack created by a confocal microscope - a
collection of optical sections (2D images) from different focal planes.
Dataset series. If the image data is four-dimensional (4D), the fourth dimension here being time and
meaning that there are several 3D image stacks from different time points, such data is referred to as a
dataset series. You can also think of a dataset as a dataset series with only one time point.
Channel. The term channel in BioImageXD refers to a “subset” in a dataset (series). One dataset may
contain several channels, all showing the same sample (and thus having the same amount of timepoints
and 2D slices per time point), but somehow differently. A typical example is a confocal microscopy
image, where the same sample has been stained with two or more different fluorescent dyes, and the
image formed from the fluorescence of each dye is saved and represented as its own data “Channel”.
Rendering. In the context of BioImageXD, rendering means the generation of an image from the dataset.
It might be a rendering of a single 2D image slice, or a 3D volume or surface rendering, or even a 3D
rendered movie.
2. BioImageXD user interface

BioImageXD has a simple user interface that was designed and intended to be intuitive and comfortable
to use, in order to achieve the best workflow. It is based on a single, large window that has four essential
parts. At the top is the main tool bar (and a traditional menu system). In the middle is the view panel,
which shows the currently chosen image. The top of the view panel holds additional settings and buttons
related to it. On the right is the task panel, which shows either settings related to the currently chosen
task or, if no task is chosen, general information about the currently chosen dataset. On the left appears
the file tree, if chosen to be displayed, and/or settings related to the visualization mode chosen. In some
situations there is nothing here, in which case the space is used to make the view panel larger. Below is a
more detailed description of each of these parts.
The animator is the function that has, due to its different nature, a different window design: the top
section with the toolbar is the same, but the other three sections are replaced by one big animator panel.
Notes:
• The BioImageXD window has been designed for a minimum screen resolution of 1024x768.
However, a higher resolution makes the software more comfortable to use.
• The various parts of the window can be resized with the mouse if required. Slight resizing may
also help if parts of the window appear garbled or not updating properly.
• The display of various items in the BioImageXD main window can be controlled from the view
menu (section 3.5.6.).
• BioImageXD supports "tooltips"; point with the mouse to an item and leave it there for a while,
and you will see a short description of the item. This feature also works with for instance file
names and eyedropper info, which may sometimes bee too long to fit into the space reserved for
them. The tooltip is then a handy way of quickly seeing for instance the full file name, without
needing to scroll or resize anything.
2.1. Main tool bar

The main tool bar at the top of the BioImageXD window contains buttons for all major functions of the
software. The buttons are divided into four categories.
1) The first six buttons are for file input/output, and are (in order from left to right): open dataset, save
dataset, open settings, save settings, save snapshot image, and file tree.
2) The next four buttons are for choosing a task, i.e. what is being done with/to your image data, and are
(in order from left to right): merge, colocalization, adjust, and process.
3) The next five buttons are for choosing the visualization mode, and they are (again from left to right):
slices view, gallery view, orthographic view, maximum intensity projection (MIP) view and 3D view.
4) The last category is special or miscellaneous functions. These are the animator and quick help.
Most of these functions can also be found from the corresponding drop-down menus at the very top of the
window/screen. Unlike the main toolbar, however, the menus may be slightly different when different
tasks or modes are chosen, and contain also additional functions and settings that cannot be found from
the main tool bar.
2.2. View panel

This largest part of the BioImageXD window shows a rendered image of the channel or channels chosen
from the file tree. If no task is chosen, the image shown is the “original”. If a task is chosen, the image
shown is the one processed according to the settings of the task. (In many situations you can, however,
control what is displayed. For instance, you can choose whether to display the original or the processed
image when a task is chosen.)
2.2.1. Top and right side buttons and settings

On top of the actual image as well as on the right hand side of it are settings and buttons related to how it
is displayed.
On the top are the following:
Viewing angle. A drop-down menu for fast choosing of the display angle. You can choose to view your
rendering along any of the three spatial axes (X, Y and Z) and from either direction (+ or -). Choosing
any of these settings also returns the object to the center of the scene, if it wasn’t there already. This
option is only available in 3D mode.
Zoom controls. There are five different zoom controls: a zoom-out button on the left, then a drop-down
menu for choosing the zoom level and then a zoom-in button. The third button zooms the image so that it
is the largest possible allowed by the available window space (“zoom to fit”). The last of the zoom
control buttons on the right allows you to zoom to any chosen area of the image. After clicking this
button you can draw a rectangle onto the image, and releasing the mouse button zooms in to this area.
Notes:
• “Zoom to fit” does not work in 3D mode.
Original. This button is handy when you are using some task that alters the image. If you wish to
compare your alteration to the original image, click and hold down this button. As long as you keep
pressing it, you will see the original, unprocessed image in the view panel. Releasing the button restores
the display to the processed image.
Perspective. This button is used to toggle between perspective and parallel viewing in the view panel.
Works only in 3D mode.
Six arrow buttons. These are used for rotating a rendered scene along any of the three spatial axes, X, Y
or Z. They are useful in situations where it is difficult to find the appropriate angle by grabbing the image
with the mouse. This function is only available in 3D mode.
Notes:
• The current release of BioImageXD does not yet support these six arrow buttons. Instead, there
are eight small arrow buttons for adjusting the pitch, yaw, roll and elevation of the image.
Sampling info. This text field displays the dimensions (in X, Y and Z) currently in use for the image
being displayed/processed as well as the original dimensions of the image. If no resampling has been
done (either automatically or manually), both numbers are the same.
On the right are the following:
Annotation/ROI drawing tools. This group of buttons on the right side of the view panel are used to
create annotations or ROIs (ROI = region of interest), in other words things that you can "draw" onto the
image. The first button draws a circle: click to the desired center of the circle and hold down the mouse
button, then move away to the desired perimeter of the circle and release the button. The second one
draws a rectangle similarly; click and hold with the left mouse button at one corner and drag, then release
at the opposite corner. The third button is a polygon drawing tool: click with the mouse to as many
polygon corners in the image as you want, and the program connects these points with straight lines.
When finished, right click, and the ROI is “closed” and ready for use. The next button adds a scale bar to
the image: click at the location where you want your bar to begin, and drag the bar to a suitable length
with the mouse. The last of the drawing tools allows you to add text to the image.
Annotation/ROI controls. The next group of buttons on the right are used to control existing annotations
and ROIs. The first button converts a ROI into a mask, i.e. a binary image that acts like a “vignette”
through which the original image is viewed. Several masks can be created into memory and activated for
use with the “Mask selection” feature (section 3.5.6.). Masks can also be created with the Process task
(section 3.2.5). The next button, the "X", deletes the ROI/annotation being clicked, and the last button
opens a dialog box for choosing the color of an annotation or ROI.
Notes:
• Any object (ROI or annotation) in the image can be selected by clicking it. A selected object can
be moved around within the image by left click, hold and drag, and it has control nodes from
which it can be resized and reshaped.
• All ROI/annotation tools work in such a way that you first choose the tool and then interact with
the view panel accordingly, never the other way around. Also, the tool only remains selected until
the first subsequent interaction with the view panel occurs. Thereafter the "normal" mode in
which objects can be selected as explained above is restored.
• Double clicking a ROI opens a dialog box for specifying a name for it. The name is also shown on
screen.
• Annotations and ROIs are only visible in the "Slices" and "Maximum intensity projection" view
modes.
• Only the scale bar annotation is currently supported. Other annotations, such as lines and text, will
be implemented in future versions.
• The scale bar function is not supported in 3D mode. However, the required effect can be achieved
with the "Distance measurement" module.
• With the Process task ROIs can be used to cut away unwanted parts of the image and thus make
images smaller.
Resample. This button toggles on or off the resampling of the image. If no resampling has been defined
when pressing this button, the "Resample dataset" dialog box (see section 3.5.4.) appears, allowing you to
specify a resampling. If a resampling has already been defined this way, by choosing "Resample dataset"
from the "Tasks" menu or by automatic resampling, the button simply switches between the resampled
and original dimensions.
Notes:
• If a resampling has already been defined, you cannot access the "Resample dataset" dialog with
this button. You must then use the "Tasks" menu.
• When resampling is used, all procedures done to the image data, visualizations and analyses alike
are carried out using the resampled dimensions. It may be a good idea to use resampling to make
the program faster with large datasets, but to calculate final analyses and visualizations using the
original data.
Resample to fit. This button resamples the image so that its size is optimal for the current resolution and
size of the view panel, taking also the zoom level into account. Especially for visualizing large images
this feature is handy, as you always get the best possible resolution but do not slow the computer down
with the processing of image detail you cannot see. Note that this feature is primarily for visualization
purposes, and using it for analyses is not recommended.
Channel display controls. This last group of buttons is available if you work with a task that processes
more than one channel simultaneously, i.e. Merge, Colocalization or Process. The buttons have borders
colored according to the color chosen for the channel they represent, and display a small maximum
intensity projection (MIP) of the channel data for easy identification. Several buttons may be selected at
the same time. In Merge you use these buttons to choose whether to view any of the original channels or
some combination. In Colocalization you use these buttons to choose whether you wish to view the
original channels, the colocalization map or some combination. In Process these buttons allow you to
choose between any of the original channels being processed (one or more) or the output channel of the
Procedure list, the default color of which is gray.
Notes
• These buttons only control what is being displayed, not what is being processed. If you wish to
change the channel being processed, you must use the file tree as usual.
2.2.2. Sliders
Below the image is a time point slider. Irrespective of the visualization mode selected you can use this
slider to choose the time point being shown (and operated upon, if such a task is selected). Note that the
time slider has no purpose and is absent if you have only one time point (i.e. only 3D data, not 4D).
To the right of the image is another slider, the Z slider, which you use to choose the image slice (Z slice,
optical section) being viewed (or operated upon, if such a task is selected). Note that the Z slider is not
available in the maximum intensity projection and 3D views, because by definition in these modes all
slices are always included in the rendered image shown.
2.2.3. Eyedropper
In all other view modes except the 3D view, there is an “eyedropper” tool available. Make sure no button
requiring successive actions (like some annotation or zoom buttons) has been pressed, and then click on
any part of the image. At the bottom you will see a text showing the original scalar value of that pixel (for
8-bit data the intensity value between 0 and 255), the location of the pixel you pointed (also the Z slice),
and the RGB value the scalar maps to (the “actual color” being displayed; the three numbers represent the
red, green and blue components of the RGB value). If multiple channels are selected, you will see this
information for all channels.
2.2.4. Interpolation method
By right-clicking on the image in slices or maximum intensity projection view, you can choose the
method of interpolation used to render the image when the number of pixels in the original image data is
less than the size of the image displayed on screen. In other words, you can choose how the program
“fills in the gaps”. This feature is usually meaningful only when images are either very small or you have
zoomed in a lot. Switching on interpolation will make the image look smoother. You can also switch the
interpolation off altogether.
Changes in the interpolation setting are applied instantly. There are basically two interpolation methods
to choose from: linear and cubic. The “Interpolation depends on size” option means that the program
automatically chooses the interpolation method based on the zoom level. This is the recommended
setting.
2.3. Task panel

If no task is chosen, this panel, on the right side of the window, shows basic information (“Channel info”)
on the currently chosen channel. At the bottom is the palette of the channel, in other words, a color strip
demonstrating the look-up table (color transfer function) for each of the 256 intensity values (8-bit image)
that a single pixel (or voxel) can have. For instance in the case of Carl Zeiss .lsm and Olympus FV1000
.oif files, channel color information is stored in the .lsm or .oif file, and BioImageXD reads this
information and automatically modifies the palette accordingly. The palette can be changed by clicking
on the color strip.
If a task is chosen, this panel shows the settings related to that task and the channel info is hidden. If you
wish to see the channel info again you can do so at any time by deselecting the task that is currently
selected (do this simply by clicking on its button in the main tool bar).
2.4. File tree & configuration panel

Depending on which buttons are pressed down in the main toolbar, there can be either one or two
additional sections to the left of the view panel. These are the file tree and the configuration panel. If
neither is displayed, the view panel extends to the left edge of the window and is at its largest. Showing
either the file tree or the configuration panel makes the view panel narrower, and it is narrowest when
both are displayed.
The file tree (see more information below) is at the far left when displayed. It can be switched on and off
at any time, irrespective of the task or visualization mode chosen. However, switching to a task
automatically hides the file tree, and opening new files automatically displays it.
To the right of the file tree (or to the far left of the window if the file tree is hidden) appears the
configuration panel if either gallery view or 3D view is chosen as the visualization mode. The panel
contains settings for configuring these visualization modes. Other visualization modes do not have any
settings, so there is more space available for the view panel when these modes are selected.
Notes
• The display of various items in the BioImageXD main window can be controlled from the view
menu (section 3.5.6.).
3. BioImageXD features and functions

Below are brief descriptions of all functions of the BioImageXD software. They are presented in the same
order and groups as their buttons in the main toolbar (described above). Other features, such as those
accessible from the menus only, are presented last.
Before looking at the features, however, a few remarks about the operational logic of the software:
• Unlike some other similar programs, BioImageXD prefers to always handle the channels of a
dataset series separately. Multiple channels are processed together only when required, for
instance when analyzing colocalization or combining the channels with the merge task.
• The basic work flow in BioImageXD, when creating visualizations, is designed to be as follows:
1) Load all channels you wish to process into the internal file tree. 2) Process each channel
separately with the “Adjust” and “Process” tasks if required, for instance to remove noise or to
correct for intensity changes due to dye bleaching. Save the processed channels as new dataset
series. 3) Combine the (processed) channels using either “Merge” or “Colocalization” tasks,
depending on what your final goal is. Save the results (the merged dataset series or the
colocalization map) as new dataset series. If necessary, combine the resultant dataset series from
steps 2 and 3 further with each other, so that in the end you have all the channels and their
combinations you wish to visualize loaded into the file tree. 4) Make 3D renderings of all the
required dataset series, and save as still images or create movies with the animator.
• When your goal is to obtain colocalization analysis results in addition to or instead of

visualizations, the basic work flow is similar to the one described above, except that in step 3 you
will in addition save colocalization statistics and/or maps into separate files for further analysis. If
you want to do segmentation, tracking and/or measure various things from either ROIs or the
segmentation results, all these are done with the “Process” task (step 2).
3.1. File input/output functions

3.1.1. Open dataset
The first button in the main toolbar (top left) is “open dataset (series)”. Usually you begin working with
BioImageXD by choosing this function. A more or less standard open file dialog box appears. The
dataset series formats that can be directly opened without any conversion steps currently are: Olympus
Image Format datasets from FV1000 (*.oif), Leica TSC-NT and TCS-SP2 datasets (*.txt), BioRad PIC
datasets (*.pic), BioImageXD datasets (*.bxd), Interfile files (*.hdr) and Zeiss LSM 510 datasets (*.lsm).
More file formats will be supported in the future, as user demand requires. “BioImageXD datasets” refers
to BioImageXD’s own file format for multidimensional image data.
When a file is opened, information about it is read into memory and the file appears to the internal file
tree of BioImageXD. The actual image data, however, is not stored in memory for obvious memory limit
reasons (the data is read from the file one time point at a time only when needed, but all slices of the
selected timepoint are always read into memory simultaneously). You can load several dataset series at
the same time, or add more to the file tree at any time later on. Datasets that are open at the same time do
not have to be of the same type or format.
Notes:
• BioImageXD supports up to 16-bit image data, but 8-bit should be a sufficient bit depth for most
practical purposes. From "Preferences" you can set up BioImageXD to automatically rescale
higher than 8-bit data to 8-bit upon loading.
• Large file sizes significantly slow down BioImageXD. From "Preferences" you can set
BioImageXD to automatically resample large files to a smaller size upon loading.
• Other than the above mentioned file types may be loaded with the “Import” feature (section
3.5.1.).
• BioImageXD does not support compressed LSM files or Olympus FV1000 .oib files, which are
also compressed. (Both of these file types use a proprietary Windows API, so they are difficult to
read in free software.)
3.1.2. Save dataset
Writes a (processed) dataset to the disk. Whenever you have done something to a dataset, you can save
the processed dataset as a new dataset series in the BioImageXD file format (*.bxd, see below). For
instance, when merging multiple channels and satisfied with the settings, clicking “Save dataset” writes a
new file of the merged dataset.
After clicking “Save dataset” a new window appears. This is where you choose the timepoints that you
wish to include in the new file. Each time point is represented by a small button. All time points are
chosen by default. You can freely select or deselect time points by clicking with the mouse (deselected
time points appear bright, selected ones dark). You can also specify an automatic selection of every nth
time point by typing a number into the corresponding field. After selecting the desired time points click
“Process Time Points” and a normal save file dialog box appears. The saved file automatically loads in
the file tree to simplify further processing, such as 3D rendering. Clicking “Close” cancels the procedure.
Notes:
• By removing unnecessary time points at the “Process” stage you can make your datasets smaller
and faster to process. This function may come in handy especially in the near future, when faster
live imaging systems become available and the number of recorded time points may increase
greatly.
• The “Merge” task always creates 24-bit RGB datasets, and the resultant saved datasets are also
24-bit RGB. In all other situations the saved datasets have the same bit depth as the original data.
• Although it is not necessary to write, for instance, the merging result of multiple channels into a
new file before creating a 3D animation of it, this is highly recommended, because rendering an
RGB dataset is much faster from an RGB file than if the program needs to combine 8-bit channels
"on the fly". The same applies for many other types of processing as well.
• If you are working with a resampled dataset and either the "Resample" or the "Resample to fit"
button (section 3.4.2.) is pressed down, the dimensions of the saved dataset correspond to the
resampled dimensions. Release the "Resample" button if you want to save the dataset using its
original dimensions.
BioImageXD file format. BioImageXD has its own file format. Although the program can read numerous
other file formats, datasets are always written in the BioImageXD format only (of course, exporting as
2D images series or movies is also possible). This file format consists of three different types of files, and
a single dataset can consist of hundreds of files. A separate directory is automatically created for all these
files, and the name of the directory is the same as that given for the "file" in the "Save dataset" dialog
box. The directory will contain VTI files (one for each time point, containing the actual image data, VTK
XML Image format files), BXC files (one for each channel, containing settings and metadata) and one
BXD file (the "mother file", basically just a list of BXC files). In BioImageXD the user always handles
only the BXD files. The other two file types are always created and read automatically and in the
background. However, if handling the files outside the BioImageXD program, for instance when copying
or moving them, be sure to always handle the entire directories, not the individual files they contain. (If
the contents of a BioImageXD dataset directory is spread to different locations, the files no longer work.)
In addition to the dataset file format, there is another BioImageXD file format, BXT, which is reserved
for settings alone. These files are handled through the "Open settings" and "Save settings" commands
(see below). Note that when a BioImageXD dataset file is created, it contains the settings of the task that
was used to make the file, as well as general settings (or general settings only, if no task was selected).
Settings can therefore be recalled with the "Open settings" command from BXT and BXD files alike. Yet
another file type used by BioImageXD is LUT (color Look Up Table). These can be saved and loaded in
the Edit Color Transfer Function window.
3.1.3. Open settings
You can use this function to recall settings that you have saved earlier (see 3.1.4.).
Handy if you need to process the results of several experiments all in the same way, or if you need to
continue working with a previously unfinished project. Uses standard Open file dialog box.
When recalling settings first go to the desired task, then recall its settings (also recalls the general
settings). If you try to recall settings that were not made for that task, only general settings are recalled
and an info message is displayed. If no task is selected, then only general settings are recalled (even if the
settings file contains settings for a task). Settings can be recalled either from the designated settings files,
*.BXT (see below), or from BioImageXD dataset files (*.BXD).
Notes:
• The settings for all channels of the currently active dataset are loaded.
• There are some other settings that can be (saved and) recalled separately, such as palettes (color
look up tables or LUTs) and procedure lists for the "Process" task.
3.1.4. Save settings
At any time during the operation of BioImageXD you can use this feature to save current settings. It is
advisable to use this function regularly while working. This way, in case the program crashes or
something else unexpected occurs, it will be easy to resume working later on. Uses a standard Save file
dialog box.
Save settings makes a BXT (settings) file, which contains the settings for the currently chosen task and
general settings (which currently include annotations only). If no task is chosen, only general settings are
saved. Remember that settings are automatically similarly saved into BioImageXD dataset files.
Notes:
• The settings for all channels of the currently active dataset are saved.
• There are some other settings that can be saved (and recalled) separately, such as palettes (color
look up tables or LUTs) and procedure lists for the "Process" task.
3.1.5. Save snapshot image
This function saves a snapshot image of the currently rendered scene. This means that whatever is
displayed in the middle part of the window, the view panel, is saved as a conventional 2D image. A
normal save file dialog box appears, and you can choose from four different file formats: PNG, JPEG,
TIFF and BMP. This feature is handy for saving a still image of the screen whenever you see something
that pleases your eye. Note that this function saves single 2D images only, not entire dataset series or
movies. You can set the default file format in which the images are saved in “Preferences” (section
3.5.3.). Note that by default the still image encoding/compression quality is always the best possible and
cannot be adjusted.
3.1.6. File tree
You can use this button to toggle on and off the display of the internal file management tree of
BioImageXD. When the tree is displayed, it is at the far left of the window.
The “root directory” is termed “Datasets” and each file type has its own “folder” (for instance “LSM
Files” or “BioImageXD Files”). Each dataset (series) file is represented by a “sub folder” inside the
appropriate file type folder (the name of the folder is the same as that of the file it represents), and each
channel is represented as a “file” inside the sub folder. Note that the folder for a given file type is visible
only if files of that type have been loaded.
The file tree is used for choosing the channel which is being processed, analyzed or visualized using
BioImageXD, so choosing a channel from the list is usually the second step (after opening a file) when
beginning work with BioImageXD. Channels are chosen simply by clicking them with the mouse, and
multiple channels can be chosen by keeping the CTRL key pressed. All channels of a file can be chosen
by clicking the name of the file.
A dataset can be removed from the file tree by right-clicking it and then choosing "Close dataset".
When a task is active (selected), you cannot change the channel(s) being processed simply by choosing
new ones from the file tree. You either have to close the task first, select the new channel(s) and then
open the task again, or use the "Switch dataset" button at the bottom of the file tree. When a task is
selected, choose the new channel(s) to process and then click "Switch dataset". This switches the
dataset(s) being processed to the new ones without switching off the task or affecting its settings. This
feature is handy if you have several similar datasets that you want to process similarly with a particular
task.
Notes:
• Both automatically and manually resampled datasets appear red in the file tree and with an
asterisk (*) after their name.
• If BioImageXD is abnormally terminated, the file tree is automatically restored the next time the
program starts. (If some of the files can no longer be found at that stage, they will not be restored,
and no error or notification message of this appears.) This feature can be switched off in the
"Preferences".
• After using a task that operated on several chosen channels, it may be difficult to choose a single
one of those channels. In that case double-clicking the channel name may help.
3.2. Tasks
3.2.1. Merge
The merge task is used to combine several channels into one RGB dataset. Begin by choosing at least two
channels from the file tree and then click the “Merge” button in the main tool bar. The settings for the
task appear into the task panel on the right.
In the settings there are first buttons from which you choose the channel you wish to adjust. Below the
list is a color strip illustrating the palette (aka. LUT) used for the selected channel. You can change the
palette by clicking on the color strip. Below the color selection is one of the key components of
BioImageXD, the intensity transfer function (ITF), explained in detail in section 3.2.2.
Alpha. In volume rendering objects appear translucent. When creating a volume rendering the computer
needs to determine how transparent/opaque a given voxel should be (not all voxels are rendered equally
transparent, as that would produce meaningless results). An opacity transfer function (OTF) is used for
this purpose. It uses intensity values as input and produces opacity values as output. This is very
straightforward when rendering a grayscale image, as there is only one intensity value per voxel, and
these intensity values (ranging from 0 to 255 for an 8-bit image) can directly be used as input for the
OTF. However, RGB images contain three different intensity values per voxel (one for the red (R), one
for the green (G) and one for blue (B) component of the image), and thus there is no clear input for the
OTF. This input needs to be created during merging by somehow combining the three intensity values,
and the resulting value is called alpha. Hence an RGB image in BioImageXD actually contains four, not
just three, scalar values per each voxel: R, G, B and alpha. Such images are sometimes called RGBA
images. In practice the alpha component has the same range as the other three components. BioImageXD
offers three different methods for creating the alpha component during merging, found under the ITF
graph under the heading "Alpha construction":
• In “Maximum mode” the alpha value is the value of the brightest of the three intensity components
(R/G/B).
• In “Average mode” the alpha value is the arithmetic average of the values of all three intensity
components (R/G/B), and this mode also allows you to specify a threshold. If the value of an
intensity component is lower than this threshold, it is not counted in.
• In “Image luminance” mode the alpha value is the luminance of the combination of all three
intensity components. Image luminance is calculated using the vtkImageLuminance class, and it
is a weighted average of the R, G and B components of the combined channels, the weighting
being based on how bright different colors are considered to be by the eye. The weights are:
0.30R, 0.59G and 0.11B.
Notes:
• BioImageXD does not use the original intensity values of the channels being combined, but rather
the intensity values produced by the ITFs, for calculating the alpha component.
• For grayscale images there is no alpha component (or it can be considered to be the same as the
"intensity component"), but for RGB images a separate alpha component is required for 3D
volume rendering.
• The R, G, B and alpha components of an RGBA image are sometimes called "channels", but it is
important to note that such channels are not the same as the dataset channels in BioImageXD (see
section 1.5). RGBA "channels" merely describe the different components of an RGBA image, but
not the original channels that were used to create it. (The only exception is, if the original
channels had palettes strictly corresponding to the components R, G and B, i.e. each channel had a
palette where the value of only one of the components changed with intensity, with the other two
remaining 0 for all intensities. Only then can the components of and RGB image be thought of as
"channels" in BioImageXD.) As BioImageXD allows the separation of an RGB(A) image into its
components, creating separate channels from the R, G and B components (section X.X), you
should be very careful not to confuse such "artificially" created channels with the "true" channels
in a dataset!
Apply. If your changes in the task panel settings are not automatically applied (for instance the view
panel is not updated), click this button at the bottom of the task panel.
Notes:
• You can switch immediate updating of the view panel on or off from the “Visualization” menu
(see section 3.5.5.)
• Remember that, as with all tasks, you can use any of the five visualization modes for previewing
the result. This means that you can even have a direct look at what a full 3D rendering of the
merged dataset series is like.
Saving the results. Using the features described above, try to find the best settings for the merging. When
satisfied, click “Save dataset” in the main toolbar (see section 3.1.2.), and BioImageXD will write a new
file, in RGB format, (*.bxd) of the merged dataset. Note that although it is not necessary to write the
channel combination into a new file before creating for instance a 3D animation of it, this is highly
recommended, because rendering an RGB dataset is much faster from an RGB file than if the program
needs to combine 8-bit channels “on the fly”.
3.2.2. Intensity Transfer Function (ITF)

The ITF is one of the key components of BioImageXD. It appears in the task panel when either the
“Merge” task (3.2.1.) or the “Adjust” task (3.2.4.) is selected. The ITF describes how the intensity values
of pixels (or voxels) are changed during processing. If you change things like image brightness or
contrast in any image processing software, in practice this means changing the ITF. In BioImageXD you
can always see a graph of the ITF (the green line), so that you know exactly how your data is being
processed. On the X axis are the input intensity values (0-255 for 8-bit data), and the green line illustrates
how these values are converted to the output values (0-255 for 8-bit data) on the Y axis. The graph is
drawn directly from the actual function, so you can be sure that it is always correct. Around the graph are
the various settings with which you can change the ITF, described below. The effects of your changes can
be seen straight away from the graph and (provided that you have chosen the channel being processed to
also be displayed) in the image in the view panel.
The purpose of the ITF in “Merge” (3.2.1.) is to be able to adjust, essentially without limitations, how the
channels look when combined together. For example, if one channel is much weaker than the other, you
can compensate by adjusting the ITFs. Careful adjustment of the ITFs can significantly improve image
quality in some situations without loss of crucial information, and you can also use the ITF to filter out
low intensity noise, or to choose only a certain intensity range to be displayed. With “Adjust” (3.2.4.) you
can do all these things also to a single channel without merging, and in addition you can interpolate ITF
adjustments between time points for instance to compensate for fluorescence bleaching.
Notes:
• The default ITF is f(x) = x, i.e. all intensity values remain unchanged. You can restore the default
ITF by clicking the “Reset” button.
• The ITF is always adjusted individually for each channel.
• In “Merge” ITF adjustments always apply to all time points.
• In “Adjust” ITF adjustments apply to the currently selected time point only.
“Brightness”, “contrast” and “gamma”. Below the ITF graph are sliders for adjusting image brightness
and gamma, and on the right a slider for adjusting image contrast. You have probably adjusted these
settings before with some other programs, but did you actually know what you were doing to your data?
With BioImageXD you know - just observe the ITF graph when you make adjustments. Observing the
graph is the best way to understand what brightness, contrast and gamma actually mean, but they can be
roughly explained as follows:
• Changing brightness adds or subtracts a certain value to or from all pixel intensities, the same
value for all pixels.
• Changing contrast changes the slope of the ITF graph.
• Changing gamma converts the graph from a straight line to a curved one.
Notes:
• Making these adjustments easily changes your data irreversibly so, that information is lost. This is
sometimes forgotten when making these adjustments, but with the BioImageXD ITF graph you
can always monitor the situation and know what is lost and what is not.
• The “brightness”, “contrast” and “gamma” adjustments are in quotation marks. Due to the special
nature of the BioImageXD ITF, in some situations these settings may be somewhat different from
what “normal” brightness, contrast and gamma are considered to be. However, in practice the
effects of these adjustments are always very similar to “normal” brightness, contrast and gamma.
Other ITF adjustments. Below the ITF graph and the brightness, contrast and gamma sliders are eight
input boxes (and two sliders) for specifying various other values and thresholds that add further flexibility
to the ITF. In order to gain an understanding of these features, please take your time to observe how the
ITF graph changes when making these adjustments.
• The “Min value” and “Max value” boxes are used for specifying minimum and maximum values
for the output pixel intensities.
• The “Min threshold” and “Max threshold” boxes are used for specifying minimum and maximum
values for the input pixel intensities.
• The “Smooth start” and “Smooth end” boxes are used for specifying thresholds for smooth start
and smooth end, which modify the ITF so that the graph begins from zero and rises as a straight
line up to the smooth start threhold, and drops again as a straight line to zero starting at the
smooth end threshold. The ITF values at the smooth start and end thresholds, i.e. the values which
the straight line rises to and descends from, are determined by all the other ITF settings.
• To the right of the smooth start and end threshold boxes are sliders (and boxes) for specifying
gamma (the “curvature”) for the smooth start and end lines.
3.2.3. Colocalization
For the colocalization task two channels need to be selected from the file tree. The task is used to
visualize and analyze bright voxels that occupy the same spatial and temporal location in both channels,
i.e. colocalize. This type of analysis is commonly used in biological microscopy to find out if two
proteins might be co compartmentalized or possibly interact with one-another. Such analysis is far too
often very non-scientific and imprecise, based only on a subjective visual inspection of superimposed
images of the two channels, which is not reproducible. BioImageXD offers robust colcoalisation analysis
methods in an easy-to-use tool for both the visualization of colocalized voxels as well as their numerical
and statistical analysis, based on a number of published approaches.
Notes:
• Unlike in merge, where the number of channels that can be simultaneously chosen is not limited,
in colocalization only two channels can (and must) be chosen.
• Colocalization analysis is always performed for one timepoint at a time but for the whole 3D
dataset, not just for the currently chosen slice (if in slices or orthographic view mode).
As with “Merge”, switching on the task shows its settings in the task panel on the right. At the top is a
channel selection just like in “Merge”. Below that are threshold sliders, a colocalization scattergram (2D
histogram), buttons for threshold and statistics calculations, point spread function calculation settings, a
list of colocalization statistics with an export button and finally colocalization color settings. At the
bottom is the same “Apply” button as in “Merge” (section 3.2.1.).
Threshold sliders. These are used to manually set the lower and upper threshold for the channel currently
selected. Only voxels with intensities between the specified thresholds in both channels are considered
while calculating many of the colocalization statistics (some statistics are calculated independently of the
thresholds). These voxels are also used to create a new dataset series, consisting of the colocalized voxels
only, called the colocalization map. If switched on from the small channel selection buttons, it can be
seen in the view panel. Remember that, as with all tasks, you can use any of the five visualization modes
to display the contents of the view panel. Note that you can specify the thresholds also by interacting with
the scattergram (see below).
2D histogram (scatterplot). The scatterplot has axes with values ranging from 0 to 255 (for 8-bit data),
and it shows all the voxels of the current time point plotted with the intensity scalars of one channel on
the X axis against those of the other channel on the Y axis. The blue, shaded rectangle in the scattergram
shows the region specified by the four thresholds. In addition to using the sliders described above, you
can change the thresholds by interacting with the scattergram: simply drag any of the the white lines
outlining the blue box to change the corresponding threshold. Dragging a corner changes both of the
corresponding thresholds at the same time. By right-clicking on the scattergram you can choose whether
to use a logarithmic scale for the axes or save it as an image.
Notes
• In addition to the 2D histogram, individual histograms for both of the selected channels can be
shown at the top of the view panel by choosing “Histograms” from the view menu (see section
3.5.8.).
• "Calculate statistics" (see below) also calculates and displays the linear regression of the
scattergram.
• You can save the scattergram as an image by right-clicking it and choosing "Save as"
Automatic thresholding. Thresholds are crucial in colocalization analysis, because the results very much
depend on them. However, setting them manually either by using the sliders or by drawing on the
scattergram is always more or less subjective and error-prone. A method has been published for obtaining
the thresholds automatically (Costes et al., 2004), and BioImageXD offers you this possibility. We
recommend using it whenever possible. The automatic threshold calculation can be initiated by clicking
the “Auto-Threshold” button. The rectangle in the scattergram and the sliders at the top are updated once
the calculation is complete. For this method to work, the background/offset of the images must be set to
zero, or images with background intensity must have that background subtracted. The method relies on
looking for spatial correlation between the 2 colour channels (see more about that below), and as such
can fail if there is very little or no spatial correlation or even anti correlation (exclusion – green is never
where red is) between the images.
P-value. An essential part of any statistical analysis is the calculation of the P-value. The P-value in this
case tells you the probability of the detected colocalization being real and not a mere coincidence. The
value is obtained by comparing the correlation of Channel 1 and Channel 2 with that of Channel 1 and a
randomly created Channel 2. BioImageXD offers three different methods for the randomization of
Channel 2: Fay, Costes and van Steensel. You can choose the appropriate method by clicking the
corresponding radio button in the “Calculate P-value” box. When you then click “Calculate statistics”,
also the P-value (the 8th item in “Coloc volume statistics”) is calculated. If you choose “None”, no P-
value is calculated. If you choose the Costes algorithm, you also need to specify additional parameters:
PSF radius is used to select a block size for randomizing the Channel 2 image. This Point Spread
Function size can be approximated by a calculation requiring knowledge of the Numerical Aperture of
the objective lens and the wavelength of the fluorescence emission light detected in Channel 2. If these
can be read from the dataset file loaded, then they are used, and the PSF radius is calculated. If they
cannot be read from the dataset file, you will need to input the Numerical Aperture and Ch2
wavelength. With correctly spatially sampled images the PSF size should be 2.3-3 pixels. For the Costes
method you also need to specify the number of iterations used - the highger the number the more accurate
the result, but the longer the calculation takes.
Colocalization statistics. The “Coloc volume statistics” lists various numerical details about the detected
colocalization. These are calculated when you click the "Calculate statistics" button, considering current
thresholds (set either automatically or manually) (and PSF radius/Numerical Aperture/Ch2 wavelength
settings for the Costes method). Some of the items in the list are self-explanatory, but all are briefly
described below. As for most of the settings in BioImageXD, the colocalization statistics have been
organized into three color coded categories: basic, intermediate and expert. We recommend using the
statistics in the "basic level" category for most colocalization analyses, and caution that even though
some of the statistics (such as % of volume colocalized) in the other categories may seem simpler, they
can actually be quite difficult to interpret correctly, and their use requires more expertise in the field. Use
the “Export” button to save the statistics as a CSV (Comma Separated Values) file. Remember that the
statistics are calculated (and the scattergram drawn) for the currently chosen time point only! However,
you can of course change the time point from the slider in the view panel, and then repeat the
calculations.
Basic level:
• Ch1 threshold (lower/upper): the lower and upper thresholds used for channel 1, set either
manually or automatically
• Ch2 threshold (lower/upper): the lower and upper thresholds used for channel 2, set either
manually or automatically
• Correlation: The spatial correlation (Pearson's correlation coefficient) between the intensities of
the two channels over the whole 2D/3D image. The thresholds do not affect this number.
Pearson's correlation coefficient is one the most frequently used methods for describing the degree
of overlap/correlation between two "patterns" in images (the two channels in this case). It is a
simple estimator of colocalization, but the values range from -1 to 1, with the negative values
being difficult to interpret when quantifying colocalization (0 means no correlation, and negative
values something like "anti-correlation" or exclusion). In addition, if the proportion of each
protein colocalized with the other is not the same, which is often the case, no single number can
accurately describe colocalization. Instead, two numbers, such as M1 and M2 (see below), are
needed.
• Correlation (voxels > threshold): The spatial correlation (Pearson's correlation coefficient)
between the intensities of the two channels in voxels above the lower thresholds.
• Correlation (voxels < threshold): The spatial correlation (Pearson's correlation coefficient)
between the intensities of the two channels in voxels below the lower thresholds. For well set
(lower) thresholds this value should be close to zero and/or slightly negative.
• Coloc coefficient 1 (M1/tM1): Colocalization coefficient 1 as defined by Manders et al., 1993.
The first number, M1, is the proportion of the total channel 1 intensity which is colocalized with
channel 2. The second number, tM1, is otherwise the same but calculated using the thresholds.
These coefficients are independent of the relative intensities of the two channels.
• Coloc coefficient 2 (M2/tM2): Colocalization coefficient 2 as defined by Manders et al., 1993.
The first number, M2, is the proportion of the total channel 2 intensity which is colocalized with
channel 1. The second number, tM2, is otherwise the same but calculated using the thresholds.
These coefficients are independent of the relative intensities of the two channels.
• P-value: The probability with which the detected colocalization is not a coincidence. A value
higher than 0.95 is generally regarded as an indication of the colocalization being “real”.
(Calculated only if something other than "None" is selected in the "Calculate P-value" box.) Note
that as in Costes et al., 2004, the P-values given by BioImageXD are "inverted" compared to the
traditional statistical p-value.
Intermediate level:
• Sum of ch1 (total/thresholds): The first number is the sum of the intensities of all channel 1
voxels. The second number is the sum of the intensities of the channel 1 voxels which are
between the lower and upper threshold.
• Sum of ch2 (total/thresholds): The first number is the sum of the intensities of all channel 2
voxels. The second number is the sum of the intensities of the channel 2 voxels which are
between the lower and upper threshold.
• # of non-zero voxels: The total amount of voxels having a non-zero intensity.
• # of voxels > threshold: The total amount of voxels that are above the lower threshold in both
channels.
• # of colocalized voxels: The number of colocalized voxels within the ranges specified by the
thresholds of both channels.
• % of volume colocalized: The percentage of colocalized voxels of all voxels in the image volume.
• % of ch1 colocalized (voxels/intensity): The first number is the percentage of colocalized channel
1 voxels within the thresholds; the second number is the percentage of colocalized channel 1
intensity within the thresholds.
• % of ch2 colocalized (voxels/intensity): The first number is the percentage of colocalized channel
2 voxels within the thresholds; the second number is the percentage of colocalized channel 2
intensity within the thresholds.
• % of ch1 colocalized (total voxels/intensity): The first number is the percentage of colocalized
channel 1 voxels in the whole 2D/3D image; the second number is the percentage of colocalized
channel 1 intensity in the whole 2D/3D image.
• % of ch2 colocalized (total voxels/intensity): The first number is the percentage of colocalized
channel 2 voxels in the whole 2D/3D image; the second number is the percentage of colocalized
channel 2 intensity in the whole 2D/3D image.
Expert level:
• Differ. stain of ch1 to ch2 (voxels/intensity): In differential staining a certain molecule, which is
found both on the cell surface and in the cytoplasm, is stained before and after permeabilization
with different colors. Such staining can be used for instance to study the internalization of a virus.
If channel 1 is the stain before permeabilization, only molecules on the cell membrane are visible.
Channel 2 is then stained after permeabilization, and shows both all internalized molecules as well
as some (but not necessarily all) of the molecules on the membrane. Here the first number gives
the ratio of membrane molecules to internal ones and is calculated as follows: ch1 voxel amount /
(ch2 voxel amount - colocalized voxel amount). The second number is calculated similarly, but is
based on intensity sums, not voxel amounts.
• Differ. stain of ch2 to ch1 (voxels/intensity): Like the above but channels “reversed”.
• % of diff. stain of ch1 (voxels/intensity):
• % of diff. stain of ch2 (voxels/intensity):
• R(obs): Pearson's correlation coefficient for the original 2 channel image data.
• R(rand) (mean +-sd): Mean Pearson's correlation coefficient of all the randomized datasets
created in confidence p-value calculation with standard deviation of the coefficients for all the
ramdomized images.
• R(rand) > R(obs): Number of randomized images giving a greater Pearson's correlation
coefficient than the original image data. Confidence P-value is 1; (no. of R(rand) > R(obs) / no. of
randomized images or iterations)
Notes
• You should not be intimidated by this long list of numbers. In most cases only a few are needed,
but we want to offer the possibility to do also very complex analyses to those who need them.
• Colocalization analyses are not always that straightforward, and it may be difficult to decide on
the best method to measure colocalization in a particular situation. However, in general we
recommend the following: use auto-thresholding and analyze colocalization using three numbers:
a colocalization coefficient (M or tM) for channel 1, the same colocalization coefficient (M or tM)
for channel 2 and the confidence P-value.
Colocalization color. The default color for the colocalization map is white. The color can be changed by
clicking the color strip under the heading “Colocalization color”. Just like with any other channel in
BioImageXD, also palettes can be specified. Palettes enable the creation of a colocalization map where
stronger colocalization is red and weaker blue, for example. The colocalization map is 8-bit by default,
but sometimes a 1-bit map may also be useful. Tick the appropriate check box below the color strip to
convert the map to 1-bit. In the field below you can type how bright you want the single color map to be.
Notes:
• When when all the settings are done, statistics saved and you are ready to write the colocalization
map into a new *.bxd file, click the “Save dataset” button in the main toolbar (see section 3.1.2.).
• In colocalization analyses usually the lower thresholds are the meaningful ones, but for additional
flexibility BioImageXD offers also adjustable upper thresholds. For most (but not all)
colocalization calculations only voxels with intensities between the lower and upper thresholds
are included. In most cases it makes sense to leave the upper threshold at the maximum value.
3.2.4. Adjust
The “Adjust” task is used for adjusting the ITF of a single channel. Begin by choosing the channel you
wish to adjust from the file tree. As with “Merge” and “Colocalization”, switching on the task shows its
settings in the task panel on the right.
In the settings there is first a color strip illustrating the palette used for the selected channel. You can
change the palette by clicking on the color strip. Below the color selection are two tabs, “Transfer
function” and “Interpolation”, explained below. At the bottom is the same "Apply" button as in “Merge”
(section 3.2.1.).
Notes:
• As with all tasks, when finished adjusting, you can (and often should) write the processed channel
into a new *.bxd file by clicking “Save dataset” in the main toolbar (see section 3.1.2.).
• Remember that, as with all tasks, you can use any of the five visualization modes to display the
contents of the view panel.
Transfer function. This is the intensity transfer function. The ITF here is exactly the same as in “Merge”,
except that changes made into it apply only to the time point currently selected, not to all time points as in
“Merge”. (See section 3.2.2. for a more detailed description of the ITF.) Below the ITF are three buttons
for managing ITF changes. The “Reset current” button restores the ITF for the currently chosen time
point to the default one, which is f(x) = x. The “Reset all” button restores the ITFs of all time points to
the default one. The “Copy to all” button copies the ITF of the currently chosen time point to all time
points.
Interpolation. This feature is used to linearly interpolate ITF adjustments between time points. It is used
together with the ITF settings, and is handy for instance for compensating intensity fluctuations over
time, such as fading/bleaching of fluorescence towards the end of a long time series. In the interpolation
tab you can specify a maximum of five and a minimum of two time points to be used as references in the
interpolation. The interpolation procedure is as follows:
1) Type the number of the interpolation starting point into the “from timepoint” input box.
2) Click the corresponding “goto” button, and the time point specified in the input box will be
chosen for ITF adjustment and for display in the view panel.
3) Click the “Transfer Function” tab and adjust the ITF for the starting time point if necessary. Then
go back to the “Interpolation” tab.
4) Type the number of the interpolation ending point into the “to timepoint” input box.
5) Click the corresponding “goto” button, and the time point specified in the input box will be
chosen for ITF adjustment and for display in the view panel.
6) Click the “Transfer Function” tab and adjust the ITF for the ending time point. Then go back to
the “Interpolation” tab.
7) You can now click the “Interpolate” button, and the ITFs of the time points between the specified
starting and ending points will be linearly interpolated.
8) Alternatively, you can create more complex interpolation patterns by specifying a maximum of
three more reference timepoints (that are between the starting and ending timepoints) into the
three “thru” input boxes, before clicking “Interpolate”. Use the “goto” buttons and switching
between tabs as above to adjust the ITF for the “thru” reference points. When ready, click
“Interpolate”.
9) After clicking “Interpolate” you can check the result simply by flipping through the time points
with the slider in the view panel and observing the images shown. If you have the “Transfer
Function” tab selected while doing this, you can also observe how the ITF changes.
10) If you are not satisfied with the results, simply readjust the ITF of any of the reference timepoints
and try “Interpolate” again. Clicking “Reset all timepoints” in the “Interpolate” tab resets the ITF
of all timepoints to the default, f(x) = x. (The “Reset all” button in the “Transfer Function” tab
does the same.)
Notes:
• The interpolation will be carried out within the range specified by the “from timepoint”
and “to timepoint” input boxes, and timepoints outside this range are not affected. Thus
typing something into these two input boxes is required for interpolation.
• Interpolation between any two successive timepoints typed into the five input boxes is
always linear. The three “thru” boxes are for the purpose of creating more complex
interpolation patters, for instance to compensate for non-linear fading of fluorescence. The
“thru” input boxes may also be left empty.
3.2.5. Process
The "Process" task is the newest and most versatile of the BioImageXD tasks, and can be used to perform
complex sets of tasks in one go. The "Process" task panel consists of a "Procedure list", to which
procedures can be added by selecting them from the four menu buttons below the list (Filtering,
Arithmetics, Segmentation and Tracking). A procedure on the list can be selected by clicking it, in which
case settings for it appear below the list (if there are any setting that can be changed). A selected
procedure can also be removed from the list (the "X" button on the right) or its position changed (the
arrow buttons on the right). When clicking "Apply", all the procedures on the list are carried out in the
order in which they appear.
The fifth menu button below the "Procedure list" is "Presets", and contains complete predefined
procedure lists for performing typical tasks such as watershed segmentation. Presets are a handy way of
learning about the Process task, particularly about doing more complicated tasks, and they can be used as
bases for creating new procedure lists. Any procedure list currently set up can also be saved as a new
preset for easy future use (choose "Save as preset..." from the "Presets" menu). Note that also the settings
for each procedure on the list are saved.
The settings for each procedure are very different, but typically contain one common element at the top; a
drop-down menu for selecting the input(s) or "source dataset(s)" for the procedure. Typical options
available are the currently chosen original channels as well as "Input from the procedure list", which is
the default setting and means that the input is the output of the preceding procedure. (For the first
procedure on the list the input is always the original channel(s), irrespective of what is chosen from the
drop-down menu.)
Notes:
• The final output of the Procedure list is by default displayed on the view panel as a "new channel"
with gray color. Depending on the last procedure, a list of numerical results may also appear
below the procedure list.
• Clicking "Apply" only processes the currently selected time point. Use the "Save dataset" feature
if you wish to process multiple time points in one go. Use this feature also, if you wish to save the
output dataset as a new image file.
3.2.6. Segmentation and tracking overview
The BioImageXD "Procedure list" can be used to perform advanced segmentation and object tracking
tasks, which are becoming very popular. Segmentation means separating objects of interest from the rest
of the image data (background), i.e. allocating parts of the image into objects that can be handled by the
computer as independent entities with their own parameters such as volume and average intensity. When
segmentation has been carried out for all time points, the same objects can be identified in successive
time points. This is called tracking. Once done successfully, parameters such as speed, distance and
direction of movement can be obtained. Tracking can only be performed with segmented data.
Doing segmentation or tracking is always a multistep process requiring fairly complex procedure lists to
be set up. Also, segmentation and tracking cannot be performed with a single procedure list, but rather
two are needed, one for segmentation and one for tracking. The segmentation procedure list typically
begins with image preprocessing such as filtering or noise removal, followed by two procedures that
carry out the segmentation (one prepares it and the other produces the actual segmented objects). The
next procedure analyzes the segmentation results, providing readouts for each object separately as well as
a summary. If continuing to tracking, the segmentation results need to be saved and processed with a
second procedure list for tracking. Now the first procedure creates the tracks (this is probably the most
complex and demanding procedure in BioImageXD), the second procedure visualizes them and the third
procedure analyzes them (again providing readouts for each track separately as well as a summary).
Segmentation. BioImageXD offers three basic classes of segmentation methods:
1) Connected component labeling. This is a simple segmentation method that needs a binary image (1-
bit) as input. Typically the binary image is created with basic thresholding, i.e. all voxels with intensities
higher than the specified threshold are considered objects and given the value 1 (white), while everything
else is 0 (black). Connected component labeling then analyzes this data to see which of the white areas
are connected to each other and thus determines the boundaries of the objects and their number. The
objects are then given different colors (also known as "blob coloring"), ordered from small to large and
numbered. A threshold can be specified for excluding small objects. Example procedure list:
• Noise filtering (e.g. Hybrid Median 2D)
• Threshold
• Connected component labeling
• Analyze objects
2) Watershed segmentation. This is a so called edge based method, and more sophisticated than
connected component labeling. It uses 8-bit grayscale images as input and is based on edges (high
contrast gradient areas) being first detected from the image data. Example procedure list:
• Noise filtering (e.g. Hybrid Median 2D)
• Feature Detection (e.g. Gradient Magnitude)
• Morphological Watershed Segmentation
• Analyze objects
3) Region growing (a region based method)

Notes:
• No segmentation method is perfect or copes with all images (e.g. noisy images are problematic for
watershed)
• Best results are often obtained by using a combination of techniques (for instance thresholding
first, then watershed)
• Segmenting touching objects is particularly challenging.
Tracking. BioImageXD tracking is based on the general principle of finding the closest object in the next
time point. However, this is often not accurate enough, so it is possible to specify additional conditions
that the nearest object candidate needs to fulfill in order to be accepted as the same object. These
conditions are specified as thresholds and weights for:
• maximum change in distance
• maximum change in size
• maximum change in average intensity
• maximum change in direction of movement
When segmentation has been successfully carried out and the saved dataset chosen for tracking, a
procedure list such as this is set up:
• Create tracks (set this up carefully, including the loading of the segmentation analysis results, the
specification of seed objects and the specification of the parameters listed above)
• Visualize tracks
• Analyze tracks
Notes:
• Error-free tracking is practically impossible for the computer. For each experimental setup the
above mentioned tracking parameters need to be found out by detailed tests, possibly including
verification with manual tracking and comparisons to artificial test pictures. This is the only way
to obtain truly reliable results.
3.2.7. Procedure descriptions
Below are brief descriptions of each of the available procedures and their settings.
Filtering - Filtering
Median 3D
Anisotropic diffusion 3D
Solitary filter
Hybrid median 2D
Gradient anisotropic diffusion (ITK)
Sigmoid filter (ITK)
Extract a subset
Gaussian smooth
Extract components
Filtering - Feature detection

Sobel 3D
Gradient magnitude
Gradient magnitude (ITK)
Canny edge detection
Find local maxima
Arithmetics - Image arithmetic

Shift ans Scale
Add
Subtract
Multiply
Divide
Sin
Cos
Log
Exp
Sqrt
Gradient
Arithmetics - Logical operations

And
Or
Xor
Not
Nand
Nor
Segmentation - Segmentation
Threshold
Otsu threshold
Mask
Connected component labeling
Threshold for maximum object number
Segmentation - Region growing

Connected threshold
Neighborhood connected threshold
Confidence connected threshold
Segmentation - Watershed
Watershed segmentation (old)
Morphological watershed segmentation
Re-label image
Invert intensity
Segmentation - Measurements
Analyze objects
Timepoint correlation
Analyze ROIs
Segmentation - Registration
Versor rigid 3D registration
Tracking - Tracking
Create tracks
View tracks
Analyze tracks
3.3. Visualization modes
BioImageXD offers five different modes to visualize image data. They are selectable from the main tool
bar, and any one of them can be used with any task, and the results are shown in the view panel and can
be saved as images or videos. (Refer to section 2.2. for a description of the various tools and buttons in
the view panel.)
3.3.1. Slices view
This most basic of the visualization modes shows the individual two dimensional images (Z-slices,
optical sections) of a three or four dimensional image stack (dataset series), one at a time. The vertical Z
slider at the right is used for choosing the image slice being viewed, and the time slider below for
choosing the time point. The slices view is perhaps the best visualization mode for observing the image
data “as it is”, without any kind of projection or rendering, and is often a good starting point for working
with new dataset series. Sometimes exported slice views may also be useful in presentations or
publications, but often you want to use the more advanced visualization modes for producing your final
images.
Notes:
• With some tasks the settings of the Z and time point sliders determine which part of the dataset
series is being processed.
• Slices view is the default visualization mode of BioImageXD.
3.3.2. Gallery view
This is like slices view, but all the two dimensional slices of a three dimensional image stack are shown
at once as smaller images (recall that in slices view only one slice at a time is displayed, and often in
larger size).You can change the time point being viewed from the slider below the image, but the Z slider
on the right is unnecessary, because all Z slices are shown simultaneously.
You also have the option to view all time points of a single slice, by choosing “Timepoints” in stead of
“Slices” from the “View in gallery” panel. In this case the time slider becomes unnecessary, but now you
use the Z slider to select which slice you wish to observe.
3.3.3. Orthographical view
In this mode you can see three different slices of the object, each cut along a different one of the three
spatial axes, X, Y and Z. The top-left image shows a cut along the Z axis, i.e. it’s like the regular “slices
view”. Below it is a cut along the X axis and on the right a cut along the Y axis. The white crosses in
each of the three images determine the planes along which the cuts are made. You can reposition any of
the crosses by clicking at the location where you want it. If you keep the mouse button pressed, you can
move the cross around with the images being constantly updated. You can also move the fourth cross in
the bottom right corner, outside any of the three images. The effect is the same as moving the Z slider.
3.3.4. Maximum Intensity Projection view

In this visualization mode, which could also be referred to as “simple 3D view”, a maximum intensity
projection (MIP) of all the Z slices of a dataset is made along an observational axis emanating from
directly above the sample. For each pixel of the MIP the intensity is determined by the highest intensity
encountered along the observational axis, hence the name “maximum intensity”. In this case that means
that for every lateral pixel position of the projection (which is actually two dimensional), the intensity is
taken from that Z slice where it happens to be highest. Although the MIP is two dimensional, it can be
considered a volume rendering technique, where the view angle is an orthographic projection along the Z
direction from the top view.
The Z slider is hidden in this visualization mode, because all Z slices are always included in the image.
Time points, however, can be changed from the relevant slider as usual.
Notes:
• This view mode is designed for quick and easy visualization of the whole 3D dataset as one
image, a feature often required while working with complex dataset series. For this reason there
are no adjustments, and there is only one, fixed projection angle.
• You can use also the 3D mode for creating MIPs, and there you can freely rotate the projection to
view it from any angle.
• Even though the MIP is very easy and quick to do and is used a lot, even in publications and
presentations, you should not necessarily settle for it when making your final images. Explore full
3D rendering also; in many cases it produces much better quality images than this simple MIP
view.
• MIPs highlight different structures disproportionately, and in most cases should not be used for
conducting measurements.
• Remember that you “lose” most of the 3D information when you make a 2D projection of a 3D
dataset. This is why interactive 3D volume rendering or making a movie of the object rotating is
more informative than a single MIP from one angle (the top).
3.3.5. 3D view
This is the most advanced visualization mode of BioImageXD and offers a wealth of methods and
settings. As the name implies, the visualizations created are three dimensional and can therefore be freely
rotated on screen by grabbing and dragging them with the left mouse button. All Z slices are always
included in the image, so the Z slider is hidden, but timepoints can be changed from the time point slider.
In addition the following manipulation methods are available:
• right-click or middle button scroll: zoom in or out (the regular zoom functions of the view panel
also work, except for “Zoom to fit”)
• CTRL-click: rotate clockwise or counter-clockwise
• SHIFT-click or middle button click: laterally reposition or “translate” the object (move it away
from the origin, the center of the rendered scene)
Certain keys control the behavior of the display panel in 3D mode:

• J and T toggle between “joystick mode” and “trackball mode”, respectively. These determine
how mouse actions control the movement of the object. In joystick mode if you do something like
drag the object with the middle mouse button or make it rotate with the left button, the movement
continues until you release the button. In addition, if you click the right mouse button while
holding down the left one, the movement continues indefinitely even after you release the mouse,
and by just moving the mouse (no clicking!) you can adjust the speed and direction of the
movement. A left click stops the movement. In trackball mode (default) the movement follows
the mouse exactly, so the rotation only occurs when you move the mouse, and the object moves
only as far as you move the mouse. Also in this mode you can keep the “link” to the mouse
movement active even after releasing mouse buttons. As in joystick mode, this is accomplished by
clicking the right button while holding down the left one.
• C and A toggle between “camera mode” and “actor mode”, respectively. These determine
whether you control the camera or the object (actor) with your mouse. You can see the difference
if you move the object away from the origin (center) and then rotate it. When in camera mode
(=default) the rotation will still be around the origin and the object will move accordingly. In actor
mode you always rotate the object itself, no matter where it is located. (Remember that you can
use the “Isometric” drop-down menu in the view panel to reset the object to the center, see 2.2.1.)
See section 2.2. for a description of other view panel features.
When 3D mode is selected, a “Visualizer” settings panel appears to the left of the view panel. First in this
panel is a drop-down menu from which you can select different rendering modules. Choose the desired
module and then click “Load”. The module will appear into the list below the drop-down menu. By
default the list contains the “Volume rendering” module. You can toggle loaded modules on or off by
clicking their check boxes. Multiple modules can be selected simultaneously, but please note that some
modules may behave oddly together.
When you select a loaded module by clicking its name (not its check box), the settings for that module
appear below the module list. The settings are different for each module, except for the “Advanced”
settings (see below), which are the same for all modules. Also common to all is an “Apply” button at the
bottom of the settings list. Clicking it updates the rendered scene according to the specified settings. A
selected module can be removed from the list of loaded modules by clicking “Remove”.
Advanced lighting settings. Clicking the “Advanced” button to be found in the settings of all rendering
modules displays OpenGL lighting settings that determine how the rendered scene is lit. These settings
allow you to specify the intensity of four different lighting parameters used when OpenGL shading is
enabled: ambient lighting, diffuse lighting, specular lighting and specular power. Experiment a little to
see how they each affect the rendered scene. If you tick the “Use shading” check box, then the settings in
the “Lights” configuration window (available from the “Visualization” menu and described below) also
affect how the rendered scene is lit.
Now let’s have a look at the available rendering modules and their specific settings:
Angle.
Axes renders an illustration of the axes of the three spatial dimensions, X, Y and Z, into the image. The
axes are shown in red, green and yellow. There are no specific settings for this module.
Clipping plane. With this module you can specify an arbitrary plane into the 3D image space. The image
will be cut away on the other side of that plane, allowing you to for instance observe internal structures of
your sample. Use together with some other module that renders the actual image. If you tick the “Show
plane controls” check box, the plane will be represented in the image by a white rectangle with an arrow
in the middle showing its directionality and orientation. You can adjust the plane and its position by
grabbing these controls with the mouse.
Distance.
Frame renders simple white frames around your object, indicating the edges of the three-dimensional
volume you are viewing. Like “Axes”, this may help you orient yourself within the three-dimensional
space. There are no specific settings for this module.
Orthogonal slices renders three cross section slices of the image, also within an image created by other
modules if so desired. It is like a more advanced version of the orthographic visualization mode described
earlier. In the settings there are first sliders for adjusting the position of each of the three slices, X, Y and
Z. Below are buttons for choosing a viewing angle of the image along either the X, Y or Z axis. You can
change the orientation of the slices with the middle mouse button. If you click on any of the planes with
the left mouse button, you will see a red cross indicating your location within that plane and the
coordinates (X, Y, Z) of your mouse pointer in the bottom left corner of the view panel, as well as the
intensity scalar of that particular voxel. You can keep the left button pressed and move the red cross
around within the plane you’re in. Note that “normal” image handling like rotating only works outside the
object. Middle click and dragging one edge of a plane changes its angle, and middle click and dragging a
corner of a plane rotates the plane about its perpendicular axis.
Surface rendering. There are two basic families of rendering methods available for creating 3D images,
surface rendering and volume rendering (see below). In surface
rendering an “iso-value surface” or “iso contour” (or several) is determined from the data, (often based on
an intensity threshold), and only that surface is rendered into the scene. Small geometric primitives like
triangles are used to construct the surface, and the result is usually completely opaque, so you can’t see
through it. In addition, all other information in the dataset, i.e. voxels with intensities below or above the
specified iso contour, is excluded from the image. “Reducing” a complex dataset to a surface like this
often makes rendering much faster than volume rendering, and the result may look very impressive. By
experimenting with the settings you may also be able to bring out a clearly visible surface from data in
which no surfaces are otherwise apparent. Nonetheless, surface rendering is not suitable for all
applications, and before choosing which family of rendering methods to use, you should always carefully
consider the type of data you are processing and the things you wish to illustrate from it. The available
BioImageXD surface rendering settings are:
• First in the settings is a drop-down list from which you can choose between three different surface
rendering methods: Contour Filter, Marching Cubes (default) and Iso-Surface Volume Rendering.
“Contour Filter” is capable of producing also isolines (2D data) and isopoints (1D data) in
addition to isosurfaces (3D data), whereas “Marching Cubes” is an isosurface generation tool
optimized for volumes, and the recommended setting here. “Iso-Surface Volume Rendering” is a
volume ray cast function that intersects a ray with an analytic isosurface in a scalar field.
• The surfaces created from complex datasets like images of cells often appear too detailed to be
useful. Ticking either “Smooth surface with gaussian smoothing” or “Smooth surface with surface
normals” helps in such situations. With the latter you can also specify the feature angle of the
normals.
• The “Simplify surface” slider can be used to reduce the number of the geometric primitives used
in rendering the surface, thus simplifying it. The higher the setting, the simpler the surface.
• If you want to make sure that connectivity and other topological features (i.e. features that cannot
be altered by deformations) are preserved despite smoothing and other processing, tick the
“Preserve topology” check box.
• The “Iso value” slider is the most important setting, as it controls the intensity iso value (between
0-255) used in creating the surface. Experiment with this slider to find out which setting most
accurately brings out the surface you are looking for.
• If you want to create several surfaces with different iso values, you can specify the range and the
amount of surfaces to be created within it by using the input boxes provided. Note: with the “Iso-
Surface Volume Rendering” method it is possible to create a single surface only, so this feature is
not available.
• The last slider controls transparency of the surface(s) and is especially useful when you have
several surfaces, because making them somewhat transparent allows you to see them all
(remember that otherwise the surfaces created with surface rendering methods are completely
opaque).
• Note: the color of an isosurface is determined by the channel palette. In other words, if the
threshold is 100, the color of the surface corresponds to the color of the scalar 100. If you want to
change the color of a surface, change the palette of the channel.
Visualize tracks.
Volume rendering. There are two basic families of rendering methods available for creating 3D images,
surface rendering (see above) and volume rendering. In volume rendering the whole 3D dataset is imaged
as a semi-transparent volume. All the data is (or at least can be) included in the rendering, and there is no
attempt to impose any geometric structure on it, unlike in surface rendering. Volume rendering is
therefore particularly useful for visualizing fluid-like biological structures such as cells, but rendering
times may be longer than with surface rendering, and sometimes skill is required to adjust the rendering
settings so that the desired details are highlighted. Before choosing which family of rendering methods to
use, you should always carefully consider the type of data you are processing and the things you wish to
illustrate from it. The available BioImageXD volume rendering settings are:
• First there is a black screen similar to the ITF graph described in 3.2.2, located in the volume
rendering settings pane. With this graph you control both the color transfer function (CTF) and
the opacity transfer function (OTF) used in the rendering. The red, green and blue buttons
represent the red, green and blue components of the RGB color system, and the corresponding
red, green and blue graphs make up the three-component CTF. The white “Alpha” button
represents the alpha, or opacity, component, and the corresponding white graph is the one-
component OTF. Both the CTF and the OTF are adjusted by changing their graphs with the
mouse. (If you can't see the graph for a particular color, then the function of that component is y =
0. Changing that will make it visible.) First choose the component you wish to adjust by clicking
its button. There are two modes for adjusting the graphs: node editing and freehand. In the node
editing mode the line consists of a number of nodes connected by straight lines. The node amount
for the currently selected component is specified in the number box at the right. The graph is
changed by dragging the nodes to the desired locations with the mouse, and more nodes can be
added by right-clicking. Clicking the “pencil” tool (on the right side of the “Alpha” button)
switches on the freehand mode, in which there are no nodes and you just draw the graph freely
with the mouse. Clicking the pen tool again restores node editing mode. With the “Open LUT”
and “Save LUT” buttons you can open and save color look-up tables (LUT), i.e. the CTF (alpha
channel is not included). Now let us have look at how the CTF and OTF behave with different
dataset types:
• If you are rendering an 8-bit (grayscale) dataset, then for the CTF the X-axis of the graph
represents the 255 possible intensity values of the dataset (input values), and the Y-axis the 255
possible values of the RGB components (output values). In other words the three colored lines
describe how the 8-bit input values are converted to a 24-bit colored RGB image. Note that when
you load a volume rendering module, the CTF is automatically modified to correspond to the
current channel palette (LUT). You can change it, but in many cases that will not be necessary.
By clicking the color button (on the right side of the freehand “pencil” button), you can choose a
single color for the dataset, and the CTF is altered accordingly. For the OTF the X-axis also
represents the 255 possible intensity values, but now the Y-axis represents the 255 possible
opacity values, with 0 being completely transparent (invisible) and 255 being completely opaque.
Generally the OTF is adjusted so that the brighter a voxel, the less transparent it is. The default
OTF is also like this.
• If you are rendering a 24-bit (color) dataset, such as a .bxd file created with the “Merge” task,
then the three components of the CTF are all of the form y = x and cannot be modified. The color
for each rendered voxel is determined directly by its R, G and B components. For the OTF the X-
axis represents the 255 possible alpha component values that were created from the original
combined channels according to the settings in the Merge task (see 3.2.1.). The Y-axis, in turn,
represents the 255 possible opacity values, just as with 8-bit datasets. Unlike the CTF, the OTF
can be modified.
• To summarize the above two points: 1) For single component images ("grayscale" images,
containing an intensity component only) the color of each voxel is determined by processing the
intensity value through the CTF. The transparency is determined by processing the intensity value
through the OTF. 2) For RGBA images the color of each voxel is determined directly from the R,
G and B components. The transparency is determined by processing the A component through the
OTF (see section 3.2.1. for a description of the A component).
• For single component ("grayscale") images the input for the OTF during volume rendering is
always the original intensity component, even if a complex multicolor palette is in use.
• Below the CTF/OTF settings is a drop-down box for selecting the volume rendering method from
among four alternatives.
o Ray Casting is the principal volume rendering method in which imaginary light rays are
cast through the volume, and reflections of those rays to the presumed camera calculated
on the basis of the CTF and OTF. If you rotate the rendering with the mouse it is often
temporarily reduced in quality to allow interactive rotating speeds. The higher quality
rendering is calculated after you release the mouse and may take several seconds to
complete.
o Texture Mapping renders several texture planes on top of each other using the OpenGL
capabilities of the graphics card, thus creating an image similar in appearance to Ray
casting, but often much faster. (The actual speed depends on the graphics card
performance and the size of its texture memory. Larger images take more time to render,
and very large images might be very slow.) In Texture mapping the image quality is not
temporarily reduced during rotations. The default number of texture planes is that of the
number of pixels/slices in the x, y or z plane that is most perpendicular to the camera. The
quality slider can be used to reduce the number of planes to make the interactive rotation
speed higher.
o 3D texture mapping renders the whole 3D image dataset as a 3D texture loaded into the
graphics card texture memory. This is a very fast rendering method, but the speed is
obtained at the expense of image quality - the 3D image is subsampled to fit into the
texture memory of the graphics card. The larger the texture memory, the more detail will
be rendered.
o Maximum Intensity Projection is a special case of the ray casting method, producing a
result similar to the Maximum Intensity Projection View (described in 3.3.4.), but freely
viewable from any angle, as all other 3D renderings.
• When the image is rendered, some method of interpolation is used to “fill in the gaps” between
calculated voxels when they are displayed on the screen. You can choose from two different
methods: Nearest Neighbor is faster than Linear interpolation, but the latter yields smoother
image rendering quality. With Nearest Neighbor the edges of real voxels are visible when zoomed
in, but with Linear interpolation these edges are smoothed out, giving what appears to be a higher
resolution rendered image (of course, this does not increase the real resolution of the image data,
just smoothes out the harsh edges of voxels).
• If you are using Ray casting, the “Rendering quality” slider controls the sample distance, i.e. the
distance between the light rays that are cast. The smaller the distance, the better the image quality,
but the slower the rendering. You can also type the desired sample distance directly into the
appropriate input box. Note that it can be less than 1.
• If you are using Texture mapping, the “Rendering quality” slider controls the maximum number
of textured planes that are rendered. A lower number means poorer image quality but faster
rendering. If the number is too low, you can see black stripes in the object while turning it. In
addition to using the slider, you can type the desired sample distance directly into the appropriate
input box.
Warp scalar. Here a single slice of a 3D image is selected for rendering. The intensity of each pixel is
linearly transformed (using the “scale factor for warping” input box) into a “height” on a 3D surface and
the original image is then pasted onto that 3D surface, which is rendered interactively using the OpenGL
capabilities of the graphics card. Warp Scalar is useful for making height maps of 2D images such as
AFM height images.
When 3D mode is active the “Visualization” menu contains two additional functions, “Lights...” and
“Render window”, briefly explained below:
Lights. This opens a small window in which you can configure how the rendered scene is lit (also the
“Advanced” lighting settings found in the settings of each rendering module affect this). You can switch
on a maximum of eight different lights be ticking the check boxes. Activated lights appear as cones in the
black screen. The tip of the cone represents the origin of the light, and the light is always pointed toward
the object, i.e. the center of the black screen. If you want to adjust a light, first select it by clicking its
cone. A red arc and a green circle appear, helping you understand the three dimensional space
represented by the black screen. You can reposition the selected light with the two sliders next to the
screen, and adjust its brightness with the “Intensity” slider below. You can change the color of the light
by clicking the color box button (white by default) and set the light to emanate from camera by clicking
“Set to headlight”. Two lighting presets, “VTK” and “Raymond”, are available by clicking their
respective buttons. Click “Apply” to see the effects of your changes in the view panel (doesn’t close the
configuration window). Readjust if necessary and click “Apply” again. When satisfied with the results,
click “OK” (closes the configuration window). If you wish to abort without changing anything, click
“Cancel” (closes the configuration window). Note: you must have the “Use shading” check box in
“Advanced” lighting settings ticked in order for the adjustments in the Lights window to have an effect!
Render window. This opens a small “Configure Render Window”, in which you can set the background
color (by clicking the color box button, which is black by default) and the size of the rendering window
in the view panel. You can also choose the stereo rendering mode of the window from 7 alternatives: No
stereo, Red-Blue, Crystal Eyes, Dresden, Interlaced, Left and Right. Click “Apply” to see the effects of
your changes in the view panel (doesn’t close the configuration window). Readjust if necessary and click
“Apply” again. When satisfied with the results, click “OK” (closes the configuration window). If you
wish to abort without changing anything, click “Cancel” (closes the configuration window). About stereo
viewing: To use Crystal Eyes type quad buffered openGL hardware stereo viewing, you need LDC
shutter glasses and an IR emitter box (NuVision or Stereographics corp.), and the emitter box must be
connected to the stereo sync port of a stereo capable graphics card such as an nVidia Quadro series card.
Red-Blue works with red-blue colored glasses, which are inexpensive. This method only works for
grayscale images, not color images. Left and Right are useful for making stereo pairs of 3D images for
publication, where the images are placed side by side, just less than eye width apart, and your eyes
defocus to infinity so that the left eye looks at the left image and the right eye looks at the right image,
similar to some 3D posters popular a few years ago.
Notes:
• When beginning work with a new dataset series, it is often a good idea to use the simpler
visualization modes first, before making 3D renderings, to get an understanding of the “actual”
image data, which helps in determining the appropriate 3D rendering settings.
• If volume rendering feels sluggish, use nearest neighbor interpolation when you're adjusting the
rendering settings, and then switch to linear interpolation for the final rendering. You can also use
a sample distance larger than 1 to increase interactive speed, but then you also lose some detail.
• Some rendering methods and settings require extensive computational power, and obtaining the
final image may take a long time, especially with slower computers. Ray casting is multithreaded,
so the more processors your computer has, the faster the image is computed.
3.4. Special or miscellaneous functions
3.4.1. Animator
The BioImageXD animator is one of the most versatile available in its class, and allows easy and flexible
creation of impressive movies from three or four dimensional image data. The animator uses a traditional
video editor type interface with a timeline and tracks, and supports the use of two different methods for
creating animations: keyframe control and camera path control.
Note:
• It is strongly recommended to create a movie with the animator in addition to creating still
images, since only a movie with motion can truly illustrate the three dimensional composition of
your sample. Most scientific publications today accept movies as “online supporting material” or
some such.
• The animator is linked to the 3D view mode in such a way that the rendering settings made in that
mode are used when rendering movies with the animator.
User interface. Due to its different nature, the animator employs a user interface slightly different from
that used by all other features of BioImageXD. When the animator is started by clicking its button in the
main toolbar, the main window below the main toolbar is changed; the view panel and the task panel are
replaced by the animator panel. At the top in this panel is the animator toolbar, below it a timeline and
tracks, and below them rendering settings and rendering screen mode settings on the left and a rendering
screen on the right. At the bottom is a time slider (not the same as the time point slider in the view panel)
and playback control buttons. The main menu also has three additional menu items: “Track”,
“Rendering” and “Camera”.
Note:
• You can resize the different parts of the animator panel by dragging the dividing line between
them with the mouse.
• When beginning work with the animator, you may need to interact with the rendering screen to
enable it.
Tracks. The first three buttons in the animator toolbar are used for adding tracks to the track list below
the timeline and are, in order from left to right: add timepoint track, add camera path track and add
keyframe track. Tracks are added simply by clicking one of these buttons. The added track appears, and
the tracks are always organized in such a way that the timepoint tracks are at the top, camera path tracks
in the middle and keyframe tracks at the bottom. Tracks can also be added with the “Add track”
command in the “Track” menu. Tracks are selected by clicking them, and the name of the selected track
appears bolded. A selected track can be removed with the “Remove track” command in the “Track”
menu.
Track items and timeline. Tracks are used to define the structure of the movie, i.e. to “edit” it. This is
done by adding items to the tracks and positioning them in the desired way with respect to the timeline at
the top. The timeline indicates the real running time of the movie (it has nothing to do with the time point
slider available in other features of BioImageXD). The drop-down menu at the far right of the animator
toolbar is used to select the zoom-level of the timeline. Items are added to tracks by dragging from the
appropriate buttons in the animator toolbar to the desired tracks and locations within them. The track item
buttons are the ones between the track buttons on the left and the timeline zoom control on the right and
are, in order from left to right: add timepoints (only to timepoint tracks), add camera path (only to camera
path tracks), add X axis rotation camera path (only to camera path tracks), add Y axis rotation camera
path (only to camera path tracks), add pause in camera movement (only to camera path tracks) and add
keyframe (only to keyframe tracks). Items can be selected by clicking them, and their length and position
changed by dragging on their vertical boundaries. A selected item can be removed with the “Remove
item” command in the “Track” menu, and the items in a selected track can be processed with the
following additional commands in the “Track” menu:
• Item sizes – expand to maximum: Resizes all items in a track such that they cover the entire
duration of the movie and are of equal length. However, does not change the starting point of the
first item.
• Item sizes – expand to track length (keep ratio): Expands all items in a track such that they cover
the entire duration of the movie, but their length ratios remain unaffected, as does the starting
point of the first item.
• Item sizes – shrink to minimum: Resizes all items in a track such that they have the shortest
possible duration, i.e. that of a single frame. Does not affect the starting point of the first item.
• Item sizes – set item size: Opens a dialog box that allows you to specify a length (in seconds) for
each item in the selected track. If a number smaller than the minimum possible (duration of a
single frame) is specified, the smallest possible value is used. Likewise, if too large a value (larger
than what “Expand to maximum” would produce) is specified, the largest possible value is used.
Does not affect the starting point of the first item.
• Item sizes – set total length: Opens a dialog box that allows you to specify the total length for all
items in a track (in seconds). Items are resized to cover this length and rescaled to be of equal
length. If a number larger than the length of the movie is specified, the largest possible value is
used. Does not affect the starting point of the first item.
• Item sizes – set physical length: Opens a dialog box that allows you to specify...
• Shift items – left: Shifts the order of items in a track clockwise, i.e. the first item becomes last.
Does not affect the starting point of the first item.
• Shift items – right: Shifts the order of items in a track counterclockwise, i.e. the last item becomes
first. Does not affect the starting point of the first item.
Keyframe animation. This is a more “traditional” animation technique where you first provide the
computer with some keyframes, i.e. frames for which you have manually specified the viewing angle,
zoom level etc., and the computer will then calculate the desired amount of frames, interpolating linearly
between the specified key frames. You can interact with the rendering screen of the animator much the
same way as you do with the view panel in 3D mode (section 3.3.5.), and thus create the keyframe of
your liking. When satisfied, drag from the “Add keyframe” button to the desired location within a
keyframe track. A keyframe item with a small MIP of the specified view appears into the track. Move the
sample in the rendering screen slightly, and add another keyframe. Continue this as long as necessary and
add as many keyframes as you like. The more you add, the better control you have over what your
animation will look like. When you add the first keyframe to a track, an “End point” item is also
automatically added. This represents the last frame of your animation, and you can treat it the same as
any other keyframe item. An existing keyframe item can be edited by selecting it; the rendering screen
will show the view specified for that frame, and you can change it by interacting with the rendering
screen as usual. The small image in the track item is changed accordingly.
Note:
• The “End point” item has a fixed length in the track, but the actual duration of the end point is
always just a single frame, located at the point in time where the “End point” item begins in the
track. In other words, a keyframe animation sequence always ends at the starting time of the “End
point” item, in the position specified in the “End point”. The “End point” is always the last item in
a keyframe track, and other keyframes cannot be added after it.
Camera path animation.

When you choose a node item in a camera path track, the corresponding node in the preview panel will
be indicated with a blue arrow.
Right-clicking on the path or on a node rescales the whole path
Left-clicking on the path (not on a node) moves the whole path
Left-clicking on a node moves the node and thus reshapes the path
explain menu items

explain number of control points dialog box
menu: maintain up direction (off for y axis rotation, otherwise on, works automatically but
changeable from here)
Note:
• It is possible to combine camera paths and keyframe animation into the same movie. Simply
position items in the tracks so that they do not overlap. For example, you can begin your movie
with a camera path rotation and continue with a keyframe sequence by moving all items in the
keyframe track forward so that they begin at the end point of the camera path.
Rendering parameters. With these parameters you can specify the desired duration, number of frames,
frame rate and frame size for your movie. For frame size you can either choose one of the presets in the
drop-down menu, or specify any size of your liking (the “custom” option). When you change either the
number of frames or the frame rate, the other one of these values is automatically re-calculated, while the
duration of the movie remains unchanged. When you have made changes to these parameters and wish to
start using them, click “Use settings”, and the tracks are resized. You may also need to use the “Item
sizes – Expand to maximum” or “Item sizes – Expand to track length” commands explained above to
rescale items within the tracks. If you want the preview panel to be of the same size as what is specified
for the movie, uncheck “Don’t resize preview, only use aspect ratio”. If it is checked, the preview panel
will always be of approximately the same size and only its aspect ratio is modified according to the
movie size setting.
Rendering screen modes and preview. The rendering screen of the animator has two modes of operation.
While editing the animation as described above, the screen is in the “Edit animation” mode. By choosing
“Preview movie” from the “Rendering screen mode” field, the rendering screen is switched to the
preview mode, in which it is possible to see what the final movie will be like. When in the “Preview
movie” mode, the time slider and the related playback buttons at the bottom of the BioImageXD window
become active. The playback buttons are, in order from left to right: Stop playback, Rewind to beginning,
Go back one frame, Play, Go forward one frame, Wind to the end. You can use these to control the
playback of the movie preview. You can also move the slider to the desired position in the timeline and
thus see a preview of that particular frame. You can switch the rendering screen back to editing mode by
selecting “Edit animation”.
Render project. When you are finished setting up your animation, choose “Render project” from the
“Rendering” menu, and a movie rendering settings panel opens to the right. This is where you determine
the exact format and quality of the movie to be rendered. By default the frame size and frame rate are the
same as those set up in “Rendering parameters”, in which case the topmost dropdown menu reads “Use
settings below”. You can change the frame size and frame rate to default DVD-quality settings (either
PAL or NTSC) from the drop-down menu, but for any other settings you need to go back to “Rendering
parameters” and the change the settings there. From the next dropdown menu (“Output format”) you can
choose whether you want your movie as a video file (“Video”) or as a set of still images (“Images”), one
image per frame. Usually video file output is the better choice here, but if you want to create the final
movie with a third party animator or video editor and/or process the frames further as stills, then output as
images may be more reasonable. If you chose “Video”, you can choose the video codec from the next
dropdown menu. If you chose “Images”, the dropdown menu is used for choosing the image format for
the stills. Last in the rendering settings is a slider for controlling encoding quality. “10” produces the best
quality, but also the largest files. Use this setting unless file size is an issue, in which case experiment a
little to find out the right setting for acceptable file size but also reasonable picture quality. Below the
rendering settings are input boxes for specifying a directory to which frames are rendered and a name for
the output file. If “Images” are chosen as the output format, a running number (frame number) will be
added to the name of each file. When done with the settings, click “Ok” to render the movie.
Notes:
• You must not have any other windows or anything else on top of the BioImageXD rendering area
while the program is performing the rendering and movie making. This is because the output to
the monitor is used for saving the rendered images, so anything shown on the monitor on top of
the rendering area will be included in the movie! So, rather than working with your computer
while the software is rendering, go have a cup of coffee, take a nap or perhaps render overnight.
• Remember that the rendering settings made in the 3D view mode are used when rendering the
movie project.
3.4.2. Quick help
The question mark, the last button in the main tool bar, can be used to obtain quick help. After clicking it,
click anything on the screen you wish to know more about. A short description is displayed, if available.
3.5. Additional features and functions
Functions that can be accessed from the menus but not the main tool bar, and some other functions, are
described in this section.
3.5.1. File menu additional functions
Import.
Note:
• If you are working with a file format not yet supported by BioImageXD, try exporting your
dataset as a stack of TIFF files or other images with the program you used to create it (most
programs have such an export feature). You can then load the exported dataset into BioImageXD
using “Import – Stack of images”.
• Only one channel can be imported at a time.
• We recommend importing 8-bit grayscale tiff files, in consecutively numbered stacks, a different
stack for each data channel.
Export. This feature allows exporting multidimensional image data in formats not supported by the “Save
dataset” feature (section 3.1.2.). From the submenu you can choose either “VTK dataset series” or “Stack
of images”. Both open up a similar dialog box, at the top of which is information of the currently selected
dataset and below that the “Target of images” section, in which you can specify target location, filename
pattern, choose the exported file format and see a list of the files that will be produced upon clicking
“OK”. Supported file formats are PNG, BMP, JPEG and TIFF for “Stack of images”, and “VTK image
data” and “XML image data” for “VTK image datasets”.
Note:
• Only one channel can be exported at a time.
• The best possible codec quality is always used when creating the images.
Quit. Quits BioImageXD. By default a verification dialogue box appears. This can be prevented in
“Preferences” (section 3.5.3). Closing the BioImageXD main window also quits the program.
3.5.2. Edit menu additional functions
Undo undoes whatever was done previously and restores everything to the state they were in before the
previous action.
Redo redoes whatever was just undone with “Undo”. (Not everything can be undone, either because they
just can’t, or reversing the action would be too complex a task to carry out.)
Command history opens a new window that displays a list of all commands executed since BioImageXD
was last started up. Any command can be selected from the list and either undone (“Undo”) or run again
(“Run”). Items appear read on the list when undone. There is no upper limit for the amount of commands
remembered.
Notes:
• Although functional, the undo, redo and command history functions are preliminary at this stage,
and do not yet cover all commands and processes.
3.5.3. Settings menu additional features
Preferences opens a window in which various settings regarding the overall function and performance of
BioImageXD can be set. The available settings are grouped into three categories selectable from the icons
on the left: “General”, “Paths” and “Performance”. In “General” you can set startup tips and quitting
verification on or off and choose the default format in which still images are saved by the “Save snapshot
image” and “Export – Stack of images” functions. In “Paths” you can choose the directory in which your
data files are most typically located, and determine whether BioImageXD by default always suggests the
last opened directory for opening or saving files. In “Performance” you can choose to limit the memory
used by any single operation to a specified value. You can also switch on or off automatic resampling
(section 3.5.4) and set the resampling threshold size and the size of the resampled image.
3.5.4. Tasks menu additional features
Resample dataset. With this feature large datasets can be resampled into smaller ones to make their
processing faster. If BioImageXD seems sluggish with a particular dataset, resampling it to a smaller one
is likely to help, and the original dataset can always be restored with the “Resampling” button in the view
panel toolbar (see section 3.4.2.), for instance right before creating the final visualization and calculating
measurements and analyses. Choosing this function opens the “Resample dataset” dialog box, in which
you can specify new dimensions ("custom size") for the dataset in X, Y and Z either directly as pixels
("dimensions") or as scale factors ("scale"). You can also choose quick resampling to half or quarter size
and choose whether to resample also in the Z dimension in these cases.
Resampled dataset are marked with red color and an asterisk (*) in the file tree, and the the original and
the resampled dimensions are always displayed on top of the view panel as well as in the dataset info box
on the right (when its visible).
Notes
• The resampling is done to the currently active timepoint only, and is repeated every time a new
timepoint is selected.
• You can also resample a dataset to larger dimensions than the original, in which case new pixels
and/or optical sections are interpolated from existing data. This is useful for instance with
confocal microscopy data (where the resolution in Z is typically much poorer than in X and Y);
creating more optical sections through resampling may improve the looks of 3D renderings
especially when viewed from the side. Please remember, however, that this type of resampling
should only be used for visualizations, not for quantitative analyses.
• BioImageXD also has an automatic resampling function that resamples datasets upon opening if
they are larger than specified thresholds. This makes use of the software more fluent with large
datasets. Automatically resampled datasets differ in no way from manually resampled ones. The
automatic resampling can be set up in “Preferences” (section 3.5.3.).
• See also section 2.2.1. for a description of the "resample" and "resample to fit" buttons.
Rescale dataset. This feature is used to change the color depth (bit depth) of a dataset. BioImageXD
supports bit depths up to 16, but the most typical and recommended depth is 8-bit (256 possible intensity
values). If you have a higher bit depth you may want to rescale it to 8-bit with this function before further
processing. Choosing the function opens a rescaling window with a view panel in slices mode, with the
appropriate time point and Z slice sliders, showing all channels of the dataset merged. Below the image
are histograms for each channel. You can specify the range to be rescaled to 8-bit by moving the vertical
yellow lines in the histograms. A preview of the resampled dataset is shown in the view panel.
Notes:
• We recommend acquiring images as 8-bit, as this is sufficient for most practical purposes, and a
higher bit depth only results in unnecessarily large files. However, there are situations where
acquiring with a higher bit depth may be in order. For example: you know that you’re interested
in a fairly narrow intensity range, but during image acquisition you don’t know exactly what that
range is. You can gather your images as 12-bit or 16-bit, and use the “Rescale dataset” function to
choose the intensity range of interest afterwards and create an 8-bit dataset from it. This way you
end up with an 8-bit dataset that still has a sufficient “intensity resolution” in the desired range.
3.5.5. Visualization menu additional features
Immediate updating. If this is checked, BioImageXD updates the display panel “on the fly”, whenever
something is adjusted or changed. For example, if you move the contrast slider, you will immediately see
the effect in the image. Un-checking this switches off immediate updating, after which you need to
always click “Preview” before the display panel is updated. It is usually better to keep immediate
updating on, but if making adjustments seems cumbersome (for instance when working with large files or
demanding processing), then switching immediate updating off may help.
3.5.6. View menu additional features
In the top section of this menu you can toggle on and off the display of the following items (displayed
items have a check mark in front of them):
• File tree (same as the corresponding button in the main tool bar)
• Configuration panel (settings for some visualization modes, on the left)
• Task panel (settings for the chosen task, on the right)

• Toolbar (the toolbar of the display panel, not the main toolbar)
• Histograms (see section 3.5.8.)
• Dataset info (on the right, normally shown in place of the task panel if no task is chosen).
Choosing “Hide info windows” (or pressing Alt-Enter) hides the file tree and the dataset info panel, and
is a quick way to maximize the size of the display panel.
The bottom section of the View menu controls the display of additional items, which are:
• Python shell: BioImageXD is largely written in the Python programming language, and from the
Python shell appearing to the bottom of the view panel you can give direct commands in Python.
For expert users only.
• Script editor: (see section 3.5.8.)
• Mask selection: Displays a window with a list of currently active masks, from which you can
choose which mask is used. Masks affect every operation of the software: if a mask is selected,
the image data is always masked before passing it on to any function. "No mask" removes any
masks from being used, and "Cancel" closes the dialog box without making any changes.
3.5.7. Help menu additional features
About BioImageXD displays information of the program, such as version number, members of the main
development team and license and library information. When reporting bugs be sure to check from here
first which version you are using! And when planning on using BioImageXD, be sure to read through the
license description first! Here you will also find our contact information as well as information for
referencing BioImageXD when using it for instance to prepare scientific publications.
Help opens the BioImageXD help window that enables you to read for instance this user manual and
other helpful information. Note that the BioImageXD help can also be opened from the “Help” button
appearing at the bottom of the task panel, in which case the section of the help describing the currently
chosen task will be automatically displayed.
3.5.8. Other additional features
Histograms. Switched on from the View menu (section 3.5.6.).
Note: histrogram may be handy to view in conjunction with adjusting ITF.

“ignore border bin” and “logarithmic scale” by right-clicking histogram
Script editor. Switched on from the View menu (section 3.5.6.).
Channel palettes.

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BioImageXD User Manual 1 / 39

• This manual is still incomplete.

2. BioImageXD user interface

3. BioImageXD features and functions

1.2. Who made BioImageXD and why?

1.4. How to start using BioImageXD?

1.5. Some terminology

2. BioImageXD user interface

2.1. Main tool bar

2.2. View panel

2.2.1. Top and right side buttons and settings

On the top are the following:

On the right are the following:

2.2.4. Interpolation method

2.3. Task panel

2.4. File tree & configuration panel

3. BioImageXD features and functions

• When your goal is to obtain colocalization analysis results in addition to or instead of

3.1. File input/output functions

3.1.2. Save dataset

3.1.3. Open settings

3.1.4. Save settings

3.1.5. Save snapshot image

3.1.6. File tree

3.2.2. Intensity Transfer Function (ITF)

3.2.6. Segmentation and tracking overview

Segmentation. BioImageXD offers three basic classes of segmentation methods:

3) Region growing (a region based method)

3.2.7. Procedure descriptions

Filtering - Feature detection

Find local maxima

Arithmetics - Image arithmetic

Arithmetics - Logical operations

Segmentation - Region growing

3.3. Visualization modes

3.3.1. Slices view

3.3.2. Gallery view

3.3.3. Orthographical view

3.3.4. Maximum Intensity Projection view

Certain keys control the behavior of the display panel in 3D mode:

See section 2.2. for a description of other view panel features.

3.4. Special or miscellaneous functions

Camera path animation.

explain menu items

3.4.2. Quick help

3.5. Additional features and functions

3.5.1. File menu additional functions

3.5.2. Edit menu additional functions

3.5.3. Settings menu additional features

3.5.4. Tasks menu additional features

3.5.5. Visualization menu additional features

3.5.6. View menu additional features

• Task panel (settings for the chosen task, on the right)

3.5.7. Help menu additional features

3.5.8. Other additional features

Histograms. Switched on from the View menu (section 3.5.6.).

Note: histrogram may be handy to view in conjunction with adjusting ITF.

Script editor. Switched on from the View menu (section 3.5.6.).

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