Count Image Objects Help
This widget counts regions in the image. It is applied
to the image after the brightness sliders from the Edit Volume
widget and after any colocalization culling from the Colocalization widget.
A region is a connected (even if only by their corners touching) group of voxels
all of which have nonzero intensity and are at least somewhat opaque.
Note: there is a separate program "countobjs" which does the same thing in
a noninteractive manner. This can be useful if you just need statistics or
if you want to analyze the data several times with different parameter
settings.
NOTE: only 253 objects can be counted and displayed in this DAVE widget.
However, if you write the statistics out to a file, all the objects (even
if greater than 253) will be written out. See "Open Stats File" below.
NOTE: I don't think this widget works with triple labelled data sets yet.
Smallest Region
Only regions which have at least this many voxels in them will be
considered valid regions. Thus, isolated voxels due to "noise" can be
eliminated from consideration by setting this number to something greater than
one.
Largest Region
Regions which have more than this many voxels are not considered valid
regions. Thus large objects such as a nucleus can be excluded from
consideration by setting the largest region size to be larger than the regions
of interest but smaller than the size of the nucleus.
Lower Threshold 1
This is usually set automatically based upon the position of the left
brightness slider. However you may want to set this manually when you are
working with 16 bit data (DAVE -t command line option), since the slider only
gives 8 bit accuracy. Type the desired threshold into the box (in the original
range of your data). Note: the displayed view will still be based upon an
8 bit approximation, but the actual analysis will be done on the 16 bit data.
Lower Threshold 2
The plan is for this threshold to apply to image 2 (if two images have
been read in). Currently threshold 1 is applied to both images.
COUNT
Once the sliders are set to pick out the regions ofinterest, pressing
COUNT will count the regions and display the results in the widget. Each
region is displayed on a line and highlighted (selected) in yellow. Summary
statistics are displayed above this listing. Statistics are based upon the
original voxel values (i.e., they will not vary with a change in the brightness
sliders unless that causes different voxels to be included in a region).
The coordinate of the brightest voxel in each region is also printed.
Coordinates are zero indexed (i.e., the first voxel has coordinates 0,0,0).
All region analysis is done at the initial (highest) level of resolution,
regardless of the resolution at which the data is displayed. In addition,
this analysis works regardless of which display mode (e.g., planes or cubes) is
currently being used.
Since DAVE only stores values scaled to 1 byte (0-255), it approximates
the original value of each voxel by rescaling the one byte number based upon
the scale and black level which was originally used to convert voxel values
into one byte. Therefore the numbers may not be exactly the same as the
original ones, and are also floating point numbers (since the scale factor can
be noninteger). You can use the 16 values by specifying -t on the command line.
NOTE: any time the selected regions changes, or the left brightness slider
or colocalization widget are changed, the displayed statistics will be
out of date. COUNT or RECALC must be pressed to update the statistics.
If the colocalization widget is active ("Show Colocalized
Voxels" is pressed on that widget) then the regions that the Count Widget
counts are based upon the Colocalization settings (but you should set the
Colocalization widget to only display the colocalized regions and not the
image1 or image2 voxels since count will count whatever is displayed).
RECALC
A region can be deselected by picking on its line (it may also be
reselected in this way). Once only the desired subset of regions is chosen,
pressing RECALC will cause the summary statistics above the region listing to
change to only reflect the currently selected regions.
NOTE: any time the selected regions changes, or the left brightness slider
or colocalization widget are changed, the displayed statistics will be
out of date. COUNT or RECALC must be pressed to update the statistics.
Deselect All Lines
Rather than picking each line separately, all lines (and hence all regions)
can be deselected at once by pushing this button. Then only those regions
which are desired can be reselected (by picking on the appropriate line) one at
a time.
Select All Lines
Rather than picking each line separately, all lines (and hence all regions)
can be selected at once by pushing this button. Then only those regions
which are not desired can be deselected (by picking on the appropriate line)
one at a time.
Inspecting Individual Regions
Select One Line
It is often useful to visually inspect individual regions. Activating this
button causes all regions to be deselected and set into a mode so that only one
region at a time can be selected (by default the first region in the object
list is selected). Selecting a second region automatically
deselects the previous region. It also causes the viewing window crosshairs
to be positioned at the brightest voxel in the selected region (Show Crosshair
in the menu must be picked for the crosshair to actually be visible). This
region can be visually highlighted by the use of the following buttons.
You can also select one region by picking near the region with an
intelligent cursor. You should first activate the Select One Line button,
and then go to the Edit Crosshair widget and pick "Intelligent Cursor - Object"
from the menu (see the help there for more details). Once this is done,
picking near a region will cause the crosshair to jump to the brightest voxel
in that region and the associated line in the object list to be highlighted.
Show Region
Pushing this button causes the region that the crosshair is positioned on
(i.e., the region specified by the currently selected line) to be filled with
a Floodfill color (default is yellow). This makes the region visually distinct
from the surrounding data volume. The color of these voxels can be changed by
choosing "Floodfill Color" from the Menu. Visualizing the extent of the region
is often useful since the region may be larger than expected due to dim,
mostly transparent voxels connecting the region. See the note below.
Note: this option does not always work well when your data is very opaque
(the Floodfill color is quickly overwritten by the data values sometimes).
Hide Region
Turns Show Region off.
The Region of Interest (ROI) Analysis widget may also aid
in examining individual regions (this widget is gotten from the Goodies menu
in the DAVE Master widget).
Menu Options
Floodfill Color
This determines the color the region will be displayed in when the
Show Region button is pressed. Note: sometimes, when data is very bright
or opaque, it is difficult to see this (the data values overwrite the
floodfill values).
Defining a Region of Interest
By default, the nonclipped data volume (at original resolution) is
examined for regions. If the data volume is clipped (using the Volume Clipping
widget gotten from the Edit Volume menu), then only the region which is not
clipped (i.e., the displayed region) is examined. Sections of the data which
are not visible because the data volume has been scaled up or translated (using
the Scale and Translate controls on the DAVE Master widget) will still be
included in the region count.
By picking "ROI Box" from the menu, the region to be examined can be
changed to be the region specified by a Region Of Interest (ROI) box. This box
is easy to move around to different areas in the image. See the HELP for the
ROI Analysis widget (gotten from the Goodies menu in the DAVE Master widget)
for more information on how to specify this box.
Using the Region of Interest
There are several ways the the Region of Interest can be employed to
restrict which data should be analyzed (in addition to just its size and
position). If "Discard objects extending outside region" is picked, then any
object which extends outside the region is not counted at all - even the part of
it which is inside the region. If "Keep objects extending outside region" is
picked then any region which has any part inside the region of interest will be
counted in its entirety. Thus voxels outside the "examined region" are counted
if they are connected to voxels inside the region. If "Eliminate points
outside region" is picked, all voxels outside of the region are conceptually
zeroed out. Thus the part of the object inside the region is counted and the
part outside the region is not counted (it may also create 2 objects where only
one existed since the two objects inside the region of interest may have been
connected by voxels outside the region of interest). This is useful for
eliminating entire out of focus z-planes for instance.
Note: sometimes when there are too many voxels in one region, DAVE dies (at least
I think that is what causes it to die). This typically shows up when
"Keep Objects Extending Outside Region" is selected since this allows objects to
get very large.
Show Crosshair
Pick this if you want to see the crosshair. You probably want to do
this when using the "Select One Line" button to examine individual regions.
The "Edit Crosshair" widget has more stuff on using the crosshair (but you
don't need to use that widget).
Center on Crosshair
Whenever you pick this entry the image will be translated in the DAVE
viewing window so that the voxel which the crosshair is positioned on will
be moved to the center of the window.
Erase Nonselected Regions
The point beind this option is to use the deselection process in the
Count Widget to delete from the display (and from statistics) regions which
are not of interest. DAVE keeps 2 views around. The default "complete"
view of the data and an "edited version". Each time this item is chosen, any
nonselected objects will be deleted from the edited version. If you want
to view the edited version instead of the complete version you must pick
"display selected regions" (see below).
Note: Once a region has been erased you cannot see it
in the edited version (even if you reselect it) until you create a
new edited version by doing a new COUNT of the objects.
Display selected regions
This item should be selected if you want to view the "edited version"
of the data set (see "erase nonselected regions" above).
You must be in cubes visualization mode in order for this to
work (Edit->Volume->(menu)->cubes).
Open Stats file
This cause all subsequently calculated statistics to be appended to
the specified file (which will be created if necessary). This also will
count all objects, even if more than 253 objects exist.
Close Stats file
Use this to close the statistics file (not necessary) or to change the
name of the file (e.g., by closing the current file and then opening a
different file).
Additional Notes
Count 3D objects
The analysis is done in 3D.
Count 2D objects
The analysis is done one z plane at a time; so connectedness in the
z direction does not count.
Rendering Speedups
If your image is fairly sparse after you have set the opacity and
brightness sliders, you may want to change display modes to render in "Cubes"
mode. Cubes mode has an internal speedup option for looking at large sparse
images quickly (assuming you are just rotating it around and not changing its
brightness or opacity values, that will still take a normal amount of time).
This can be gotten from the menu in the Edit Volume widget. See the help there
for more details.
Visually unnoticed voxels still may count
Visually you may not notice voxels which are almost totally transparent,
yet they count towards region definition as long as they are not totally
transparent. You can make these voxels more visually apparent (if you desire)
by making all voxels whose brightness has not been mapped to zero (i.e., whose
values are greater than the left brightness slider setting) to be totally
opaque. This is done by choosing the "Edit Volume Options" from the menu in
the Edit Volume widget. The choose "Opaque Data" from its menu.
You might also want to change your background color (Edit Bkgd Color from
the DAVE Master Widget) to be something other than black - since it is difficult
to see nearly black voxels against a black background.
The only slider that affects the actual definition of regions
is the left brightness slider (since voxels with a value less than this are set
to zero).
The HELP button in the Edit Volume widget explains the function of its
sliders and menu options in more detail.
Copyright 1995 by Lawrence M. Lifshitz and the University of
Massachusetts Medical School. All rights reserved.