Colocalization Analysis Widget Help

This widget aids in the visualization of colocalized voxels (i.e., those voxels with "roughly equal" amounts of label in the images being displayed, either 2 or 3 images). It does this by first dividing the multiply-labelled data set into mutually exclusive sets of voxels: those that are colocalized, those that are not colocalized and have more of image1 (the "green" image) in them than image2 or image3, and similarly for image2 (the "red" image) and image3 (if present). It then displays (or doesn't display) each of these subsets in one of several ways. By default it only displays the colocalized subset, and they are displayed in a "grayscale". Thus voxels with low colocalization can be removed from view. This is obviously only used when two or more images have been read in by DAVE (e.g., dave -z 2 -I image1.i2i -I image2.i2i). The colocalization sliders work best if you are displaying volume data in "cubes" mode ("planes" is the default). At times "planes" mode produces streaks or other artifacts. Go to the menu in the Edit Volume widget, pick Render Cubes from its menu. [Actually, this difficulty with "planes" mode may have been removed.]

After performing colocalization, the Count Widget (in the Goodies menu of the DAVE Master Widget) can be used to get statistics on the number of colocalized regions (the Colocalization Widget only gives voxel based statistics). The Count Widget can also be used to eliminate some unwanted colocalized regions from consideration. See it for more details.

NOTE: This widget is not "active" until "Show Colocalized Voxels" is pressed. When active this button is highlighted in yellow. Don't forget to deactivate the widget before you remove (HIDE) it (assuming you don't want it to affect what you see anymore).

Definition of Which Pixel Intensities are Compared

Many operations get applied to your initial data during the course of displaying it. First each image gets rescaled into one byte (0-255). These scaling factors (scale and black level) are usually different for each image (you can control these scale factors via the -S command line option). Then the RS slider in the Edit Volume widget gets applied (by default it does nothing). After these the Opacity and Brightness sliders rescale intensity values. Finally, at each voxel these resulting values for the two or three images are combined to produce a color at that location. For colocalization analysis, all intensities are compared after the initial rescaling performed when reading in the data (i.e., rescaling each image 0-255). The comparisons also occur after the RS slider on the Edit Volume Data widget is applied to the images (by default this slider changes nothing). If "Apply Before O+B Sliders" is set in the menu, then intensities are compared prior to doing the opacity and brightness mappings. In other words, what you see is not necessarily what you are analyzing. The philosophy behind this is that those sliders are being used merely to enhance the visualization of the data and should not affect the colocalization statistics. If it is desired to have the opacity and brightness sliders affect the colocalization analysis (e.g., the sliders are being used to threshold the image) then the "Apply After O+B Sliders" menu option should be chosen. Intensities are compared, for display purposes only, at the current resolution level used for displaying the image. Since lower resolution levels are created by taking the maximum (by default) of neighboring voxels, voxels may appear more colocalized at lower levels of resolution than they will at higher levels. Resolution level will NOT affect the colocalization statistics (see below).

Volume Subset

Only voxels which are not clipped out by the volume clipping widget will contribute to colocalization statistics.

REL Slider

This slider specifies the maximum relative difference in intensity which the same voxel in pairs of images can have and still be considered colocalized. Relative intensity is calculated as: absolute_value_of(image1-image2)/(min_of(image1,image2)). So it is the difference in intensities over the minimum intensity. This method of calculation will yield the same number if image1 and image2 are exchanged. If either intensity is zero the voxel will not be considered colocalized. If a voxel is totally transparent it will not be considered colocalized.

ABS Slider

This slider specifies the maximum (absolute value) difference in intensity two voxels can have and still be considered colocalized. Since the default is 255 (and all data values have been rescaled 0-255), no voxels are eliminated from the display based upon the default setting, except if either intensity is zero the voxel will not be considered colocalized. If a voxel is totally transparent it will not be considered colocalized.

AND/OR Buttons

If OR is chosen (the default) a pair of voxels will be displayed if the difference in their intensities meets the criterion specified by the REL slider (i.e., relative difference less than the specified amount) OR the criterion specified by the ABS slider (i.e., absolute difference less than the specified amount). If AND is chosen a voxel pair will be displayed only if both criteria are met. If three images are being displayed, a voxel will only be considered colocalized if all 3 pairs of images (1 vs. 2, 2 vs. 3, and 1 vs. 3) qualify as colocalized.

Typical Usage

Typically you might only want to display voxels which are within 2535520453640f each other, so REL would be set to .25. But for dim voxels you might want more leeway (since a difference of, say, 5 when the voxels are only 10 is already a 50-314419296ifference), thus you would set ABS to perhaps 5. If you don't care about dim voxels (perhaps you are not seeing them anyway because you have set the opacity slider in the Edit Volume Widget so they are transparent) you can set the ABS slider to 0. Alternatively, you may not really want to worry about the relative amounts of the two labels since their intensities depended upon the amount of fluorescent label loaded into the cell, exposure time, etc. All you really want to do is threshold the images, and any voxel with both image values above threshold should count as a colocalized voxel. In this case, pick the menu option "Apply After O+B Sliders". Now use the brightness slider (on the Edit Volume widget) to threshold the image (you may want to pick "Map - Threshold" from the Edit Volume widget's menu). Set the ABS slider on the colocalization widget to 255 and leave the OR button active. Thus any voxel with the two intensities within 255 of each other (which is all of them since the rescaled numbers only range from 0 to 255) will be colocalized. The definition of "colocalized" excludes any voxel from being colocalized if either of its two values (or its opacity) is zero. Thus all voxels with two nonzero values after the threshold will be counted as colocalized.

A note of caution: visually unnoticed voxels still may count

Visually you may not notice voxels which are almost totally transparent, yet they count towards colocalization as long as they are not totally transparent. You can make these voxels more visually apparent (if you desire) by choosing "Opaque Data" from the Edit Volume Options Widget's menu (this widget is gotten from the Edit Volume Widget's menu). Thus all voxels will either be totally transparent or totally opaque. Similarly, if you set the right brightness slider to the same level as the left brightness slider all voxels will be either totally black or totally white. Rather than do this you might also just change the Mapping function from Rescale to Threshold, this will permit "dim" objects to be somewhat more visible. You might also change the background color (Edit Bkgd Color from the DAVE Master Widget) to something other than black. The HELP button in the Edit Volume widget explains the function of its sliders and menu options in more detail.


The window shown to the right of the sliders is used to display colocalization statistics. The top line is the number of voxels colocalized. Line 4 is the fraction of colocalized voxels in the region being displayed (i.e., "total" is the number of voxels in the image at the current level of resolution unless the Volume Clipping widget is being used to display a subset of the image). Line 2 is the number of colocalized voxels divided by the number of nonzero (and nontransparent) voxels that are in the green image (image 1) in the region being displayed. Line 3 is the number of colocalized voxels divided by the number of nonzero (and nontransparent) voxels that are in the red image (image 2) in the region being displayed. Line 4 is the number of colocalized voxels divided by the number of nonzero (and nontransparent) voxels that are in the blue image (image 3) in the region being displayed. So lines 2 and 3 are the colocalization percentage by volume. The statistics are not calculated (or recalculated when the sliders change) until the "Recalc Co-Localization" button is pressed. The statistics are all based upon colocalization at the initial (highest) image resolution (even if you are viewing the data at lower resolution). Thus the statistics may disagree slightly from the rendered view. Statistics will be out of date if the left brightness slider, the volume sliders, or the colocalization sliders are moved. When the statistics are out of date, the "Recalc Co-Localization" button becomes highlighted in blue and an asterisk is added to the label to remind the user that the button needs to be pressed to update the displayed statistics.


The popup menu/toggle buttons to the far right of the sliders determine how each of the subsets - colocal, image1, image2, and image3 (see explanation at top) - are displayed. You can change this by toggling through the options (by clicking with the left mouse button) or by picking the desired option from the pop-up menu gotten by holding down the right mouse button. The options are as follows.

Note: If two or three images are on (i.e, not set "off" in this widget)

then those components of a non-colocalized voxel will be displayed

in their normal colors (e.g., RED/Bi.Op.Green will be displayed as

RED/GREEN, but Bi.Op.Red/Off will be displayed as Bi.Op.Red).

This is because it is unclear how to otherwise combine multiple colors.

Note:Opacity is the maximum of the opacities of the displayed images, so

turning a subset off may change the opacity of the remaining colors.

For the colocalized voxels

Default:the voxels will tend to look grayish. The same algorithm is used to color them as normally when both data sets are displayed. To wit: Image1 is displayed in green, image2 in red. Voxels which have similar intensities in the two images will normally appear gray (dark gray if the intensities are low and brighter white if the intensities are higher). The gray will have a reddish tint if image2 is brighter than image1; similarly, a greenish tint implies that the two voxels are similar in intensity but image 1 is slightly brighter. Red: The red value is displayed. Blue and green are set to zero. Opacity is based upon the red level (brighter => more opaque). Green: As for "Red" but the green channel is set. Blue: As for "Red" but the blue channel is set. Bin Red:Binary red. The red component is set to maximum brightness regardless of the voxel value. Green and blue are zero. Opacity is based upon voxel values as in the "Red" option above. Bin Green:As for "Bin Red" above, but the green channel is set instead. Bin Blue:As for "Bin Red" above, but the blue channel is set instead. This seems to be buggy, looking the same as just "Blue". Bin White:The voxel is set to maximum brightness (full white). Bi.Op.Red:Binary Opaque Red. Same as Bin Red, but the voxels are also set to be totally opaque. This is the option that will tend to make them maximally visible. Bi.Op.Green: As for "Bi.Op.Red" above, but the green channel is set instead. Bi.Op.Blue: As for "Bi.Op.Red" above, but the blue channel is set instead. This seems to be buggy, looking the same as just "Blue". Bi.Op.Wht:The voxels are set to full white and are set totally opaque.

For the non-colocalized voxels in image 1:

Green:The voxel will be green. The larger the data value the brighter the green (and the more opaque). This is similary to the way image1 data is normally displayed (although it is not combined with image2 data). Bin.Green: Binary Green. All nonzero (image1) voxel values (which aren't colocalized) are shown full green; but opacity is still varied depending upon the voxel value. Bi.Op.Green: the same as Binary Green but all nonzero voxels are set to be totally opaque. Grey: The red, green, and blue channels are all set to the intensity of the image1 ("green") voxel. Thus a gray voxel is produced. Opaciy is based upon the voxel intensity. OFF: Don't display the image1 data set.

For the non-colocalized voxels in image 2 or image 3:

The same as the items described above for image 1, with the obvious changes.

NOTE: Image1 and Image2 voxels (or any other pair of images) cannot be simultaneously displayed other than at their default color values. NOTE: At least 2 images must be set on in the Edit Volume widget for colocalization to work. Otherwise only one data set is examined and no colocalization is observed. These data sets are turned on by pressing their buttons in the Edit Volume widget (ON is the default). If any 2 images are on, colocalization will be done between them.

If 3 images are on, colocalization comparisons are done on

all 3 pairs of images and only if all 3 pairs colocalize is a voxel colocalized.

NOTE: All operations controlling display mode RENDERING are applied to the voxel data AFTER it is rescaled by the Opacity and Brightness slider in the Edit Volume widget (unlike dividing the data INTO the subsets, which is applied before or after the sliders depending upon whether "Apply After O+B Sliders" or "Apply Before O+B Sliders" is chosen from the menu).

Copyright 1995 by Lawrence M. Lifshitz and the University of Massachusetts Medical School. All rights reserved.