[Chimera-users] Align and average intensity images in Chimera

Tom Goddard goddard at sonic.net
Mon Sep 7 18:27:34 PDT 2015

Hi Peggy,

  To average two aligned 3d maps use the Chimera "vop add" command:

	vop add #0,1 scaleFactors 0.5,0.5

which will create a new map.  If the two maps have different intensity normalization than you probably want to normalize them to have the same intensities in getting the average.  For instance if the intensity values for map #1 are 10 times greater than map #0 I would instead use

	vop add #0,1 scaleFactors 0.5,0.05

to reduce the map #1 intensity by a factor of 10.  More info on vop add is in the Chimera manual

	http://www.cgl.ucsf.edu/chimera/current/docs/UsersGuide/midas/vop.html#add <http://www.cgl.ucsf.edu/chimera/current/docs/UsersGuide/midas/vop.html#add>

  The numbers in the reply log after using Fit Map give the motion required for the alignment.  The 3 row, 4 column matrix gives the 3x3 rotation matrix in the first 3 columns and the translation in the 4th column.  In your screen image, the translation is 0.92, 6.14, -0.04 so about 6 units in y.  Since your maps are from TIF images, Chimera may not know the pixel size (unless it is an OMETIFF), and will take it to be 1, so the shift of 6 units would be 6 pixels.  If Chimera does know the pixel size, say it is 0.2 microns, then it would have reported the shift as 1.2 in the reply log, since those numbers use physical units.  With light microscopy your z-spacing is usually different from x and y.  So you may want to set the correct numbers.  This is done in the Volume Viewer dialog, menu Features / Coordinates, the voxel size field.  For instance for x,y,z grid spacings 0.2, 0.2 and 0.3 microns enter 0.2 0.2 0.3.  This will be important if Fit Map rotates the image in doing the alignment.  More info on fitmap is in the Chimera manual.

	http://www.cgl.ucsf.edu/chimera/current/docs/ContributedSoftware/fitmaps/fitmaps.html <http://www.cgl.ucsf.edu/chimera/current/docs/ContributedSoftware/fitmaps/fitmaps.html>

Chimera does not optimize scaling, so if your images have different magnification you will have to figure out a way to get them to the same magnification.


> On Sep 7, 2015, at 5:08 PM, 許紹君 wrote:
> To whom it may concern,
> I am willing to use Chimera for aligning several images from the structure illumination microscope (SIM).
> Here is the surface maps generated by Chimera from two images. 
> <image001.jpg>
> After alignment by the tool “Fit in Map”, they are aligned very well.
> <image002.jpg>
> My next step is to average the intensity from two aligned images and generate a new map which will be used to fit other images.
> Can anyone tell me how to do it in Chimera? 
> By the way, please also tell me what the values mean in the reply log. 
> <image003.jpg>
> It helps me to use another software to complete the mission, if I can get the rotation, voxel shift, or even scaling values from the alignment.
> Thank you!
> All Best,
> Peggy Hsu, PhD
> Institute of Cellular and Organismic Biology, Academia Sinica
> 128 Academia Rd Sec 2, Nankang, 
> Taipei 11529, Taiwan
> Tel: 886-2-2787-1531 (O)
> email: peggyschsu at gate.sinica.edu.tw <mailto:peggyschsu at gate.sinica.edu.tw>
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