This document describes a 3D stabilization software called Stabi Tissue for intravital imaging. It aims to solve issues with tissue movement during imaging by stabilizing images in XYZ. The software allows users to upload images, select cycles/slices, view stabilized videos, and remove ghost images. It uses template matching and other algorithms to register images and compensate for motion. The document compares it to other stabilization software and plugins, and provides examples of its use stabilizing intravital imaging data.

1. 3D Stabilization software for
intravital imaging
Iván Gómez Conde, David Olivieri, Carlos Tadokoro
(ivangconde@uvigo.es)
March 1st, 2013

2. Outline
• What do we want to solve?
▫ 3D stabilization
▫ Ghost images
• Stabi Tissue 0.3 (software)
▫ Main options
▫ Running the software
• Other papers and software about stabilization of intravital imaging
• Discussion
▫ Software
▫ Paper

3. What do we want to solve?
Two photon microscope
Manufacturer: Prairie (Model : Ultima )
Spleen in mice

4. Doing images of intravital tissue
1
2
3
4
-> 2.19 s
-> 2.19 s
-> 2.19 s
-> 2.19 s
The tissue is static
Stack of images in each cycle
(4 slices)
Images captured in 1 cycle
(Total time: 8.9 seconds)Cycle 1

5. Stabilization in a plane (XY)
1
2
3
4
Stack of images in each cycle
(4 slices)
-> 2.19 s
-> 2.19 s
-> 2.19 s
-> 2.19 s
Images captured in 1 cycle
(Total time: 8.9 seconds)
The tissue is moving (left-right)
Cycle 1

6. Stabilization in Z
1
2
3
4
Stack of images in each cycle
(4 slices)
-> 2.19 s
-> 2.19 s
-> 2.19 s
-> 2.19 s
Images captured in 1 cycle
(Total time: 8.9 seconds)
The tissue is moving (up-down)
Cycle 1

7. Stabilization in XYZ
1
2
3
4
-> 2.19 s
-> 2.19 s
-> 2.19 s
-> 2.19 s
The tissue is moving (all directions)
Stack of images in each cycle
(4 slices)
Images captured in 1 cycle
(Total time: 8.9 seconds)Cycle 1

8. Ghost images
1
2
Cycle 1
The tissue can be contracting
1
Ghost images
Cycle 2

9. Stabi Tissue Software 0.3
• 3D Stabilization software for intravital imaging of mouse organs
▫ Command-line software (http://sourceforge.net/projects/stabitissue/)
▫ User-interface software (building…)

10. Stabi Tissue Software 0.3
• Open source (free redistribution and access to an end product's
design and implementation details)
• Cross-platform (Linux, Windows, Mac OS X)
• Friendly user interface

11. Main options of the software
Upload the images
Select some cycles
or slices
View the video of a
slice (by time)
Stabilize in XYZ
Save the stabilized
images
Remove the ghost-
images

12. Upload images
• The user only needs to introduce:
▫ Folder
▫ Type of images (tif, tiff, jpeg…)
The software uses regular expressions to solve that problem:
"Infected-2x-389_Cycle052_CurrentSettings_Ch5_000022.tif“
"File_Infected-2x-388-1_t001_z004_c0002.tif"

13. Image formats of microscopes
• LOCI Bio-Formats of University of Wisconsin-Madison lets to
convert propietary microscopy data into a standard format
▫ Problems: written for Java and bindings for C++ (not for Python)
▫ Possible solution: external library or script
http://loci.wisc.edu/software/bio-formats

14. Select images (cycles/slices)
• The user can choose the images selecting with the mouse

15. Stabilization method in XY (1)
1. Create a mask for the first image (template)
▫ Color Threshold, algorithm to detect contours, remove the small contours

16. Stabilization method in XY (2)
2. Template matching using the mask with the other image of the next
stack
𝑀𝑜𝑣𝑋𝑌𝑖𝑚𝑔1,𝑖𝑚𝑔2 = 𝑓𝑚𝑎𝑡𝑐ℎ 𝑥𝑦 𝑖𝑚𝑔1, 𝑖𝑚𝑔2 = max(
𝑝𝑖𝑥 𝑥=−𝑛
𝑛
𝑝𝑖𝑥 𝑦=−𝑚
𝑚
𝑓𝑀𝑜𝑣𝑖𝑚𝑔1,𝑖𝑚𝑔2(𝑝𝑖𝑥 𝑥, 𝑝𝑖𝑥 𝑦))
Image 1 Image 2 Mask Img 2Mask Img 1

17. Stabilization method in XY (2)
3. Find the maximum likelihood between the mask of the image 1 and
the mask of the new image
Mask 1
Mask 2

18. Stabilization method in XYZ
1. Find the best matching whith the neighbours in the next stack
(cycle t+1)
Cycle t Cycle t+1
Cycle t Cycle t+1
𝑀𝑜𝑣𝑍𝑡,𝑡+1 = max(
𝑠𝑙 𝑡=0
4
𝑠𝑙 𝑡+1=0
4
𝑓𝑚𝑎𝑡𝑐ℎ 𝑥𝑦(𝑖𝑚𝑔𝑠𝑙 𝑡
, 𝑖𝑚𝑔𝑠𝑙 𝑡+1 ))

19. Stabilize in XYZ
• Choose some initial parameters and the software can stabilize in XYZ
▫ Initial threshold, minimum area of the contours, reduction factor of the
resolution in the images and pixels of movement

20. Ghost images
• Find the images that are similar to choose only one
▫ The software can choose automatically or the user can change the choice
The software will show the
recognized ghost images

21. View video
• Selecting the slice that you want to see by time
▫ Change the speed
▫ See the different channels

22. Other papers of Stabilization (PLoS One)
• Device designated ‘microstage’ that reduces organ motions without
causing tissue lesions. Combining with an upright intravital laser
scanning microscope equipped with a unique stick-type objective lens
Cao L, Kobayakawa S, Yoshiki A, Abe K. High Resolution Intravital Imaging of Subcellular
Structures of Mouse Abdominal Organs Using a Microstage Device. PLoS ONE . 2012 May 7(3)

23. Other papers of Stabilization (Nature
Methods)
• They constructed a thoracic window (4-mm internal diameter)
• Its loose adherence to the lung surface allows for gas to enter and
expand the lung but limiting the associated movement in x, y and z.
Mark R. Looney, Emily E. Thornton, Debasish Sen, Wayne J. Lamm, Robb W. Glenny, and Matthew F.
Krummel “Stabilized Imaging of Immune Surveillance in the Mouse Lung” Nat Methods. 2011
January; 8(1): 91–96.

24. Other papers of stabilization (Intravital-
Landes Bioscience)
• A new water-immersion suctioning stabilizer that enables minimally
invasive intravital fluorescence microscopy using water-based stick
objectives
Vinegoni C, Lee S, Gorbatov R, Weissleder R, “Motion compensation using a suctioning
stabilizer for intravital microscopy” J Intravital (Landes Bioscience). 2012;1:115-121

25. Plugin for FIJI (stabilization)
• This plugin stabilizes jittery image stacks using the Lucas-Kanade
algorithm
• It creates a template from the currently shown slice in an image
stack and stimates the geometrical transformation needed to best
align
K. Li, "The image stabilizer plugin for ImageJ,"
http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html , February, 2008.

26. Image stabilizer FIJI vs Stabi Tissue
• The Stabi Tissue software had been tested with a video against the
plugin of FIJI. (The result is only with a XY stabilization)
Image Stabilizer
FIJI
Stabi Tissue
software
Video with
movement
Luciana Vieira de Moraes, Carlos Eduardo Tadokoro, Iván Gómez-Conde, David N. Olivieri, Carlos Penha-
Gonçalves “Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration
and Phagocytosis of Plasmodium-Infected Erythrocytes” PLoS Pathog. 2013 January; 9(1): e1003154

27. Other results (Flávia’s paper) (1)

28. Other results (Flávia’s paper) (2)
Stabi Tissue software
Stabilization XYZ
Video with
movement in XYZ
Flávia A Lima, Iván Gómez-Conde, Paula A Videira, Cláudio R Marinho, David N Olivieri, Carlos Eduardo Tadokoro,
“Intravital microscopy technique to study parasite dynamics in the labyrinth layer of the mouse
placenta” Submitted to Parasitology International (Elsevier)

29. Aknowledgements
• University of Vigo
▫ David Olivieri Cecchi
• Instituto Gulbenkian de Ciência
▫ Carlos Tadokoro
▫ Henrique Silva
▫ Flavia Afonso Lima
▫ Susana Caetano