3D Transformation Matrix Calculation and Pixel Intensity Normalization for the Dual Focus Tracking System

  • Kenneth Sutherland
  • Toshiyuki HamadaEmail author
  • Masayori IshikawaEmail author
  • Naoki Miyamoto
  • Masahiro Mizuta
  • Hiroyuki Date
  • Hiroki Shirato
Original Article



We provide details of the dual-focal 3D tracking and pixel intensity calibration (DuFT) system used to record gene expression in targeted areas on the skin of freely moving mice.


The accuracy of the 3D position calculation was determined by placing a scintillator on the calibration disk at various known locations within the recording cage. The height of the disk was varied from the bottom (Z = 0 cm) to the top (Z = 30 cm). The distance from the central axis range from near the center (R = 1 cm) to near to edge (R = 5 cm).


The mean deviation between the known and calculated position was .31 ± 0.16 mm. The maximum deviation was less than .86 mm.


The results indicate that the location of a scintillator within the recording cage imaged with two cameras can be calculated with submillimeter accuracy. We hope that our methods can be applied to improve automatic (even real-time) tracking of various animals in vivo.


Circadian rhythm In vivo imaging Fluorescence imaging Multi-camera tracking 3D tracking 3D interpolation 



This research was partially supported by Special Expenditures of ‘Reverse Translational Research from Advanced Medical Technology to Advanced Life Science; from Real-time Tracking Technology to Real-time Tracking Life Science’ funded by the Japanese Ministry of Education, Culture, Sports, Science and Technology and was partially supported by a research fund from Research Foundation for Opto-Science and Technology. This work was also supported in part by the Global Station for Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE), Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical Approval

Institutional Review Board approval was obtained.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

40846_2019_474_MOESM1_ESM.docx (165 kb)
Supplementary material 1 (DOCX 165 kb)


  1. 1.
    Young, M. W., & Kay, S. A. (2001). Time zones: A comparative genetics of circadian clocks. Nature Reviews Genetics, 2, 702–715.CrossRefGoogle Scholar
  2. 2.
    Abe, M., et al. (2002). Circadian rhythms in isolated brain regions. Journal of Neuroscience, 22, 350–356.CrossRefGoogle Scholar
  3. 3.
    Yamazaki, S., et al. (2000). Resetting central and peripheral circadian oscillators in transgenic rats. Science, 288, 682–685.CrossRefGoogle Scholar
  4. 4.
    Tahara, Y., et al. (2012). In vivo monitoring of peripheral circadian clocks in the mouse. Current Biology, 22, 1029–1034.CrossRefGoogle Scholar
  5. 5.
    Sakamoto, A., et al. (2005). Influence of inhalation anesthesia assessed by comprehensive gene expression profiling. Gene, 356, 39–48.CrossRefGoogle Scholar
  6. 6.
    Ohe, Y., Iijima, N., Kadota, K., Sakamoto, A., & Ozawa, H. (2011). The general anesthetic sevoflurane affects the expression of clock gene mPer2 accompanying the change of NADt level in the suprachiasmatic nucleus of mice. Neuroscience Letters, 490, 231–236.CrossRefGoogle Scholar
  7. 7.
    Zhang, W., et al. (2001). Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression. Transgenic Research, 10, 423–434.CrossRefGoogle Scholar
  8. 8.
    Hamada, T., Sutherland, K., Ishikawa, M., Miyamoto, N., Honma, S., Honma, K., et al. (2016). In vivo imaging of clock gene expression in multiple tissues of freely moving mice. Nature Communications, 7, 11705.CrossRefGoogle Scholar
  9. 9.
    Shirato, H., et al. (2000). Physical aspects of a real-time tumor-tracking system for gated radiotherapy. International Journal of Radiation Oncology Biology Physics, 48, 1187–1195.CrossRefGoogle Scholar
  10. 10.
    de Chaumont, F., Dos-Santos Coura, R., Serreau, P., Cressant, A., Chabout, J., Granon, S., et al. (2012). Computerized video analysis of social interactions in mice. Nature Methods, 9(4), 410.CrossRefGoogle Scholar
  11. 11.
    de Chaumont, F., Dallongeville, S., Chenouard, N. & Olivo-Marin, J.-C. (2010) Tracking multiple articulated objects using physics engines: Improvement using multiscale decomposition and quadtrees. Proceedings IEEE International Conference on Image Processing (Hong Kong).Google Scholar
  12. 12.
    Dankert, H., Wang, L., Hoopfer, E. D., Anderson, D. J., & Perona, P. (2009). Automated monitoring and analysis of social behavior in Drosophila. Nature Methods, 6, 297–303.CrossRefGoogle Scholar
  13. 13.
    Khan, Z., Herman, R., Wallen, K., & Balch, T. (2005). An outdoor 3D visual tracking system for the study of spatial navigation and memory in rhesus monkeys. Behavior Research Methods, 37, 453–463.CrossRefGoogle Scholar
  14. 14.
    Mitsubishi Digital Radiograph RTRT Instruction Manual revision C. (2003).Google Scholar
  15. 15.
    Matrox Electronic Systems, Matrox Imaging Library (MIL) 9 User Guide, Manual, no. Y10513-301-0900. (2008).Google Scholar
  16. 16.
    Khan, Z., Balch, T., & Dellaert, F. (2005). MCMC-based particle filtering for tracking a variable number of interacting targets. IEEE Transactions on Pattern Analysis and Machine Intelligence, 27, 1805–1819.CrossRefGoogle Scholar
  17. 17.
    Editorial. (2011). Animal rights and wrongs. Nature 470, 435Google Scholar

Copyright information

© Taiwanese Society of Biomedical Engineering 2019

Authors and Affiliations

  • Kenneth Sutherland
    • 1
  • Toshiyuki Hamada
    • 2
    Email author
  • Masayori Ishikawa
    • 1
    • 4
    Email author
  • Naoki Miyamoto
    • 1
    • 5
  • Masahiro Mizuta
    • 3
  • Hiroyuki Date
    • 4
  • Hiroki Shirato
    • 1
    • 5
  1. 1.Global Station for Medical Science and Engineering, Global Institution for Collaborative Research and Education (GI-CoRE)Hokkaido UniversitySapporoJapan
  2. 2.International University of Health and WelfareIbarakiJapan
  3. 3.Laboratory of Advanced Data Science, Information Initiative CenterHokkaido UniversitySapporoJapan
  4. 4.Graduate School of Health SciencesHokkaido UniversitySapporoJapan
  5. 5.Hokkaido University HospitalSapporoJapan

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