Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images
Version of Record online: 15 MAR 2004
Copyright © 2004 Wiley-Liss, Inc.
Cytometry Part A
Volume 58A, Issue 2, pages 167–176, April 2004
How to Cite
Meijering, E., Jacob, M., Sarria, J.-C.F., Steiner, P., Hirling, H. and Unser, M. (2004), Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry, 58A: 167–176. doi: 10.1002/cyto.a.20022
- Issue online: 25 MAR 2004
- Version of Record online: 15 MAR 2004
- Manuscript Accepted: 18 DEC 2003
- Manuscript Revised: 30 OCT 2003
- Manuscript Received: 17 JUN 2003
- cell culture;
- neurite outgrowth;
- fluorescence microscopy;
- neurite tracing;
- interactive segmentation;
- pattern recognition;
- computer-assisted image analysis;
- software validation
For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique.
The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to high-resolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance.
Both methods slightly underestimated the true neurite length, but the differences were not unanimously significant. The average deviation from the true neurite centerline was a factor 2.6 smaller with the developed technique compared to fully manual tracing. Intraobserver variability in the respective measures was reduced by a factor 6.0 and 23.2. Interobserver variability was reduced by a factor 2.4 and 8.8, respectively, and labor intensiveness by a factor 3.3.
Providing similar accuracy in measuring neurite length, significantly improved accuracy in neurite centerline extraction, and significantly improved reproducibility and reduced labor intensiveness, the developed technique may replace fully manual tracing methods. © 2004 Wiley-Liss, Inc.