Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images

E Meijering, M Jacob, JCF Sarria… - Cytometry Part A: the …, 2004 - Wiley Online Library
E Meijering, M Jacob, JCF Sarria, P Steiner, H Hirling, M Unser
Cytometry Part A: the journal of the International Society for …, 2004Wiley Online Library
Background 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.
Methods The technique was compared to fully manual delineation. Four observers
repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture …
Background
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.
Methods
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.
Results
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.
Conclusions
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.
Wiley Online Library