Iwabuchi, S., Koh, J.-Y. Y., Wang, K., Ho, K. W. D., & Harata, N. C.
Department of Molecular Physiology and Biophysics, University of Iowa, Carver College of Medicine, Iowa City, IA, USA.
Images in biomedical imaging research are often affected by non-specific background noise. This poses a serious problem when the noise overlaps with specific signals to be quantified, e.g. for their number and intensity. A simple and effective means of removing background noise is to prepare a filtered image that closely reflects background noise and to subtract it from the original unfiltered image. This approach is in common use, but its effectiveness in identifying and quantifying synaptic puncta has not been characterized in detail. We report on our assessment of the effectiveness of isolating punctate signals from diffusely distributed background noise using one variant of this approach, "Difference of Gaussian(s) (DoG)" which is based on a Gaussian filter. We evaluated immunocytochemically stained, cultured mouse hippocampal neurons as an example, and provided the rationale for choosing specific parameter values for individual steps in detecting glutamatergic nerve terminals. The intensity and width of the detected puncta were proportional to those obtained by manual fitting of two-dimensional Gaussian functions to the local information in the original image. DoG was compared with the rolling-ball method, using biological data and numerical simulations. Both methods removed background noise, but differed slightly with respect to their efficiency in discriminating neighbouring peaks, as well as their susceptibility to high-frequency noise and variability in object size. DoG will be useful in detecting punctate signals, once its characteristics are examined quantitatively by experimenters.
… “blue fluorescent beads were excited using a 400-nm light-emitting diode (LED, CoolLED-Custom Interconnect, Hampshire, UK) at 100% intensity,”…
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