3D Multicolor Super-Resolution Imaging Offers Improved Accuracy in Neuron Tracing
Figure 3
Tracing of hippocampal neurons.
(A) Z-stack showing two neural processes that cross each other at different heights in confocal (upper panels), confocal after deconvolution (middle panels), and STORM (lower panels). (B) The xz cross-sections are plotted across the three green lines in (A) for the confocal image after deconvolution (left) and the STORM (right) image. The xz cross-section of the “top” and “bottom” neural processes cannot be easily discerned in the confocal images at the crossing point (left) as they merge together. Thus the two neural processes (red and green circles) appear to merge into one process (yellow circles). On the other hand, the membrane that separates the two neural processes is clear in the STORM cross-sections and a “top” (red oval) and “bottom” (green oval) neural process can be identified at all locations. (C) xy cross-section taken from the midpoint image of a 3D confocal (left) and STORM stack (right). The middle panel shows the confocal slice after deconvolution. (D) The graphs show the intensity profile across the red rectangle shown in (C) for the confocal image after deconvolution (grey plot) and the STORM (black plot) image. Three clearly separable peaks are seen in the STORM plot. The first peak is the membrane edge of the first neuron and the last peak is the membrane edge of the second neuron. The peak in the middle is the membrane boundary that separates the two neurons. (E) The difference in tracing results for this region in confocal (left) and STORM (right). The confocal tracing leads to one parent process splitting into two branches (red) whereas the STORM tracing leads to two neural processes (red and green) in close proximity. (F) Tracing results for an identical region of neurons in confocal (left) and STORM (right). Distinct processes are assigned different colors.