Grey wall is an inevitability with current standards of sectioning and imaging. While larger objects of interest can be (mostly) cross-sectioned like dendritic shafts, there are many other objects like axons and endosomes that are not as clearly defined in a single 2D section. Because of this, it is important to place traces that respect grey wall areas to ensure accurate surface area and volume measurements.

The Test

Using the in-silico microtome, a tool developed by Tom Bartol at the Salk Institute, a dense reconstruction of neuropil was used as ground truth and re-sectioned to test different tracing methods. The results demonstrated that in order to achieve accurate surface area and volume measurements of sampled objects, grey walls must be traced.

Blending Sections

While the in-silico microtome is an extremely helpful tool, it generates much cleaner virtual sections compared to real examples. This means that the double-darkening of overlapping membrane walls is not as distinguishable in real data as in the above figure. Even though the ‘inner’ tracing method was shown to be the most accurate, it is nearly impossible to replicate. However the “outer” tracing method can be easily replicated in real EMs using the “blend” function.

Encounters with grey walls must take into account the sections above and below. More specifically, the placement of clear cytoplasm. The blend method uses traces of the cytoplasm of an object from adjacent sections to inform object boundaries.

The above example is of 2 sections taken from the in-silico microtome. On section 2 there is an area of grey wall towards the top. Tracing the cytoplasm from the preceding section (orange line) and blending it with section 2 shows the area of the grey wall that should be traced for the object of interest.

Note that this tracing method will result in overlapping traces for different objects which share grey wall space due to section thickness limitations. This is okay. We know that two membranes can occur on top of each other through thicker sections.

Example

The below example is of two obliquely sectioned objects passing one another. Between them is an ambiguous grey area. Where should the traces of each object be placed?

Looking at the preceding section, the cytoplasm of Object 1 is clearly visible. Tracing the cytoplasm and blending it with the section in question shows the extent of the grey wall belonging to Object 1.

The same can be done on the next section with the cytoplasm of Object 2.

The cytoplasm traces can be overlaid on the central section to see which areas of the ambiguous grey wall should be attributed to each object and inform the final traces.

Final

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