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. 2017 Sep 20;11:275. doi: 10.3389/fncel.2017.00275

Figure 4.

Figure 4

Intensity measurements reveal similar volume change between superficial and deep neurons. (A) Representative thresholded images of a Thy1-eGFP neuron taken from deep tissue (~65–90 μm below slice surface) at baseline (A1) and 5 min 40% hACSF (A2) time points. Magenta regions (indicated by white arrows) show areas of cell volume increase. (B) Volume change in deep Thy1-eGFP neurons exposed to 40% hACSF, measured using our standard analysis method. Inset displays average percent change across hACSF applications. The apparent reduction in volume change as compared to shallower neurons (e.g., Figure 3) is most likely an artifact of lower cellular resolution at depth, which is poorly handled by our standard analysis technique. (C) Illustration of the analysis problem posed by deeper neurons. Raw image MIPs from 5 min hACSF time point for a typical shallow (C1) and deep (C2) neuron, illustrating the analysis problem posed by the latter. Images have been brightened to enhance visibility of the cell border. Magenta lines indicate borders of thresholded area for each cell after final processing (see Figure 3B, Panel (A) for respective thresholded images), from which soma area is typically calculated. Shallower neurons (C1) have well-defined borders in the raw MIP which are almost completely preserved after thresholding, allowing for highly-accurate measurements of soma area in these cells. In contrast, the “fuzzy” borders of deeper neurons (C2) are poorly thresholded and insufficient for detecting volume changes. Scale bars = 5 μm. (D) Standard (thresholding) and microspectrofluorimetric (fluorescence intensity) methods applied to a standard-depth neuron at baseline (left) and 5 min 40% hACSF (right) time points. As in (C), magenta outlines indicate cell area calculated by thresholding. Yellow ROIs denote the 5 × 5 μm region from which average fluorescence intensity was measured. (E) Analysis methods from (D) applied to a deep neuron. While thresholded borders are inaccurate as expected, fluorescence intensity within the yellow ROI decreases to a similar degree between this deep neuron and the more superficial neuron in (D). (F) Percent change over the 1st 5 min of 40% hACSF, calculated from changes in average fluorescence intensity in both standard depth and deep neurons. ***p < 0.001, *p < 0.05, percent change vs. 0% (baseline).