The accurate physical volumes of the dissectors were carefully calibrated before being used for the synapse density measurement in HVC in both Experiment I and II.
The tissue deformation that occurs during the EM staining, embedding and ultramicrotomy were quantified by evaluating LM and EM images taken from the same brain section sample at different tissue preparation stages.
Tissue deformation resulting from EM embedding was measured by comparing the LM images of wet brain sections taken prior to embedding, with the LM images of the same brain section after embedding (Figure 2. 3 β a & b). Landmarks, such as blood vessels, ventricles, and sharp sample borders, were identified on both images.
The line distances πΊ between pairs of the identified landmarks were measured with Fiji on both images with known image pixel size obtained from the microscopes.
The ratio of the 3D physical volume deformation (shrinkage or dilation) was calculated then from the ratio of measured 1-D line distances as follows:
Equation S. 1: πΉπππππππππ π πππ= ( πΊ (ππππππππ π πππ‘πππ) πΊ (π€ππ‘ π πππ‘πππ) )3
The resulting deformation ratio of each sample are summarized in Table S. 1, and illustrated in Figure S. 1. The deformation of the tissue during EM staining and embedding was isotropic, indicating the potential dilation or shrinkage should be the same on any axis.
Figure S. 1: Tissue deformation ratio during EM embedding process of each sample.
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Table S. 1: Tissue deformation during EM embedding and ultramicrotomy.
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Similarly the tissue deformation after ultramicrotomy was quantified by comparing the LM images taken from the trimmed sample block face and the SEM images taken from the ultrathin sections cut from the same sample block (Figure S. 2). The physical cutting was always along one direction and thus introduced non-isotropic tissue deformation onto the ultrathin sections. Therefore the tissue deformation on the three orthogonal axes X, Y, and Z were measured separately. The well-trimmed trapezoidal shape of the sample block face helps to identify the X-axis, as the axis is perpendicular to the cutting direction. The Y-axis is the axis that is parallel to the cutting direction (Figure S. 2, red and blue lines). The ratio of the physical deformation for the X and Y axes resulting from ultramicrotomy was calculated as follows:
Equation S. 2: πΉπππππππβπΏ(ππ π)= πΊ (ultraβthin section) πΊ (ππππππππ π πππ‘πππ)
Figure S. 2: Calibration of tissue deformation resulting from ultramicrotomy. Left, LM image of a sample block face after ultramicrotomy. Right, SEM overview of an ultrathin section that was cut from the same sample block face. The red solid lines represent three measurements of the same distances along the X- axis in both images. The blue dashed lines represent three measurements of the same distances along the Y- axis in both images.
The resulting deformation ratio of X and Y axes for each sample are summarized in Table S. 1, and illustrated as blue and red bar plots respectively in Figure S. 4.
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Figure S. 3: Ultrathin section thickness calibration with cylindrical objects. We drew 3D ball objects with TrakEM2 onto mitochondria at given locations and let the ball inscribe the mitochondria. a - f are consecutive 2D images of a sample mitochondria longitudinally dissected. The yellow, blue and red circles are the 2D sections of the ball objects, which denote center, pre-center, and post-center Z positions.
The length along the Z-axis, which being the thickness of the ultrathin sections, was measured in a different way. A cylindrical diameter method was adopted (Fiala and Harris 2001). This method provides an estimation of the average separation distance of a given set of consecutive 2D images, based on precise measurement of the diameter of any given cylindrical object that is dissected longitudinally in the images. Mitochondria in the ssSEM images that are cylindrical shaped and longitudinally dissected, were therefore used to perform this section thickness calibration (Figure S. 3). The diameter π π of selected mitochondria π was measured, and the number of sections ππ it spans were counted. By averaging the measurements of π΅ mitochondria the mean section thickness (πΜ ) can thus be calculated as:
Equation S. 3:
πΜ =
π1β
ππ ππ
Equation S. 4: πΉπππππππβπ=ππ ππ (π ππ‘ π πππ‘πππ π‘βππππππ π )πΜ
The physical deformation ratio of the Z-axis due to ultramicrotomy were calculated with Equation S. 3 and Equation S. 4. The resulting deformation ratios of the Z-axis for each sample are summarized in Table S. 1, and illustrated as green bar plot in Figure S. 4.
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Figure S. 4: Tissue deformation ratios during ultramicrotomy of each sample. The deform ratio of the X, Y and Z axes were plotted seperately with different colors (blue, X; red, Y; green, Z).
The tissue deformation during ultramicrotomy was reconfirmed to be non-isotropic, as can be observed in the measurement results (Figure S. 4). The ultrathin sections shrunk to roughly 80% of their original size along the cutting directions (Y-axis), while the axis parallel to the cutting was almost unchanged (X-axis). The section thickness was controlled by the setup and quality of the ultramicrotome, which is independent of the cutting procedure itself. The tissue deformation of the wet brain sections to the ultrathin sections was calculated as follows:
Equation S. 5:
πΉπππππππ= πΉπππππππππ π πππ β πΉπππππππβπΏ β πΉπππππππβπβ πΉπππππππβπ
Figure S. 5: Tissue deformation ratio of the EM embedding and ultramicrotomy procedures together for each sample.
The total deformation ratio for each sample was summarized in Table S. 1, and illustrated in Figure S. 5. This measurement summarizes how much in percentage the brain tissue was physically shrink or dilate in volume from the wet brain section state to the ultrathin section state. The dissector physical volume was then calibrated for each sample according to this measurement, prior to the density estimation and analysis. The synapse density values presented in Experiment I and Experiment II (see Result section 2.3.2, 2.3.3, 2.3.4, and 3.3.3) thus reflected the value in the wet brain section state, which was closer to the living state of the bird.
In summary the embedding caused roughly 2.2 Β± 0.2 % tissue dilation in all directions. The ultramicrotomy caused 1.4 Β± 0.3 % tissue dilation in the X-axis and 17.9 Β± 0.3 % shrinkage in the Y-axis. The average thickness of the ultrathin section
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was 1.5 Β± 0.7 % thinner than the set thickness (70 nm) in the ultra-microtome. On average the final physical volume measured with the ssSEM dataset from ultrathin sections would be 16.1 Β± 0.7 % smaller than that of the wet brain sections.