Fig 1.
Comparison of the MIP and the 3D visualization of some of the main regions analyzed for colocalization. The 3D image shows the selection overlaid on the whole cell, as well as the selection isolated from the remainder of the cell. The depicted cell is from the control sample set (t = 0). A: The whole cell. B: The perinuclear region for both a cylindrical and ellipsoidal selection. C: The sampled middle region. D: The sampled membrane region. E: The freehand membrane region (no overlay shown).
Table 1.
Colocalization metrics Ri represents the intensity of voxels in channel 1 (red fluorescent label) and Gi represents the intensity of voxels in channel 2 (green fluorescent label).
and
represent the mean intensities of the respective channels [26].
Fig 2.
The ROI selection tools employed in the analysis. Combinations of these tools were used to make selections. A: The box tool. B: The cylinder tool. C: The sphere tool. D: The freehand tool.
Fig 3.
Representative cells for each considered time (t = 0, t = 6, t = 24) with the maximum intensity projection on the left and the 45° 3D view on the right. Shown from top to bottom are the acetylated α-tubulin (red), the Tau (green), the tubulin and Tau overlaid, and only the colocalized pixels/voxels.
Fig 4.
Ellipsoidal perinuclear region.
Representative cells for each considered time (t = 0, t = 6, t = 24) with the maximum intensity projection on the left and the 45° 3D view on the right. Shown from top to bottom are the acetylated α-tubulin (red), the Tau (green), the tubulin and Tau overlaid, and only the colocalized pixels/voxels.
Fig 5.
Representative cells for each considered time (t = 0, t = 6, t = 24) with the maximum intensity projection on the left and the 45° 3D view on the right. Shown from top to bottom are the acetylated α-tubulin (red), the Tau (green), the tubulin and Tau overlaid, and only the colocalized pixels/voxels.
Fig 6.
Representative cells for each considered time (t = 0, t = 6, t = 24) with the maximum intensity projection on the left and the 45° 3D view on the right. Shown from top to bottom are the acetylated α-tubulin (red), the Tau (green), the tubulin and Tau overlaid, and only the colocalized pixels/voxels.
Fig 7.
Representative cell for t = 6 with the maximum intensity projection at the top and the 45° 3D view with the ROI isolated from the whole cell at the bottom. Shown from left to right the acetylated α-tubulin (red), the Tau (green), the tubulin and Tau overlaid, and only the colocalized pixels/voxels. For the protein aggregates only the results at t = 6 were visualized and analyzed.
Table 2.
Pearson’s correlation coefficients calculated for Tau and acetylated α-tubulin in different areas of GT1-7 cells under control conditions and after exposure to chloroquine for 6 and 24 hours respectively.
(Data are presented as mean ± 95% confidence interval). * indicates significant differences relative to control (p < 0.05); A shaded block indicates that the ROI selection methods (i.e. 3D vs 2D) are statistically significantly different. In all cases statistical significance was determined using the two-sample, two-tailed t-test. Protein aggregates were only analyzed at the 6 hour time point.
Table 3.
Manders’ overlap coefficient with procedure described in Table 2.
Table 4.
Manders’ correlation coefficient for Tau (M1) with procedure described in Table 2.
Table 5.
Manders’ correlation coefficient for acetylated α-tubulin (M2) with procedure described in Table 2.