Side averaging

Side averaging is an image-processing method used to generate averaged side-view images of Golgi ministacks after alignment and normalization. Golgi ministacks are first identified by the presence of giantin double puncta, which subsequently serve as geometric references for image rotation, translation, and size normalization. Following intensity normalization, the processed images are averaged to produce a representative side-averaged Golgi ministack.

This approach enhances intrinsic structural features along both the cis–trans axis and the lateral dimension, providing detailed insights into cisternal organization. Therefore, side-averaged images incorporate complementary information derived from en face averaging.

Steps

Step 1  Prepare the cropped image

• Ensure the pixel size is 45 nm. Otherwise, use Fiji>Image>Scale (None) to resize the image so that the pixel size is 45 nm.

• Crop a 40x40 pixel 3-color image.

• Rotate so that the trans-Golgi side is up.

• Subtract background (background intensity = 0).

• One channel must show giantin double puncta; double puncta are selected in the ROI Manager (ROI1 andROI2).

• Select giantin channel before the next step.

Step 2  Translate the giantin centroid to the image center

• (x1, y1) and (x2, y2) are centers of mass of left and right giantin puncta.

• xc = (x1 + x2)/2; yc = (y1 + y2)/2;

• Note xc and yc are the geometric center, not the center of the mass, of giantin double puncta.

• Calculate θ.

• Check if a Golgi image is truncated after translation. If so, repeat Step 1 and crop a larger image, such as 60×60 pixels, to avoid the truncation.

Step 3  Rotate the image so that the giantin double puncta are horizontal

• Rotate the image by - θ.

• The radius of the giantin double-punctum is the distance from (xc, yc) to either (x1, y1) or (x2, y2).

Step 4  Lateral expansion so that the radius of giantin-punctum is 80 pixels.

• Expand the image along the x-axis to ensure the radius of the giantin double-punctum is 80 pixels.

Step 5  Axial expansion by 8×

•  Expand the image 8× along the y-axis.

  • The image height becomes 320 pixels; width ranges from 200 to 400 pixels based on giantin's lateral size.

Step 6  Enlarge the canvas to 701×701 pixels

Step 7  Normalize the total intensity of each channel to 50 million.

Step 8  Average