en face averaging

Principle and Workflow of En Face Averaging of Golgi Ministacks

En face averaging is an image-processing method used to determine the average spatial organization of proteins within Golgi ministacks. In this approach, individual Golgi ministacks are imaged in the en face orientation, and the signals from many ministacks are computationally aligned and averaged. This procedure reduces noise and biological variability while revealing the common structural features and relative protein localization within the Golgi stack.

The alignment is performed using the giantin ring as a reference landmark because giantin appears as a circular ring that reliably marks the Golgi rim. Each cropped Golgi image is first centered based on the position of the giantin ring, then rescaled so that all rings have the same radius. After normalization of signal intensity, multiple images are averaged to generate a high signal-to-noise en face representation of the average Golgi ministack organization.

Steps

Step 1 Prepare the cropped image

·         Use multi-channel 2D image.

·         Ensure square images (e.g., 40×40 or 60×60 pixels).

·         Subtract background so that the background intensity equal 0.

·         One channel must be the giantin-ring, which should be selected for the following alignment steps.

Step 2 Translate the giantin-ring centroid to the image center.

·         The geometric center or centroid of the giantin-ring is calculated as the center of mass, assuming the mass density is 1.

o    centroid (mx, my) is (xc, yc) 

·         Image translation (Bilinear) to the image center.

Step 3 Expand the image so the giantin-ring’s gyradius is 100 pixels

·         The gyradius of the giantin-ring is calculated assuming the mass density is 1. The rotation axis is at the centroid (mx, my).

·         Expansion (interpolation=None)

Step 4  Enlarge the canvas to 701x701 pixels

(interpolation=None)

Step 5  Normalize the total intensity of each channel to 200 million.

Download the Image J macro for en face averaging.

Step 6  Averaging.

All processed images are averaged to generate the final en face average.

Download

Protocol: 

• en face averaging protocol in PDF.

Macros:

1.  “Normalization and Giantin alignment for en face average_New_Center” (20251024)"

2. "Radial mean intensity profile-v1.ijm"

3. "ROI_to_image.ijm"

Training images:

• Zipped folder containing images for practice.

References

1. Tie HC, Ludwig A, Sandin S and Lu L (2018) The spatial separation of processing and trafficking functions to the interior and periphery of the Golgi stack. eLife 7:e41301.

2. Tie HC and Lu L. (2023) Studying the organization of the Golgi by super-resolution microscopy. Methods. Mol. Biol. 2557:113-125.