Part 1: Setup and Basic Image Processing

Installation and Setup¶

If you haven’t installed FIJI yet, download the latest version from https://fiji.sc.

Activate Update Sites¶

After installation, start FIJI and configure the necessary update sites:

1. Go to Help > Update

2. Click Manage Update Sites

3. Activate the following update sites:

   • Fiji Legacy

   • 3D-Image suite

   • ImageScience

   • Leiden University

4. Click Close and Apply Changes

Basic Image Processing¶

In this exercise, you’ll learn fundamental image processing operations using HeLa cell images.

1. Open Sample Image¶

Open HeLa cells: File > Open Samples > HeLa cells

2. View Image Information¶

Press I or go to Image > Show Info to see how the image is constructed.

3. Split Channels¶

1. Open the Channel tool: Image > Color > Channel tool

2. Click the More button

3. Select Split channels

4. Process the Red Channel¶

The red channel shows bright spots on a strong cytoplasmic background.

1. Duplicate the red channel: Image > Duplicate

2. Remove background: Process > Subtract background

3. Adjust settings for the best result using the Preview button

5. Process the Green Channel¶

The green channel image is blurred and needs sharpening.

1. Change the background to black: Image > Adjust > Brightness/Contrast

2. Adjust the minimum value until the background is black

3. Try two different sharpening filters:

   • Process > Sharpen, or

   • Process > Filter > Unsharp Mask

Compare the results to see which works better.

6. Process the Blue Channel¶

1. Sharpen the image using the method that worked best for the green channel

2. Remove background signal using either:

   • Brightness/Contrast adjustment (Step 5), or

   • Subtract Background (Step 4)

Which method works best?

7. Merge Channels¶

1. Merge all channels: Image > Color > Merge channels

2. Compare the result to the original image

8. Adjust Look-up Tables¶

Your colleague mislabeled the pseudocolors. Green and red should be swapped.

Adjust the look-up tables: Image > Look-up tables

9. Practice on Your Own Images¶

Try these operations on your own microscopy images.

Brightness and Contrast¶

Understanding and controlling brightness and contrast is essential for image analysis.

1. Open Galaxy Sample¶

Open File > Open Samples > M51-galaxy

2. Auto-adjust Contrast¶

1. Go to Image > Brightness/Contrast

2. Click Auto to automatically adjust contrast

You’ll see 4 sliders in the Brightness and Contrast window. Experiment with all 4 sliders to see their effects.

3. Optimize Display¶

Try to display all intensities clearly without overexposing the image.

4. Apply Settings to Multiple Images¶

1. Open the M51 galaxy image a second time

2. In the first image, click Set in the Brightness/Contrast window

3. Carefully adjust the minimum and maximum displayed values

4. Check Propagate to all other open images

5. Click OK

What happened to the second image? This feature is useful for comparing intensities between images.

5. Understanding Apply vs. Display¶

1. Select one image and click Apply in the B/C menu

2. Read the warning and click OK

3. What happened to the pixel values?

4. Try to lower the contrast

Close the images when finished.

Look-up Tables¶

Look-up tables (LUTs) control how pixel values are displayed as colors.

1. Explore Different LUTs¶

Re-open the M51 galaxy image and try these look-up tables (Image > Look-up tables):

1. Green

2. Fire

3. Red Hot

4. Glasbey

Which one makes the image look best?

2. Edit LUT Values¶

1. Go to Image > Color > Edit Lut

2. View all 256 assigned values of the look-up table

What is essential to see all density levels properly? (Hint: examine the Glasbey LUT)

Puzzle: Hidden Information¶

Test your skills by revealing hidden information in an image.

1. Open Plugins > LeidenUniv > Teaching > Get the answer

2. Use all the knowledge you’ve gained to reveal the answer