Part 3: Quantitative Analysis

Scaling¶

Proper scaling is essential for accurate measurements in biological images.

1. Open Embryos Sample¶

File > Open Samples > embryos

2. Set the Scale¶

1. Draw a line on the scale bar in the image

2. Go to Analyze > Set Scale

3. Enter the known distance (the number of pixels is shown in the top box)

4. Click OK

3. Measure Embryo Area¶

Now you can measure the average area of embryos:

1. Convert to 8-bit: Image > Type > 8-bit

2. Apply threshold: Image > Adjust > Threshold

3. Fill holes: Process > Binary > Fill Holes

4. Separate touching embryos: Process > Binary > Watershed

5. Analyze particles: Analyze > Analyze Particles

   • Set a minimum size (e.g., 10 µm²)

   • Check Display results

6. Click OK

What is the average embryo area?

Histograms¶

Histograms show the distribution of pixel intensities in an image.

1. Open Sample Image¶

Open File > Open Samples > clown (or if you have coulrophobia, use The New lenna)

2. View Histogram¶

Go to Analyze > Histogram

3. Understand the Information¶

What do these values mean?

• Count: Total number of pixels

• Mean: Average pixel intensity

• StdDev: Standard deviation of pixel intensities

4. RGB Histograms¶

1. Click the RGB button

2. Click it again

What changes do you see?

5. Color Analysis¶

Which color is least represented in this picture?

Counting and Segmenting¶

Learn how to count and segment objects in fluorescence images.

1. Open HeLa Cells¶

Open the HeLa cells sample image again: File > Open Samples > HeLa cells

2. Prepare the Red Channel¶

1. Split the channels: Image > Color > Channels Tool > More > Split channels

2. Select the red channel (mitochondria)

3. Subtract background: Process > Subtract background

3. Threshold and Count¶

1. Apply threshold: Image > Adjust > Threshold

2. Adjust the threshold to your liking

3. Click Apply

4. Count particles: Analyze > Analyze Particles

5. Check Add to manager

6. Click OK

How many objects were detected?

4. Separate Merged Objects¶

If you zoom in, some objects are merged together.

1. Separate them: Process > Binary > Watershed

2. Count particles again: Analyze > Analyze Particles

How many foci do you get now?

5. Find Maxima Method¶

An alternative method based on local intensity differences:

1. Load the red channel again (before thresholding)

2. Go to Process > Find Maxima

3. Adjust the Noise tolerance until no foci are missed

4. Check Preview to verify

How many foci are detected now?
Why is this method better than the previous ones?

Measurements¶

1. Prepare the Blue Channel¶

1. Switch to the blue channel (nuclei)

2. Create a thresholded image: Image > Adjust > Threshold

3. Click Apply

2. Configure Measurements¶

1. Go to Analyze > Set Measurements

2. Select:

   • Area

   • Feret’s diameter (longest axis)

   • Perimeter

3. Click OK

3. Measure Nuclei¶

1. Run Analyze > Analyze Particles

2. Make sure Display results is checked

3. Click OK

The results table shows measurements for each nucleus.

Segmentation and Labeling¶

1. ROI Interactive Filter¶

1. Go to Plugins > LeidenUniv > Teaching > ROI interactive filter

2. Explore the different parameters:

   • Which parameters distinguish merged blobs from single blobs?

   • Which parameter identifies homogeneous vs. variable staining?

   • What is the purpose of Exclude edges?

2. Understanding Measurements¶

When you click OK, you’ll see data for all measured objects. What do these parameters mean?

• Area: Number of pixels in the object

• Mean: Average intensity

• StdDev: Standard deviation of intensity

• Mode: Most common intensity value

• Min: Minimum intensity

• Max: Maximum intensity

• IntDen: Integrated density (sum of all pixel values)

3. Filter Embryos¶

1. Open File > Open Samples > embryos

2. Convert to 8-bit: Image > Type > 8-bit

3. Run Plugins > LeidenUniv > Teaching > ROI interactive filter

Answer these questions:

• What settings can you use to filter out doubles (touching embryos)?

• What can you do to filter out empty embryos?

The Necessity of Quantitative Measurements¶

Visual perception can be misleading. This exercise demonstrates why quantitative measurements are essential.

1. Test Visual Perception¶

1. Copy this image to FIJI

2. Measure the length of both robots:

   • Draw a line along each robot

   • Hold Shift to keep it straight

   • Record the measurements (Ctrl+M)

2. Compare Measurements¶

How big is the difference?
Does this match your visual perception?