This protocol outlines the end-to-end workflow for quantifying symbiont density, from microscope settings to automated image analysis using ImageJ/Fiji. Version: Feb 6, 2026**

1. Information and Purpose

The goal of this protocol is to standardize the calculation of algae cell concentrations (cells/mL) across different coral research projects. By using a specific “Middle Big Square” crop logic, we ensure that the area analyzed always represents a known, fixed volume of the hemocytometer (0.1 µL), regardless of the camera’s full field of view.

2. Phase 0: Imaging Protocol (Critical)

To ensure the automated script calculates volume correctly, all images must be taken using these exact settings.

Setting	Requirement
Magnification	10X Objective (Total Mag: 100X). Do not use 20X or 40X for counting.
Fluorescence Channel	Use the Chlorophyll / Red channel (Excitation: ~450-480nm, Emission: >600nm).
Exposure	Adjust so cells are bright but not saturated. Background should be dark.

Camera Settings (Binning)

• Hemocytometer (Brightfield): Full Resolution (Binning 1x1). Expected Size: ~1600 x 1600 pixels.
• Algae (Fluorescence): High Sensitivity (Binning 3x3). Expected Size: ~536 x 536 pixels.

  Note: If you change the binning, you must update the boxSize in the script (e.g., 512 for 3x3 binning, 1360 for 1x1).

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3. Mandatory File Naming Convention

The script uses String Parsing to group replicates and calculate averages. If the naming is wrong, the CSV output will be incorrect.

The Rule: SampleID_ReplicateNumber.extension

• The Underscore (_) is the Divider: The script reads everything before the first underscore as the Sample Name.
• Replicate Number: Everything after the underscore identifies the specific image.
• Example of Correct Naming:
  • S1_01.nd2, S1_02.nd2, S1_03.nd2 → Result: One row for “S1” with an average of 3 counts.
  • Coral-Alpha_01.tif, Coral-Alpha_02.tif → Result: One row for “Coral-Alpha”.

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4. The Logic: Why It Works

The script relies on a precise spatial calibration to convert “pixels” into “volume”.

Parameter	Setting/Value	Rationale
The “Cut” (Volume)	512x512 box	Represents exactly 0.1 µL. Based on Standard Neubauer Central Square = 1mm x 1mm.
The Threshold	“Triangle” Method	Optimized for “dim” cells; ignores background static better than standard “Otsu”.
The Size Filter	> 50 pixels	Removes small dust/noise (~14px) while keeping small algae (~240px).
Watershed	Active	Automatically cuts lines between cells that are touching to count them as two objects.

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5. Running the Scripts

A. Universal Automated Counter (F4)

Best for high-throughput analysis of clean images (no scale bars).

• File Name: Auto_Algae_Counter.ijm ⬇️ View/Download the ImageJ Macro File (.ijm)
• Installation: In ImageJ/Fiji, go to Plugins > Macros > Run... and select the downloaded file.

1. Launch: Open ImageJ and press F4.
2. Select Folders: Choose your Input (raw images) and Output (where CSV/JPEGs go).
3. Process: The script runs in Batch Mode. You won’t see the images opening; wait for the “Success” message.

B. Scale Bar Eraser Script (F2)

Use if your images contain a scale bar, date stamp, or any text burned into the image.

• File Name: Auto_Algae_Counter_with_Scalebar.ijm ⬇️ View/Download the ImageJ Macro File (.ijm)
• Installation: In ImageJ/Fiji, go to Plugins > Macros > Run... and select the downloaded file.

1. Launch: Press F2.
2. Manual Step: The script will open each image and beep.
3. Eraser Action:
   • Use the Rectangle tool (it selects automatically) to draw a box over the scale bar.
   • Click OK in the “waitForUser” box.
4. Result: The script paints that area black (value = 0) so the software ignores it during the count.

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6. Final Output & Verification

The CSV Results (Algae_Results_...csv)

• Conc (Cells/mL): The final concentration calculated as: $Average \times 10,000$.
• Average: The mean count of all replicates for that Sample ID.
• Counts: Raw data for every individual image to allow for outlier detection.

“Checked” Evidence Images

Every image is saved as a .jpg with Cyan Outlines around detected cells.

[!WARNING] Check the JPEGs! If you see a large cyan box around your scale bar, you forgot to use the Eraser script, and your concentration data is incorrect.

5. The Script Code (Downloadable)

To ensure the ImageJ macro runs correctly, the entire script is provided as a downloadable file. This prevents errors from broken line breaks during copy-paste.

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