🔬 SOP: Automated Algae Counting (ImageJ)

Field	Detail
Date	November 21, 2025
Scope	Fluorescence Microscopy & Hemocytometer Analysis

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1. Purpose

To automate the counting of algal cells (e.g., Symbiodiniaceae, Phytoplankton) from fluorescence microscope images.

This protocol uses a custom ImageJ macro to:

• Standardize the Count Area: Automatically crops the image to the exact volume of the Hemocytometer Central Square (0.1 µL).
• Remove Bias: Uses mathematical thresholding (“Triangle”) rather than human eye estimation.
• Handle Variable Replicates: Automatically adjusts to any number of photos per sample.

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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.

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3. The Logic: Why It Works

Before running the script, understanding the core parameters is critical.

Parameter	Setting/Value	Rationale
The “Cut” (Volume)	512x512 box	Anything inside represents exactly 0.1 µL of sample. (Derived from Standard Neubauer Central Square = 1mm x 1mm, Calibrated Brightfield Width = 1535 px, Fluorescence Width / 3 = 512 px).
The Threshold (Detection)	“Triangle” Method	This is optimized for “dim” cells and ignores background static better than the standard “Otsu” method.
The Size Filter (Noise)	> 50 pixels	This removes dust (typically ~14px) while keeping small algae (~240px).

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4. Procedure

Phase 1: Preparation

1. Organize Files: Place all your .tif, .jpg, or .png images into a single Input Folder.
2. Naming Convention: SampleID_Replicate.tif (e.g., 401_1.tif, 401_2.tif). The script groups replicates automatically.
3. Create Output Folder: Create an empty folder named “Results” or “Counted_Images”.

Phase 2: Running the Script

1. Open Fiji / ImageJ.
2. Drag the script file (AlgaeCounter.ijm) into the Fiji bar (or go to Plugins > Macros > Run).
3. Select Input Folder: Choose the folder with your images.
4. Select Output Folder: Choose your results folder.

Phase 3: The “Eraser” Step (Interactive)

The script will open images one by one and pause. For EACH image:

1. Look for the Scale Bar (or any large debris clumps).
2. Use the mouse to Draw a Box over the text/scale bar.
3. Click OK on the prompt window.
4. The script will paint the box black (ignoring it), count the algae, and move to the next image.

Phase 4: Results & Quality Control

Extracting Data to Excel

1. When finished, a window named “Log” will appear.
2. Click inside the “Log” window.
3. Press Ctrl + A (Select All) then Ctrl + C (Copy).
4. Paste into Excel.
5. Column Layout: Sample Name -> Concentration -> Average -> Count 1 -> Count 2….

   Note: Column B is always the Final Result.

Visual Verification

1. Open the output folder and check images named Checked_.....
2. Cyan Numbers indicate valid cells.
3. Verify that the scale bar is gone and faint cells are numbered.

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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.

• File Name: AlgaeCounter.ijm
• Installation: In ImageJ/Fiji, go to Plugins > Macros > Run... and select the downloaded file.

⬇️ View/Download the ImageJ Macro File (.ijm)

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