pHfit

Estimate intracellular pH from fluorescent indicator data using bidirectional sigmoid curve fitting.

The relationship between pH and fluorescence signal (intensity or ratio) of pH-sensitive indicators is inherently sigmoidal, governed by the Henderson-Hasselbalch equation. While a linear approximation may appear adequate over a narrow pH range near the pKa, it systematically misestimates pH at both ends of the dynamic range — precisely where many biological samples fall. A sigmoid fit faithfully captures the saturation behavior at extreme pH values, yielding accurate estimates across the full range of the indicator.

Overview

pHfit fits a Henderson-Hasselbalch-derived sigmoid function to standard curve data from fluorescent pH indicator reagents (e.g., LysoSensor, Oregon Green, pHrodo) and estimates sample pH values from the fitted curve.

Sigmoid function:

$$F(\text{pH}) = y_{\min} + \frac{y_{\max} - y_{\min}}{1 + 10^{n \cdot (pK_a - \text{pH})}}$$

Parameter	Description
y_min	Minimum signal (fully protonated)
y_max	Maximum signal (fully deprotonated)
pKa	Apparent acid dissociation constant (inflection point)
n	Hill coefficient (positive = ascending, negative = descending)

pHfit supports both ascending indicators (pH↑ → fluorescence↑, e.g., Oregon Green, BCECF) and descending indicators (pH↑ → fluorescence↓, e.g., pHrodo, LysoSensor). The direction is auto-detected from the data or inferred from the chosen preset.

Installation

pip install pHfit

For development:

git clone https://github.com/Sika-Zheng-Lab/pHfit.git
cd pHfit
pip install -e ".[dev]"

Usage

Quick start with example data

Example input files and expected output are included in the example/ directory. Try it out:

phfit -i example/standard_curve.tsv -s example/sample.tsv -o example/output --preset oregongreen488

Basic usage

phfit -i standard_curve.tsv -s sample.tsv -o output/

With a reagent preset

phfit -i standard_curve.tsv -s sample.tsv -o output/ --preset oregongreen488

Descending indicator (e.g., pHrodo)

pHrodo and similar descending indicators produce higher signal at lower pH. pHfit auto-detects the direction, or you can use a preset:

# Auto-detection
phfit -i phrodo_standard.tsv -s phrodo_sample.tsv -o output/

# With preset
phfit -i phrodo_standard.tsv -s phrodo_sample.tsv -o output/ --preset phrodo_red

A pHrodo example is included in the repository:

phfit -i example/phrodo_standard_curve.tsv -s example/phrodo_sample.tsv -o example/phrodo_output

Standard curve fitting only (no samples)

phfit -i standard_curve.tsv -o output/

List available presets

You can check all available presets and their directions with:

phfit --preset list

Estimate Hill coefficient from data

phfit -i standard_curve.tsv -s sample.tsv -o output/ --hill fit

Input File Format

standard_curve.tsv

Tab-separated file with columns pH and value. Replicates (rows with the same pH) are automatically averaged.

The value column accepts any scalar fluorescence readout — single-wavelength intensity, fluorescence ratio, or any other derived metric. The same sigmoid model applies to all cases.

pH	value
3.0	105.2
3.0	110.8
3.0	98.5
4.0	150.3
4.0	145.7
4.0	155.1
5.0	450.2
5.0	448.9
5.0	455.3
6.0	720.1
6.0	715.8
6.0	725.4
7.0	790.5
7.0	785.2
7.0	792.8

sample.tsv

Tab-separated file with columns sample and value. Replicates (rows with the same sample name) are automatically averaged.

sample	value
sampleA	250.3
sampleA	245.7
sampleA	255.1
sampleB	520.4
sampleB	518.9
sampleB	525.3

Creating TSV files from Excel

If you are not familiar with TSV files, you can easily create them from Microsoft Excel or Google Sheets:

1. Open Excel (or Google Sheets) and enter your data with the column headers (pH and value, or sample and value) in the first row.
2. Excel: Go to File → Save As, choose Text (Tab delimited) (*.txt) as the file format, and save. Then rename the file extension from .txt to .tsv.
3. Google Sheets: Go to File → Download → Tab-separated values (.tsv).

Note

Make sure the first row contains exactly the column headers pH and value (for the standard curve) or sample and value (for sample data). Do not include extra blank rows or columns.

Output Files

File	Description
fit_params.tsv	Fitted parameters (y_min, y_max, pKa, n, R²)
standard_curve.pdf	Standard curve plot (PDF)
standard_curve.png	Standard curve plot (PNG)
estimated_pH.tsv	Estimated pH for each sample (averaged)
estimated_pH_all.tsv	Estimated pH for each individual replicate
sample_estimates.pdf	Sample estimates plot (PDF)
sample_estimates.png	Sample estimates plot (PNG)
report.html	Self-contained HTML report with interactive plots
summary.json	Machine-readable run summary (version, timestamp, fit parameters, out-of-range statistics)

Interactive HTML Report

report.html is a self-contained, single-file HTML report that can be opened in any web browser. It includes:

• Fitted parameters — y_min, y_max, pKa, n (Hill coefficient), R², and sigmoid direction
• Interactive standard curve — Plotly-powered chart with hover tooltips showing exact pH and signal values; zoom, pan, and export are built-in
• Sample pH estimates — Interactive chart mapping each sample's fluorescence to estimated pH on the fitted curve (when -s is provided)
• Sigmoid equation — The fitted model equation displayed in the report header

No internet connection is required after generating the report — all Plotly assets are loaded via CDN at generation time and the chart data is embedded inline.

See an example report: example/output/report.html

CLI Options

Option	Description
-v, --version	Show version and exit
-i, --input	Path to standard curve TSV (required)
-s, --sample	Path to sample TSV (optional)
-o, --output	Output directory (required)
--preset	Reagent preset name or list
--pka	Fix pKa value
--ymin	Fix y_min value
--ymax	Fix y_max value
--hill	Hill coefficient: number to fix, or fit to estimate
--dpi	PNG resolution (default: 300)
--verbose	Enable verbose (DEBUG-level) logging output
--include-out-of-range	Include out-of-range replicates in sample mean/SD calculation (default: excluded)

Available Presets

Preset name	Reagent	pKa	Direction
oregongreen488	Oregon Green 488	4.7	↑ ascending
oregongreen514	Oregon Green 514	4.8	↑ ascending
phrodo_red	pHrodo Red	6.5	↓ descending
phrodo_green	pHrodo Green	6.5	↓ descending
lysosensor_green	LysoSensor Green DND-189	5.2	↓ descending
lysosensor_blue	LysoSensor Blue DND-167	5.1	↓ descending
lysosensor_yellowblue	LysoSensor Yellow/Blue DND-160	4.2	↓ descending
bcecf	BCECF	6.98	↑ ascending
fitc	FITC / Fluorescein	6.4	↑ ascending
snarf1	SNARF-1	7.5	↑ ascending
cypher5e	CypHer5E	7.3	↓ descending
hpts	HPTS (Pyranine)	7.3	↑ ascending

Note on Ratiometric Indicators

Some pH indicators (e.g., BCECF, SNARF-1, HPTS, LysoSensor Yellow/Blue) are measured ratiometrically — the ratio of fluorescence at two excitation or emission wavelengths is used instead of a single intensity value. The Henderson-Hasselbalch sigmoid model applies equally to ratio data; users simply provide the pre-computed fluorescence ratio in the value column of the input TSV.

Tip

For ratiometric indicators, the preset pKa represents a typical apparent pKa that may vary depending on your optical setup (filter sets, detector gains, etc.). Consider omitting --pka or using --hill fit to let pHfit estimate all parameters freely from your calibration data.

License

MIT License

Citation

If you use pHfit in your research, please cite it via its Zenodo DOI:

Kubota, N., & Zheng, S. (2026). pHfit. Zenodo. https://doi.org/10.5281/zenodo.19687851

The DOI above is the concept DOI and always resolves to the latest version. A per-version DOI is also minted automatically for each GitHub Release. Machine-readable citation metadata is provided in CITATION.cff.

Contributing

Thank you for wanting to improve pHfit! If you have any bugs or questions, feel free to open an issue or pull request. See CONTRIBUTING.md for development workflow and release process.

Authors

• Naoto Kubota (0000-0003-0612-2300)
• Sika Zheng (0000-0002-0573-4981)