# fluorojip Executive Summary

## Executive Summary

`fluorojip` is an R package for calculating chlorophyll fluorescence OJIP / JIP-test parameters, visualizing them with heatmaps and 3D plots, and exporting normalized parameter tables for downstream statistical analysis.

The main purpose of this review was to verify whether the package calculates scientifically acceptable values, especially for `PI(Abs)` and the parameters that determine it. After correction and validation, the package now performs well and produces biologically plausible outputs.

## Main Conclusion

The package is now suitable for distribution as an exploratory and analytical tool.

The strongest evidence comes from the external Biolyzer validation, which remains the primary reference standard for this review. A secondary validation using FluorPen Excel exports was also implemented and supports the corrected calculation pathway.

## Primary Validation: Biolyzer

The main external validation used:

- `inst/extdata/OJIPExporttoExcelTest001-01062024at20h31.xls`

`fluorojip` was compared against Biolyzer using `Fo`, `F(K)`, `F(J)`, `F(I)`, `Fm`, and `PI(Abs)`.

Mean absolute differences were extremely small:

- `Fv/Fm`: `0.0000201`
- `Mo`: `0.0000349`
- `ABS/RC`: `0.0000855`
- `TRo/ABS`: `0.0000201`
- `ETo/ABS`: `0.0000234`
- `PI(Abs)`: `0.0002215`

This level of agreement indicates that the corrected `fluorojip` implementation is essentially matching the vendor calculation.

## Secondary Validation: FluorPen

Three FluorPen Excel exports were also reviewed:

- `FluorPen - test 1.xlsx`
- `FluorPen - test 2.xlsx`
- `FluorPen - test 3.xlsx`

This work added direct FluorPen import support and a project-local validation workflow.

Overall mean absolute differences across 197 FluorPen traces were:

- `Fv/Fm`: `0.003225903`
- `Mo`: `0.013992607`
- `ABS/RC`: `0.086349`
- `TRo/RC`: `0.066355454`
- `ETo/RC`: `0.05382725`
- `DIo/RC`: `0.081792117`
- `Psi_o`: `0.006425699`
- `Phi_Eo`: `0.005375592`
- `PI_abs`: `0.20084551`

These differences are larger than the Biolyzer differences, mainly because one FluorPen workbook contains inconsistent decimal formatting in the vendor footer and one trace is invalid flat data. Even so, the FluorPen results support the corrected import and calculation pipeline.

## Main Improvements Made

The review produced several important improvements:

- correction of the `Mo` / RC-flux / `PI_abs` calculation path
- removal of behavior that forced invalid fluorescence steps into plausible-looking outputs
- support for raw traces expressed in seconds, milliseconds, and microseconds
- correction of the O-to-K timing logic to target `O = 0.02 ms` and `K = 0.27 ms`
- addition of direct FluorPen Excel import support
- addition of reproducible Biolyzer and FluorPen validation scripts
- stronger tests, updated examples, and clearer documentation

## Remaining Limitations

The main remaining limitations are practical rather than fundamental:

- export is still text / CSV-style rather than native `.xlsx`
- plotting is functional but basic
- broader external validation is still desirable
- FluorPen footer formatting rules should be confirmed on additional files

## Recommendation

My recommendation is to distribute the corrected package, while continuing to:

1. keep Biolyzer as the main external validation reference
2. maintain FluorPen as a secondary validation and supported import format
3. expand validation with additional real-world datasets
4. improve export and usability features in future versions

For the full technical review, see:

- [FINAL_REVIEW_REPORT.md](D:/FLUOROJIP/FINAL_REVIEW_REPORT.md)
